US20220193247A1 - Novel Codon-Optimized CFTR MRNA - Google Patents

Novel Codon-Optimized CFTR MRNA Download PDF

Info

Publication number
US20220193247A1
US20220193247A1 US17/570,352 US202217570352A US2022193247A1 US 20220193247 A1 US20220193247 A1 US 20220193247A1 US 202217570352 A US202217570352 A US 202217570352A US 2022193247 A1 US2022193247 A1 US 2022193247A1
Authority
US
United States
Prior art keywords
mrna
cftr
codon optimized
seq
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/570,352
Inventor
Anusha Dias
Jonathan Abysalh
Brian Bettencourt
Frank DeRosa
Michael Heartlein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Translate Bio Inc
Translate Bio MA Inc
Original Assignee
Translate Bio Inc
Translate Bio MA Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Translate Bio Inc, Translate Bio MA Inc filed Critical Translate Bio Inc
Priority to US17/570,352 priority Critical patent/US20220193247A1/en
Publication of US20220193247A1 publication Critical patent/US20220193247A1/en
Assigned to TRANSLATE BIO MA, INC. reassignment TRANSLATE BIO MA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BETTENCOURT, BRIAN, ABYSALH, Jonathan, DEROSA, FRANK, DIAS, Anusha, HEARTLEIN, MICHAEL
Assigned to TRANSLATE BIO, INC. reassignment TRANSLATE BIO, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TRANSLATE BIO MA, INC.
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions

Definitions

  • Cystic fibrosis is an autosomal inherited disorder resulting from mutation of the CFTR gene, which encodes a chloride ion channel believed to be involved in regulation of multiple other ion channels and transport systems in epithelial cells. Loss of function of CFTR results in chronic lung disease, aberrant mucus production, and dramatically reduced life expectancy. See generally Rowe et al., New Engl. J. Med. 352, 1992-2001 (2005).
  • the present invention provides, among other things, pharmaceutical compositions comprising messenger RNA (mRNA) encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and methods of making and using thereof.
  • mRNA messenger RNA
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the present invention provides pharmaceutical compositions for treating cystic fibrosis, comprising an mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1.
  • the mRNA encoding the CFTR protein comprises SEQ ID NO: 1.
  • the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4.
  • the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the mRNA encoding the CFTR protein is encapsulated within a nanoparticle.
  • the nanoparticle is a liposome.
  • the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • the liposome comprises no more than three distinct lipid components.
  • one distinct lipid component is a sterol-based cationic lipid.
  • the liposome has a size less than about 100 nm.
  • the present invention provides methods for large scale production of mRNA encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
  • a method according to the present invention comprises in vitro synthesizing mRNA encoding a CFTR protein using a SP6 RNA polymerase, wherein at least 80% of the synthesized mRNA molecules are full-length and wherein at least 100 mg of mRNA is synthesized at a single batch.
  • the in vitro synthesized mRNA encoding CFTR is substantially free of a secondary polynucleotide species of approximately 1800 nucleotides in length. In some embodiments, the in vitro synthesis of mRNA results in a secondary polynucleotide species that constitutes less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total mRNA synthesized.
  • the synthesized mRNA molecules are full-length. In some embodiments, the synthesized mRNA molecules are substantially full-length.
  • At least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA is synthesized at a single batch.
  • the CFTR protein comprises the amino acid sequence of SEQ ID NO: 3.
  • the mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1.
  • the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4.
  • the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the method further comprises a step of capping and/or tailing of the synthesized CFTR mRNA.
  • the present invention provides mRNA encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) synthesized using various methods described herein and pharmaceutical compositions containing the same.
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the present invention provides methods of delivering mRNA encoding CFTR described herein for in vivo protein expression and/or for treatment of Cystic Fibrosis.
  • the present invention provides methods of treating cystic fibrosis, comprising administering to a subject in need of treatment a composition comprising an mRNA encoding an Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein wherein the mRNA comprises a polynucleotide sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1.
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the mRNA encoding the CFTR protein comprises SEQ ID NO: 1.
  • the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4.
  • the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the mRNA encoding the CFTR protein is encapsulated within a nanoparticle.
  • the nanoparticle is a liposome.
  • the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • the liposome comprises no more than three distinct lipid components.
  • one distinct lipid component is a sterol-based cationic lipid.
  • the sterol-based cationic lipid is the imidazole cholesterol ester “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate.
  • the liposome has a size less than about 100 nm.
  • the mRNA is administered to the subject via pulmonary delivery.
  • the pulmonary delivery is nebulization.
  • FIG. 1 depicts an exemplary gel showing that synthesis of the novel codon-optimized Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) sequence using an SP6 promoter eliminated the secondary polynucleotide species (lane 2), as compared to a previous codon-optimized CFTR sequence (lane 3). Arrow indicates a secondary polynucleotide species approximately 1800 nucleotides in length.
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the term “batch” refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing order during the same cycle of manufacture.
  • a batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and/or a single aliquot of DNA template for continuous synthesis under one set of conditions.
  • a batch would include the mRNA produced from a reaction in which not all reagents and/or components are supplemented and/or replenished as the reaction progresses.
  • the term “not in a single batch” would not mean mRNA synthesized at different times that are combined to achieve the desired amount.
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).
  • patient's circulation system e.g., serum
  • systemic distribution also referred to as “systemic distribution” or “systemic delivery.
  • delivery is pulmonary delivery, e.g., comprising nebulization.
  • Encapsulation As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining an mRNA molecule within a nanoparticle.
  • expression refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody).
  • expression and “production,” and grammatical equivalents, are used interchangeably.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • the terms “improve,” “increase” or “reduce,” or grammatical equivalents indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • Impurities refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • messenger RNA As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated.
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • nucleic acid “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.
  • peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence.
  • Nucleotide sequences that encode proteins and/or RNA may include introns.
  • Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated.
  • a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaaden
  • the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.
  • nucleic acids e.g., polynucleotides and residues, including nucleotides and/or nucleosides
  • the nucleotides T and U are used interchangeably in sequence descriptions.
  • a patient refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.
  • pharmaceutically acceptable refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • the present invention provides, among other things, improved methods and pharmaceutical compositions for treating cystic fibrosis based on codon optimized messenger RNA (mRNA) encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.
  • mRNA messenger RNA
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • these codon optimized mRNA may be synthesized efficiently at a large scale by, e.g., SP6 RNA polymerase.
  • Certain codon optimized mRNA may be particularly useful for producing homogenous, safe and efficacious clinical product.
  • the present invention provides methods of producing a pharmaceutical composition comprising an mRNA, wherein the mRNA is an in vitro transcribed mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, wherein the in vitro transcribed mRNA is synthesized from a DNA template using an SP6 RNA polymerase, and wherein the synthesis of the in vitro transcribed mRNA does not result in the production of a secondary polynucleotide species of approximately 1800 nucleotides in length.
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the present invention may be used to treat a subject who is suffering from or susceptible to cystic fibrosis.
  • Cystic fibrosis is a genetic disorder characterized by mutations in the gene for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
  • the CFTR protein functions as a channel across the membrane of cells that produce mucus, sweat, saliva, tears, and digestive enzymes.
  • the channel transports negatively charged particles called chloride ions into and out of cells.
  • the transport of chloride ions helps control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus.
  • Mucus is a slippery substance that lubricates and protects the lining of the airways, digestive system, reproductive system, and other organs and tissues.
  • Respiratory symptoms of cystic fibrosis include: a persistent cough that produces thick mucus (sputum), wheezing, breathlessness, exercise intolerance, repeated lung infections and inflamed nasal passages or a stuffy nose.
  • Digestive symptoms of cystic fibrosis include: foul-smelling, greasy stools, poor weight gain and growth, intestinal blockage, particularly in newborns (meconium ileus), and severe constipation.
  • the present invention provides methods and compositions for delivering codon optimized mRNA encoding CFTR to a subject for the treatment of cystic fibrosis.
  • a suitable codon optimized CFTR mRNA encodes any full length, fragment or portion of a CFTR protein which can be substituted for naturally-occurring CFTR protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with cystic fibrosis.
  • a suitable codon optimized mRNA sequence is an mRNA sequence encoding a human CFTR (hCFTR) protein.
  • hCFTR human CFTR
  • Exemplary codon optimized CFTR mRNA coding sequence and the corresponding amino acid sequence are shown in Table 1:
  • SEQ ID NO: 7 and SEQ ID NO: 8 both of which include 5′ and 3′ untranslated regions framing a codon-optimized hCFTR-encoding mRNA and SEQ ID NO: 27 to SEQ ID NO: 40.
  • a suitable mRNA sequence may be an mRNA sequence encoding a homolog or an analog of human CFTR (hCFTR) protein.
  • hCFTR human CFTR
  • a homolog or an analog of hCFTR protein may be a modified hCFTR protein containing one or more amino acid substitutions, deletions, and/or insertions as compared to a wild-type or naturally-occurring hCFTR protein while retaining substantial hCFTR protein activity.
  • an mRNA suitable for the present invention encodes an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 3.
  • an mRNA suitable for the present invention encodes a protein substantially identical to hCFTR protein.
  • an mRNA suitable for the present invention encodes an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 3.
  • an mRNA suitable for the present invention encodes a fragment or a portion of hCFTR protein. In some embodiments, an mRNA suitable for the present invention encodes a fragment or a portion of hCFTR protein, wherein the fragment or portion of the protein still maintains CFTR activity similar to that of the wild-type protein. In some embodiments, an mRNA suitable for the present invention has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 8.
  • an mRNA suitable for the present invention has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • a suitable mRNA encodes a fusion protein comprising a full length, fragment or portion of an hCFTR protein fused to another protein (e.g., an N or C terminal fusion).
  • the protein fused to the mRNA encoding a full length, fragment or portion of an hCFTR protein encodes a signal or a cellular targeting sequence.
  • mRNAs according to the present invention may be synthesized according to any of a variety of known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7, or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • CFTR mRNA is produced using SP6 RNA Polymerase.
  • SP6 RNA Polymerase is a DNA-dependent RNA polymerase with high sequence specificity for SP6 promoter sequences.
  • the SP6 polymerase catalyzes the 5′ ⁇ 3′ in vitro synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter; it incorporates native ribonucleotides and/or modified ribonucleotides and/or labeled ribonucleotides into the polymerized transcript. Examples of such labeled ribonucleotides include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotope-labeled nucleotides.
  • An SP6 RNA polymerase suitable for the present invention can be any enzyme having substantially the same polymerase activity as bacteriophage SP6 RNA polymerase.
  • an SP6 RNA polymerase suitable for the present invention may be modified from SEQ ID NO: 9.
  • a suitable SP6 RNA polymerase may contain one or more amino acid substitutions, deletions, or additions.
  • a suitable SP6 RNA polymerase has an amino acid sequence about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, or 60% identical or homologous to SEQ ID NO: 9.
  • a suitable SP6 RNA polymerase may be a truncated protein (from N-terminus, C-terminus, or internally) but retain the polymerase activity.
  • a suitable SP6 RNA polymerase is a fusion protein.
  • An SP6 RNA polymerase suitable for the invention may be a commercially-available product, e.g., from Aldevron, Ambion, New England Biolabs (NEB), Promega, and Roche.
  • the SP6 may be ordered and/or custom designed from a commercial source or a non-commercial source according to the amino acid sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 as described herein.
  • the SP6 may be a standard-fidelity polymerase or may be a high-fidelity/high-efficiency/high-capacity which has been modified to promote RNA polymerase activities, e.g., mutations in the SP6 RNA polymerase gene or post-translational modifications of the SP6 RNA polymerase itself. Examples of such modified SP6 include SP6 RNA Polymerase-PlusTM from Ambion, HiScribe SP6 from NEB, and RiboMAXTM and Riboprobe® Systems from Promega.
  • a suitable SP6 RNA polymerase is a fusion protein.
  • an SP6 RNA polymerase may include one or more tags to promote isolation, purification, or solubility of the enzyme.
  • a suitable tag may be located at the N-terminus, C-terminus, and/or internally.
  • Non-limiting examples of a suitable tag include Calmodulin-binding protein (CBP); Fasciola hepatica 8-kDa antigen (Fh8); FLAG tag peptide; glutathione-S-transferase (GST); Histidine tag (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N-utilization substance (NusA); small ubiquitin related modifier (SUMO) fusion tag; Streptavidin binding peptide (STREP); Tandem affinity purification (TAP); and thioredoxin (TrxA).
  • CBP Calmodulin-binding protein
  • Fh8 Fasciola hepatica 8-kDa antigen
  • FLAG tag peptide e.g., hexahistidine tag (His6)
  • maltose-binding protein (MBP) N-utilization substance
  • NusA small ubiquitin related modifier
  • STREP Tandem affinity
  • an SP6 promoter comprises 5′ ATTTAGGTGACACTATAG-3′ (SEQ ID NO: 10). Variants of the SP6 promoter have been discovered and/or created to optimize recognition and/or binding of SP6 to its promoter.
  • Non-limiting variants include but are not limited to: 5′-ATTTAGGGGACACTATAGAAGAG-3′; 5′-ATTTAGGGGACACTATAGAAGG-3′; 5′-ATTTAGGGGACACTATAGAAGGG-3′; 5′-ATTTAGGTGACACTATAGAA-3′; 5′-ATTTAGGTGACACTATAGAAGA-3′; 5′-ATTTAGGTGACACTATAGAAGAG-3′; 5′-ATTTAGGTGACACTATAGAAGG-3′; 5′-ATTTAGGTGACACTATAGAAGGG-3′; 5′-ATTTAGGTGACACTATAGAAGGG-3′; 5′-ATTTAGGTGACACTATAGAAGNG-3′; and 5′-CATACGATTTAGGTGACACTATAG-3′ (SEQ ID NO: 11 to SEQ ID NO: 20).
  • a suitable SP6 promoter for the present invention may be about 95%, 90%, 85%, 80%, 75%, or 70% identical or homologous to any one of SEQ ID NO: 10 to SEQ ID NO: 20.
  • an SP6 promoter useful in the present invention may include one or more additional nucleotides 5′ and/or 3′ to any of the promoter sequences described herein.
  • a CFTR DNA template is either entirely double-stranded or mostly single-stranded with a double-stranded SP6 promoter sequence.
  • Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzyme and/or physical means), PCR products, and/or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with SP6, provided that they contain a double-stranded SP6 promoter upstream (and in the correct orientation) of the DNA sequence to be transcribed.
  • the linearized DNA template has a blunt-end.
  • the DNA sequence to be transcribed may be optimized to facilitate more efficient transcription and/or translation.
  • the DNA sequence may be optimized regarding cis-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and/or other elements relevant to transcription;
  • the DNA sequence may be optimized regarding cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability motif, and/or other elements relevant to mRNA processing and stability;
  • the DNA sequence may be optimized regarding codon usage bias, codon adaptability, internal chi sites, ribosomal binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and/or other elements relevant to translation; and/or the DNA sequence may be optimized regarding codon context, codon-anticodon interaction, translational
  • the DNA template includes a 5′ and/or 3′ untranslated region.
  • a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
  • a 5′ untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer.
  • Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule.
  • a 5′ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • IE1 immediate-early 1
  • hGH human growth hormone
  • modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
  • the present invention relates to large-scale production of codon optimized CFTR mRNA.
  • a method according to the invention synthesizes mRNA at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more at a single batch.
  • the term “batch” refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing setting.
  • a batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and/or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized at a single batch would not include mRNA synthesized at different times that are combined to achieve the desired amount.
  • a reaction mixture includes SP6 RNA polymerase, a linear DNA template, and an RNA polymerase reaction buffer (which may include ribonucleotides or may require addition of ribonucleotides).
  • At least 5 mg of SP6 polymerase is used to produce at least 1 gram of mRNA. In some embodiments, at least 500 mg of SP6 polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of SP6 polymerase is used to produce at least 1 kilogram of mRNA. In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA is used per gram of mRNA produced. In some embodiments, about 10-30 mg of plasmid DNA is used to produce about 1 gram of mRNA.
  • about 1 to 3 grams of plasmid DNA is used to produce about 100 grams of mRNA. In some embodiments, about 10 to 30 grams of plasmid DNA is used to about 1 kilogram of mRNA. In some embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.
  • the concentration of the SP6 RNA polymerase in the reaction mixture may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. In certain embodiments, the concentration of the SP6 RNA polymerase is from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM.
  • a concentration of 100 to 10000 Units/ml of the SP6 RNA polymerase may be used, as examples, concentrations of 100 to 9000 Units/ml, 100 to 8000 Units/ml, 100 to 7000 Units/ml, 100 to 6000 Units/ml, 100 to 5000 Units/ml, 100 to 1000 Units/ml, 200 to 2000 Units/ml, 500 to 1000 Units/ml, 500 to 2000 Units/ml, 500 to 3000 Units/ml, 500 to 4000 Units/ml, 500 to 5000 Units/ml, 500 to 6000 Units/ml, 1000 to 7500 Units/ml, and 2500 to 5000 Units/ml may be used.
  • the concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in a reaction mixture is between about 0.1 mM and about 10 mM, e.g., between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about 10 mM, between about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, between about 3 mM and about 8 mM, between about 3 mM and about 6 mM, between about 4 mM and about 5 mM.
  • each ribonucleotide e.g., ATP, UTP, GTP, and CTP
  • each ribonucleotide is at about 5 mM in a reaction mixture.
  • the total concentration of rNTPs for example, ATP, GTP, CTP and UTPs combined
  • the total concentration of rNTPs used in the reaction range between 1 mM and 40 mM.
  • the total concentration of rNTPs used in the reaction range between 1 mM and 30 mM, or between 1 mM and 28 mM, or between 1 mM to 25 mM, or between 1 mM and 20 mM.
  • the total rNTPs concentration is less than 30 mM.
  • the total rNTPs concentration is less than 25 mM. In some embodiments, the total rNTPs concentration is less than 20 mM. In some embodiments, the total rNTPs concentration is less than 15 mM. In some embodiments, the total rNTPs concentration is less than 10 mM.
  • the RNA polymerase reaction buffer typically includes a salt/buffering agent, e.g., Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate sodium phosphate, sodium chloride, and magnesium chloride.
  • a salt/buffering agent e.g., Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate sodium phosphate, sodium chloride, and magnesium chloride.
  • the pH of the reaction mixture may be between about 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, and in some embodiments, the pH is 7.5.
  • Linear or linearized DNA template (e.g., as described above and in an amount/concentration sufficient to provide a desired amount of RNA), the RNA polymerase reaction buffer, and SP6 RNA polymerase are combined to form the reaction mixture.
  • the reaction mixture is incubated at between about 37° C. and about 42° C. for thirty minutes to six hours, e.g., about sixty to about ninety minutes.
  • RNA polymerase reaction buffer final reaction mixture pH of about 7.5
  • a reaction mixture contains linearized double stranded DNA template with an SP6 polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT and a reaction buffer (when at 10 ⁇ is 800 mM HEPES, 20 mM spermidine, 250 mM MgCl 2 , pH 7.7) and quantity sufficient (QS) to a desired reaction volume with RNase-free water; this reaction mixture is then incubated at 37° C. for 60 minutes.
  • the polymerase reaction is then quenched by addition of DNase I and a DNase I buffer (when at 10 ⁇ is 100 mM Tris-HCl, 5 mM MgCl 2 and 25 mM CaCl 2 , pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification.
  • DNase I a DNase I buffer (when at 10 ⁇ is 100 mM Tris-HCl, 5 mM MgCl 2 and 25 mM CaCl 2 , pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification.
  • This embodiment has been shown to be sufficient to produce 100 grams of mRNA.
  • a reaction mixture includes NTPs at a concentration ranging from 1-10 mM, DNA template at a concentration ranging from 0.01-0.5 mg/ml, and SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg/ml, e.g., the reaction mixture comprises NTPs at a concentration of 5 mM, the DNA template at a concentration of 0.1 mg/ml, and the SP6 RNA polymerase at a concentration of 0.05 mg/ml.
  • an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazagua
  • the mRNA comprises one or more nonstandard nucleotide residues.
  • the nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5 mC”), pseudouridine (“ ⁇ U”), and/or 2-thio-uridine (“2sU”). See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA.
  • the mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine.
  • RNA is disclosed US Patent Publication US20120195936 and international publication WO2011012316, both of which are hereby incorporated by reference in their entirety.
  • the presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues.
  • the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications.
  • Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2′-O-alkyl modification, a locked nucleic acid (LNA)).
  • LNA locked nucleic acid
  • the RNAs may be complexed or hybridized with additional polynucleotides and/or peptide polynucleotides (PNA).
  • PNA polynucleotides and/or peptide polynucleotides
  • the sugar modification is a 2′-O-alkyl modification
  • such modification may include, but are not limited to a 2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a 2′-O-methoxyethyl modification and a 2′-deoxy modification.
  • any of these modifications may be present in 0-100% of the nucleotides—for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.
  • a 5′ cap and/or a 3′ tail may be added after the synthesis.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • a 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. Additional cap structures are described in published US Application No. US 2016/0032356 and U.S. Provisional Application 62/464,327, filed Feb. 27, 2017, which are incorporated herein by reference.
  • a tail structure includes a poly(A) and/or poly(C) tail.
  • a poly-A or poly-C tail on the 3′ terminus of mRNA typically includes at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine
  • a poly A or poly C tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to
  • a tail structure includes is a combination of poly (A) and poly (C) tails with various lengths described herein.
  • a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides.
  • a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.
  • the addition of the 5′ cap and/or the 3′ tail facilitates the detection of abortive transcripts generated during in vitro synthesis because without capping and/or tailing, the size of those prematurely aborted mRNA transcripts can be too small to be detected.
  • the 5′ cap and/or the 3′ tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA).
  • the 5′ cap and/or the 3′ tail are added to the synthesized mRNA before the mRNA is purified as described herein.
  • the 5′ cap and/or the 3′ tail are added to the synthesized mRNA after the mRNA is purified as described herein.
  • mRNA synthesized according to the present invention may be used without further purification.
  • mRNA synthesized according to the present invention may be used without a step of removing shortmers.
  • mRNA synthesized according to the present invention may be further purified.
  • Various methods may be used to purify mRNA synthesized according to the present invention. For example, purification of mRNA can be performed using centrifugation, filtration and/or chromatographic methods.
  • the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means.
  • the mRNA is purified by HPLC.
  • the mRNA is extracted in a standard phenol: chloroform: isoamyl alcohol solution, well known to one of skill in the art.
  • the mRNA is purified using Tangential Flow Filtration. Suitable purification methods include those described in US 2016/0040154, US 2015/0376220, PCT application PCT/US18/19954 entitled “METHODS FOR PURIFICATION OF MESSENGER RNA” filed on Feb. 27, 2018, and PCT application PCT/US18/19978 entitled “METHODS FOR PURIFICATION OF MESSENGER RNA” filed on Feb. 27, 2018, all of which are incorporated by reference herein and may be used to practice the present invention.
  • the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.
  • the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation.
  • the mRNA is purified either before or after or both before and after capping and tailing, by filtration.
  • the mRNA is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF).
  • the mRNA is purified either before or after or both before and after capping and tailing by chromatography.
  • full-length or abortive transcripts of mRNA may be detected and quantified using any methods available in the art.
  • the synthesized mRNA molecules are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver stain, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. Other detection methods known in the art are included in the present invention.
  • the synthesized mRNA molecules are detected using UV absorption spectroscopy with separation by capillary electrophoresis.
  • mRNA is first denatured by a Glyoxal dye before gel electrophoresis (“Glyoxal gel electrophoresis”).
  • synthesized mRNA is characterized before capping or tailing.
  • synthesized mRNA is characterized after capping and tailing.
  • mRNA generated by the method disclosed herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% impurities other than full length mRNA.
  • the impurities include IVT contaminants, e.g., proteins, enzymes, free nucleotides and/or shortmers.
  • mRNA produced according to the invention is substantially free of shortmers or abortive transcripts.
  • mRNA produced according to the invention contains undetectable level of shortmers or abortive transcripts by capillary electrophoresis or Glyoxal gel electrophoresis.
  • the term “shortmers” or “abortive transcripts” refers to any transcripts that are less than full-length.
  • “shortmers” or “abortive transcripts” are less than 100 nucleotides in length, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length.
  • shortmers are detected or quantified after adding a 5′-cap, and/or a 3′-poly A tail.
  • mRNA may be provided in a solution to be mixed with a lipid solution such that the mRNA may be encapsulated in lipid nanoparticles.
  • a suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations.
  • a suitable mRNA solution may contain an mRNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml.
  • a suitable mRNA solution may contain an mRNA at a concentration ranging from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6
  • a suitable mRNA solution may contain an mRNA at a concentration up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07 mg/ml, 0.06 mg/ml, or 0.05 mg/ml.
  • a suitable mRNA solution may also contain a buffering agent and/or salt.
  • buffering agents can include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate.
  • suitable concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM.
  • suitable concentration of the buffering agent is or greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.
  • Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride.
  • suitable concentration of salts in an mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.
  • Salt concentration in a suitable mRNA solution is or greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.
  • a suitable mRNA solution may have a pH ranging from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.
  • a suitable mRNA solution may have a pH of or no greater than about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5.
  • mRNA may be directly dissolved in a buffer solution described herein.
  • an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation.
  • an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation.
  • a suitable mRNA stock solution may contain mRNA in water at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.
  • an mRNA stock solution is mixed with a buffer solution using a pump.
  • exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps.
  • the buffer solution is mixed at a rate greater than that of the mRNA stock solution.
  • the buffer solution may be mixed at a rate at least 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , or 20 ⁇ greater than the rate of the mRNA stock solution.
  • a buffer solution is mixed at a flow rate ranging between about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute).
  • a buffer solution is mixed at a flow rate of or greater than about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute.
  • an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute.
  • mRNA encoding a CFTR protein may be delivered as naked RNA (unpackaged) or via delivery vehicles.
  • delivery vehicle delivery vehicle
  • transfer vehicle nanoparticle or grammatical equivalent
  • Delivery vehicles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid to a target cell.
  • a delivery vehicle comprising CFTR mRNA is administered by pulmonary delivery, e.g., comprising nebulization.
  • the delivery vehicle may be in an aerosolized composition which can be inhaled.
  • the mRNA is expressed in the tissue in which the delivery vehicle was administered, e.g., nasal cavity, trachea, bronchi, bronchioles, and/or other pulmonary system-related cell or tissue. Additional teaching of pulmonary delivery and nebulization are described in the related international application PCT/US17/61100 filed Nov. 10, 2017 by Applicant entitled “NOVEL ICE-BASED LIPID NANOPARTICLE FORMULATION FOR DELIVERY OF MRNA”, and the U.S. Ser. No. 62/507,061, each of which is incorporated by reference in its entirety.
  • mRNAs encoding a CFTR protein may be delivered via a single delivery vehicle.
  • mRNAs encoding a CFTR protein may be delivered via one or more delivery vehicles each of a different composition.
  • suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinylene
  • a delivery vehicle comprising CFTR mRNA may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration (e.g., local and systemic, including oral, pulmonary, and via injection), the scheduling of administration, the subject's age, sex, body weight, and other factors relevant to clinicians of ordinary skill in the art.
  • the “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts.
  • the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art.
  • a suitable amount and dosing regimen is one that causes at least transient protein production.
  • delivery vehicles are formulated such that they are suitable for extended-release of the mRNA contained therein.
  • extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • a suitable delivery vehicle is a liposomal delivery vehicle, e.g., a lipid nanoparticle.
  • liposomal delivery vehicles e.g., lipid nanoparticles
  • lipid nanoparticles are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).
  • Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
  • a nanoparticle delivery vehicle is a liposome.
  • a liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • a liposome comprises no more than three distinct lipid components.
  • one distinct lipid component is a sterol-based cationic lipid.
  • liposomes may comprise one or more cationic lipids.
  • cationic lipid refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH.
  • Several cationic lipids have been described in the literature, many of which are commercially available.
  • An example of suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (for example, CI 2-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference.
  • compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (
  • provided liposomes include a cationic lipid described in WO 2013/063468 and in U.S. provisional application entitled “Lipid Formulations for Delivery of Messenger RNA” filed concurrently with the present application on even date, both of which are incorporated by reference herein.
  • a cationic lipid comprises a compound of formula I-c1-a:
  • each R 2 independently is hydrogen or C 1-3 alkyl
  • each q independently is 2 to 6;
  • each R′ independently is hydrogen or C 1-3 alkyl
  • each R L independently is C 8-12 alkyl.
  • each R 2 independently is hydrogen, methyl or ethyl. In some embodiments, each R 2 independently is hydrogen or methyl. In some embodiments, each R 2 is hydrogen.
  • each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.
  • each R′ independently is hydrogen, methyl or ethyl. In some embodiments, each R′ independently is hydrogen or methyl. In some embodiments, each R′ independently is hydrogen.
  • each R L independently is C 8-12 alkyl. In some embodiments, each R L independently is n-C 8-12 alkyl. In some embodiments, each R L independently is C 9-11 alkyl. In some embodiments, each R L independently is n-C 9-11 alkyl. In some embodiments, each R L independently is C 10 alkyl. In some embodiments, each R L independently is n-C 10 alkyl.
  • each R 2 independently is hydrogen or methyl; each q independently is 3 to 5; each R′ independently is hydrogen or methyl; and each R L independently is C 8-12 alkyl.
  • each R 2 is hydrogen; each q independently is 3 to 5; each R′ is hydrogen; and each R L independently is C 8-12 alkyl.
  • each R 2 is hydrogen; each q is 4; each R′ is hydrogen; and each R L independently is C 8-12 alkyl.
  • a cationic lipid comprises a compound of formula I-g:
  • each R L independently is C 8-12 alkyl. In some embodiments, each R L independently is n-C 8-12 alkyl. In some embodiments, each R L independently is C 9-11 alkyl. In some embodiments, each R L independently is n-C 9-11 alkyl. In some embodiments, each R L independently is C 10 alkyl. In some embodiments, each R L is n-C 10 alkyl.
  • provided liposomes include a cationic lipid cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).
  • cKK-E12 a cationic lipid cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).
  • the structure of cKK-E12 is shown below:
  • Additional exemplary cationic lipids include those of formula I:
  • the one or more cationic lipids may be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA” (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355).
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells.
  • suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc.
  • Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”, 1, 2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”, N-dioleyl-N,N-dimethylammonium chloride or “DODAC”, N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en
  • one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • the one or more cationic lipids may be chosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraaza
  • sterol-based cationic lipids are dialkylamino-, imidazole-, and guanidinium-containing sterol-based cationic lipids.
  • certain embodiments are directed to a composition comprising one or more sterol-based cationic lipids comprising an imidazole, for example, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (II) below.
  • imidazole cholesterol ester or “ICE” lipid 3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,
  • a lipid nanoparticle for delivery of RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (II).
  • imidazole cholesterol ester or “ICE” lipid 3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-
  • the percentage of cationic lipid in a liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.
  • cationic lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight.
  • the cationic lipid e.g., ICE lipid
  • provided liposomes contain one or more non-cationic (“helper”) lipids.
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE
  • non-cationic lipids may be used alone, but are preferably used in combination with other lipids, for example, cationic lipids.
  • the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipid present in a liposome.
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • provided liposomes comprise one or more cholesterol-based lipids.
  • suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.
  • the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • PEG polyethylene glycol
  • PEG-CER derivatized ceramides
  • C8 PEG-2000 ceramide C8 PEG-2000 ceramide
  • Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to S kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
  • Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18).
  • the PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.
  • the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the MCNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.
  • a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein.
  • liposomal delivery vehicles as used herein, also encompass nanoparticles comprising polymers.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI).
  • PEI polyethylenimine
  • a suitable liposome for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids and/or polymers described herein at various ratios.
  • a suitable liposome formulation may include a combination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, and DMG-PEG2K.
  • cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio.
  • the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) is or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome by molar ratio.
  • the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5, respectively.
  • the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.
  • the ratio of total lipid content i.e., the ratio of lipid component (1):lipid component (2):lipid component (3)
  • x:y:z the ratio of lipid component (1):lipid component (2):lipid component (3)
  • each of “x,” “y,” and “z” represents molar percentages of the three distinct components of lipids, and the ratio is a molar ratio.
  • each of “x,” “y,” and “z” represents weight percentages of the three distinct components of lipids, and the ratio is a weight ratio.
  • lipid component (1) is a sterol-based cationic lipid.
  • lipid component (2) is a helper lipid.
  • lipid component (3) represented by variable “z” is a PEG lipid.
  • variable “x,” representing the molar percentage of lipid component (1) is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • variable “x,” representing the molar percentage of lipid component (1) is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%.
  • variable “x,” representing the molar percentage of lipid component (1) is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • variable “x,” representing the weight percentage of lipid component (1) is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • variable “x,” representing the weight percentage of lipid component (1) is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%.
  • variable “x,” representing the weight percentage of lipid component (1) is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • variable “z,” representing the molar percentage of lipid component (3) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.
  • variable “z,” representing the molar percentage of lipid component (3) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.
  • variable “z,” representing the weight percentage of lipid component (3) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.
  • variable “z,” representing the weight percentage of lipid component (3) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.
  • variables “x,” “y,” and “z” may be in any combination so long as the total of the three variables sums to 100% of the total lipid content.
  • the liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques which are presently known in the art.
  • the liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art.
  • multilamellar vesicles may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs.
  • Unilamellar vesicles can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles.
  • unilamellar vesicles can be formed by detergent removal techniques.
  • compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome.
  • cationic liposomes may associate with the mRNA through electrostatic interactions.
  • cationic liposomes may associate with the mRNA through electrostatic interactions.
  • the compositions and methods of the invention comprise mRNA encapsulated in a liposome.
  • the one or more mRNA species may be encapsulated in the same liposome.
  • the one or more mRNA species may be encapsulated in different liposomes.
  • the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands and/or combinations thereof.
  • the one or more liposome may have a different composition of sterol-based cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof.
  • the one or more liposomes may have a different molar ratio of cholesterol-based cationic lipid, neutral lipid, and PEG-modified lipid used to create the liposome.
  • the process of incorporation of a desired mRNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference.
  • the liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane.
  • the incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome.
  • a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA to the target cell or tissue.
  • Suitable liposomes in accordance with the present invention may be made in various sizes.
  • provided liposomes may be made smaller than previously known mRNA encapsulating liposomes.
  • decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.
  • an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA.
  • a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • the size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • QELS quasi-electric light scattering
  • CFTR Codon-optimized Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) messenger RNA was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides in length as determined by gel electrophoresis. 5′ and 3′ untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated (vide infra).
  • An exemplary codon-optimized human CFTR mRNA sequence includes SEQ ID NO: 1 as described in the detailed description section.
  • a previous hCFTR sequence (SEQ ID NO: 2) was codon-optimized using a T7 promoter.
  • “cleaner” mRNA was synthesized with respect to pre-aborted sequences, but a second species of approximately 1800 nt (“longmer”) was being produced in low quantities. This was visualized by gel electrophoresis as depicted in FIG. 1 .
  • lane 1 contains an RNA ladder
  • lane 2 contains mRNA of SEQ ID NO: 1
  • lane 3 contains mRNA of SEQ ID NO: 2.
  • a secondary polynucleotide species approximately 1800 nucleotides in length is present in lane 3.
  • Several new sequences (relative to SEQ ID NO: 2) were designed with site mutations to remove suspected cryptic promoters, but that did not result in the disappearance of the ⁇ 1800 nt secondary species.
  • Complete codon-re-optimization was performed to create SEQ ID NO: 1, which successfully led to an mRNA product without the additional production of the second species at ⁇ 1800 nt (lane 1).
  • SEQ ID NO: 1 is particularly useful in a homogenous, safe and efficacious pharmaceutical composition.

Abstract

The present invention provides, among other things, improved methods and pharmaceutical compositions for treating cystic fibrosis based on codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.

Description

    RELATED APPLICATIONS
  • This application claims priority to U.S. Provisional Application Ser. No. 62/464,215, filed Feb. 27, 2017, the disclosures of which are hereby incorporated by reference.
    • SEQUENCE LISTING
  • The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named MRT-2001 US_ST25 on Feb. 27, 2018). The .txt file was generated on date and is 166,293 bytes in size. The entire contents of the sequence are herein incorporated by reference.
    • BACKGROUND
  • Cystic fibrosis is an autosomal inherited disorder resulting from mutation of the CFTR gene, which encodes a chloride ion channel believed to be involved in regulation of multiple other ion channels and transport systems in epithelial cells. Loss of function of CFTR results in chronic lung disease, aberrant mucus production, and dramatically reduced life expectancy. See generally Rowe et al., New Engl. J. Med. 352, 1992-2001 (2005).
  • Currently there is no cure for cystic fibrosis. The literature has documented numerous difficulties encountered in attempting to induce expression of CFTR in the lung. For example, viral vectors comprising CFTR DNA triggered immune responses and CF symptoms persisted after administration. Conese et al., J. Cyst. Fibros. 10 Suppl 2, S114-28 (2011); Rosenecker et al., Curr. Opin. Mol. Ther. 8, 439-45 (2006). Non-viral delivery of DNA, including CFTR DNA, has also been reported to trigger immune responses. Alton et al., Lancet 353, 947-54 (1999); Rosenecker et al., J Gene Med. 5, 49-60 (2003). Furthermore, non-viral DNA vectors encounter the additional problem that the machinery of the nuclear pore complex does not ordinarily import DNA into the nucleus, where transcription would occur. Pearson, Nature 460, 164-69 (2009).
    • SUMMARY OF THE INVENTION
  • The present invention provides, among other things, pharmaceutical compositions comprising messenger RNA (mRNA) encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and methods of making and using thereof. These pharmaceutical compositions can be used for improved treatment of cystic fibrosis.
  • In one aspect, the present invention provides pharmaceutical compositions for treating cystic fibrosis, comprising an mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises SEQ ID NO: 1. In some embodiments, the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4. In some embodiments, the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • In some embodiments, the mRNA encoding the CFTR protein is encapsulated within a nanoparticle. In some embodiments, the nanoparticle is a liposome. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In some embodiments, the liposome comprises no more than three distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. In some embodiments, the liposome has a size less than about 100 nm. In another aspect, the present invention provides methods for large scale production of mRNA encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In some embodiments, a method according to the present invention comprises in vitro synthesizing mRNA encoding a CFTR protein using a SP6 RNA polymerase, wherein at least 80% of the synthesized mRNA molecules are full-length and wherein at least 100 mg of mRNA is synthesized at a single batch.
  • In some embodiments, the in vitro synthesized mRNA encoding CFTR is substantially free of a secondary polynucleotide species of approximately 1800 nucleotides in length. In some embodiments, the in vitro synthesis of mRNA results in a secondary polynucleotide species that constitutes less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total mRNA synthesized.
  • In some embodiments, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the synthesized mRNA molecules are full-length. In some embodiments, the synthesized mRNA molecules are substantially full-length.
  • In some embodiments, at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA is synthesized at a single batch.
  • In some embodiments, the CFTR protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. In some embodiments, the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4. In some embodiments, the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • In some embodiments, the method further comprises a step of capping and/or tailing of the synthesized CFTR mRNA.
  • Among other things, the present invention provides mRNA encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) synthesized using various methods described herein and pharmaceutical compositions containing the same.
  • In yet another aspect, the present invention provides methods of delivering mRNA encoding CFTR described herein for in vivo protein expression and/or for treatment of Cystic Fibrosis. In some embodiments, the present invention provides methods of treating cystic fibrosis, comprising administering to a subject in need of treatment a composition comprising an mRNA encoding an Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein wherein the mRNA comprises a polynucleotide sequence at least 85% (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1.
  • In some embodiments, the mRNA encoding the CFTR protein comprises SEQ ID NO: 1. In some embodiments, the mRNA further comprises a 5′ untranslated region (UTR) sequence of SEQ ID NO: 4. In some embodiments, the mRNA further comprises a 3′ untranslated region (UTR) sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • In some embodiments, the mRNA encoding the CFTR protein is encapsulated within a nanoparticle. In some embodiments, the nanoparticle is a liposome. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In some embodiments, the liposome comprises no more than three distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. In some embodiments, the sterol-based cationic lipid is the imidazole cholesterol ester “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate. In some embodiments, the liposome has a size less than about 100 nm.
  • In some embodiments, the mRNA is administered to the subject via pulmonary delivery. In some embodiments, the pulmonary delivery is nebulization.
  • Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.
    • BRIEF DESCRIPTION OF THE DRAWING
  • The drawings are for illustration purposes only not for limitation.
  • FIG. 1 depicts an exemplary gel showing that synthesis of the novel codon-optimized Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) sequence using an SP6 promoter eliminated the secondary polynucleotide species (lane 2), as compared to a previous codon-optimized CFTR sequence (lane 3). Arrow indicates a secondary polynucleotide species approximately 1800 nucleotides in length.
    •  Definitions
  • In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
  • Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • As used herein, the term “batch” refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing order during the same cycle of manufacture. A batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and/or a single aliquot of DNA template for continuous synthesis under one set of conditions. In some embodiments, a batch would include the mRNA produced from a reaction in which not all reagents and/or components are supplemented and/or replenished as the reaction progresses. The term “not in a single batch” would not mean mRNA synthesized at different times that are combined to achieve the desired amount.
  • Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.
  • Encapsulation: As used herein, the term “encapsulation,” or grammatical equivalent, refers to the process of confining an mRNA molecule within a nanoparticle.
  • Expression: As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms “expression” and “production,” and grammatical equivalents, are used interchangeably.
  • Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life: As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • Impurities: As used herein, the term “impurities” refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.
  • In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • In Vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated: As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated.
  • Nucleic acid: As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguano sine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.
  • Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.
  • Pharmaceutically acceptable: The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
    • DETAILED DESCRIPTION
  • The present invention provides, among other things, improved methods and pharmaceutical compositions for treating cystic fibrosis based on codon optimized messenger RNA (mRNA) encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. In particular, these codon optimized mRNA may be synthesized efficiently at a large scale by, e.g., SP6 RNA polymerase. Certain codon optimized mRNA may be particularly useful for producing homogenous, safe and efficacious clinical product.
  • In some embodiments, the present invention provides methods of producing a pharmaceutical composition comprising an mRNA, wherein the mRNA is an in vitro transcribed mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, wherein the in vitro transcribed mRNA is synthesized from a DNA template using an SP6 RNA polymerase, and wherein the synthesis of the in vitro transcribed mRNA does not result in the production of a secondary polynucleotide species of approximately 1800 nucleotides in length.
    • Cystic Fibrosis
  • The present invention may be used to treat a subject who is suffering from or susceptible to cystic fibrosis. Cystic fibrosis is a genetic disorder characterized by mutations in the gene for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The CFTR protein functions as a channel across the membrane of cells that produce mucus, sweat, saliva, tears, and digestive enzymes. The channel transports negatively charged particles called chloride ions into and out of cells. The transport of chloride ions helps control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus. Mucus is a slippery substance that lubricates and protects the lining of the airways, digestive system, reproductive system, and other organs and tissues.
  • Respiratory symptoms of cystic fibrosis include: a persistent cough that produces thick mucus (sputum), wheezing, breathlessness, exercise intolerance, repeated lung infections and inflamed nasal passages or a stuffy nose. Digestive symptoms of cystic fibrosis include: foul-smelling, greasy stools, poor weight gain and growth, intestinal blockage, particularly in newborns (meconium ileus), and severe constipation.
  • Codon Optimized mRNA Encoding CFTR
  • In some embodiments, the present invention provides methods and compositions for delivering codon optimized mRNA encoding CFTR to a subject for the treatment of cystic fibrosis. A suitable codon optimized CFTR mRNA encodes any full length, fragment or portion of a CFTR protein which can be substituted for naturally-occurring CFTR protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with cystic fibrosis.
  • In some embodiments, a suitable codon optimized mRNA sequence is an mRNA sequence encoding a human CFTR (hCFTR) protein. Exemplary codon optimized CFTR mRNA coding sequence and the corresponding amino acid sequence are shown in Table 1:
  • TABLE 1
    Exemplary Codon-Optimized Human CFTR
    SEQ ID AUGCAACGCUCUCCUCUUGAAAAGGCCUCGGUGGUGUCCAAGCUCUU
    NO: 1 CUUCUCGUGGACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCU
    UGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGAC
    AACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUC
    AAAGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUC
    UGGCGGUUCAUGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCAC
    CAAGGCCGUGCAGCCCCUGUUGCUGGGACGGAUUAUUGCCUCCUACG
    ACCCCGACAACAAGGAAGAAAGAAGCAUCGCUAUCUACUUGGGCAUC
    GGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCUUGUUGCAUCCUGC
    UAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUGCCAUG
    UUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCU
    UGACAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUC
    UGAACAAGUUCGACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUC
    GCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAUCUGGGAGCUGCU
    GCAAGCCUCGGCAUUCUGUGGGCUUGGAUUCCUGAUCGUGCUGGCAC
    UGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGAAGUACAGGGACCA
    GAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGGAAAUG
    AUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAU
    GGAAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCC
    GCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUC
    UCCGGGUUCUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAU
    UAAGGGAAUCAUCCUCAGGAAGAUCUUCACCACCAUUUCCUUCUGUA
    UCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAG
    ACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCU
    UCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCG
    AGGUCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGG
    CGAACUGUUCGAGAAGGCCAAGCAGAACAACAACAACCGCAAGACCU
    CGAACGGUGACGACUCCCUCUUCUUUUCAAACUUCAGCCUGCUCGGG
    ACGCCCGUGCUGAAGGACAUUAACUUCAAGAUCGAAAGAGGACAGCU
    CCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAGACUUCCCUGCUGA
    UGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUCAAGCA
    CUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCG
    GAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUAC
    CGCUACCGGUCCGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUC
    AAAGUUCGCGGAGAAAGAUAACAUCGUGCUGGGCGAAGGGGGUAUU
    ACCUUGUCGGGGGGCCAGCGGGCUAGAAUCUCGCUGGCCAGAGCCGU
    GUAUAAGGACGCCGACCUGUAUCUCCUGGACUCCCCCUUCGGAUACC
    UGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGUGCAA
    GCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGC
    ACCUGAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCC
    UACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUU
    CUCAUCGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCG
    AAAGAAGGAACUCGAUCCUGACGGAAACCUUGCACCGCUUCUCUUUG
    GAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUU
    CAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUG
    AACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCC
    ACUGCAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGA
    GGCGCCUGUCCCUGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUG
    CCUCGGAUUUCCGUGAUCUCCACUGGUCCGACGCUCCAAGCCCGGCG
    GCGGCAGUCCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCC
    AAAACAUUCACCGCAAGACUACCGCAUCCACCCGGAAAGUGUCCCUG
    GCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAGACU
    GUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAG
    GAUCUGAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGU
    GACGACUUGGAACACUUAUCUGCGGUACAUCACUGUGCACAAGUCAU
    UGAUCUUCGUGCUGAUUUGGUGCCUGGUGAUUUUCCUGGCCGAGGU
    CGCGGCCUCACUGGUGGUGCUCUGGCUGUUGGGAAACACGCCUCUGC
    AAGACAAGGGAAACUCCACGCACUCGAGAAACAACAGCUAUGCCGUG
    AUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGUCGG
    AGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGC
    UGGUCCACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUG
    UUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACUCUGAA
    GGCCGGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCCUGG
    ACGAUCUCCUGCCGCUUACCAUCUUUGACUUCAUCCAGCUGCUGCUG
    AUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACA
    UUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGG
    GCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGA
    GGGACGAUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGAC
    UGUGGACCCUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUC
    UUCCACAAGGCCCUGAACCUCCACACCGCCAAUUGGUUCCUGUACCU
    GUCCACCCUGCGGUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGUCA
    UCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACUACCGGAGAG
    GGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUUAU
    GAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCC
    UGAUGCGAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACU
    GAGGGAAAACCCACUAAGUCCACUAAGCCCUACAAAAAUGGCCAGCU
    GAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAGAAGGACGAU
    AUUUGGCCCUCCGGAGGUCAAAUGACCGUGAAGGACCUGACCGCAAA
    GUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAGCUUCUCCA
    UUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGG
    AAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGG
    GGAAAUCCAAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGC
    AGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUC
    UCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAGUGGAGCGA
    CCAAGAAAUCUGGAAGGUCGCCGACGAGGUCGGCCUGCGCUCCGUGA
    UUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGACGGGGG
    AUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGU
    CCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCC
    CACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCA
    GGCCUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGG
    CCAUGCUGGAGUGCCAGCAGUUCCUGGUCAUCGAGGAGAACAAGGUC
    CGCCAAUACGACUCCAUUCAAAAGCUCCUCAACGAGCGGUCGCUGUU
    CAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUC
    GGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAG
    GAAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAA
    SEQ ID AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUU
    NO: 2 CUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGC
    UUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAU
    AACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUC
    UAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCU
    GGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCAC
    AAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACG
    ACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUC
    GGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGC
    AAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGU
    UUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUU
    GGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAAC
    CUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGA
    UUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUG
    UUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGG
    CAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGA
    CCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAA
    UGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGC
    UAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUG
    ACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUU
    CUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCU
    UGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUC
    UGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGU
    GCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACU
    UCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACU
    ACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUU
    UUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAA
    GACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGC
    UCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGG
    ACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCC
    UCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAU
    UAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCA
    UGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGA
    UGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAG
    GACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAG
    GGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCG
    AGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGU
    UUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUG
    CGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCA
    AAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGA
    AGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGC
    AGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAG
    UUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCG
    AUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGA
    AGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAA
    CAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCC
    AGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGA
    ACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGG
    AGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUU
    CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGU
    AAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA
    AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUAC
    AGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAA
    UCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUC
    AAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGG
    UGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUU
    CUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAA
    UACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUU
    CCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUAC
    AUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCG
    AGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCC
    ACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUG
    AAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUA
    UUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUC
    CAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCC
    UCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUU
    AUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCA
    ACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCA
    GUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUAC
    UUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUG
    GUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAG
    AUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCU
    UGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUC
    GCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGA
    UUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUU
    CAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCU
    AUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCA
    CGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUG
    AAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA
    AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCG
    GGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAG
    ACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGG
    GAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCC
    CCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUC
    CUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGA
    GGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGAC
    UUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGC
    AGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUU
    CUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAU
    CAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUC
    UCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUU
    GUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCU
    GCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUA
    GGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAA
    ACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAA
    GACACGCGUCUUUAA
    Human MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEK
    CFTR LEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLL
    Protein GRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIA
    Sequence MFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQ
    VALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKIS
    ERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFN
    SSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQT
    WYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFE
    KAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTG
    AGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDE
    YRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKD
    ADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKI
    LILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHR
    FSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQ
    MNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNL
    MTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEI
    NEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAA
    SLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLL
    AMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSK
    DIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAY
    FLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALN
    LHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLA
    MNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQ
    LSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQ
    RVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIP
    QKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVD
    GGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAF
    ADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPS
    DRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 3)
  • Additional exemplary codon optimized mRNA sequences are described in the Examples section below, for example, SEQ ID NO: 7 and SEQ ID NO: 8, both of which include 5′ and 3′ untranslated regions framing a codon-optimized hCFTR-encoding mRNA and SEQ ID NO: 27 to SEQ ID NO: 40.
  • In some embodiments, a suitable mRNA sequence may be an mRNA sequence encoding a homolog or an analog of human CFTR (hCFTR) protein. For example, a homolog or an analog of hCFTR protein may be a modified hCFTR protein containing one or more amino acid substitutions, deletions, and/or insertions as compared to a wild-type or naturally-occurring hCFTR protein while retaining substantial hCFTR protein activity. In some embodiments, an mRNA suitable for the present invention encodes an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 3. In some embodiments, an mRNA suitable for the present invention encodes a protein substantially identical to hCFTR protein. In some embodiments, an mRNA suitable for the present invention encodes an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 3. In some embodiments, an mRNA suitable for the present invention encodes a fragment or a portion of hCFTR protein. In some embodiments, an mRNA suitable for the present invention encodes a fragment or a portion of hCFTR protein, wherein the fragment or portion of the protein still maintains CFTR activity similar to that of the wild-type protein. In some embodiments, an mRNA suitable for the present invention has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 8.
  • In some embodiments, an mRNA suitable for the present invention has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • In some embodiments, a suitable mRNA encodes a fusion protein comprising a full length, fragment or portion of an hCFTR protein fused to another protein (e.g., an N or C terminal fusion). In some embodiments, the protein fused to the mRNA encoding a full length, fragment or portion of an hCFTR protein encodes a signal or a cellular targeting sequence.
  • Synthesis of mRNA
  • mRNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
  • In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • Synthesis of mRNA Using SP6 RNA Polymerase
  • In some embodiments, CFTR mRNA is produced using SP6 RNA Polymerase. SP6 RNA Polymerase is a DNA-dependent RNA polymerase with high sequence specificity for SP6 promoter sequences. The SP6 polymerase catalyzes the 5′→3′ in vitro synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter; it incorporates native ribonucleotides and/or modified ribonucleotides and/or labeled ribonucleotides into the polymerized transcript. Examples of such labeled ribonucleotides include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotope-labeled nucleotides.
  • The sequence for bacteriophage SP6 RNA polymerase was initially described (GenBank: Y00105.1) as having the following amino acid sequence:
  • (SEQ ID NO: 9)
    MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSE
    LIAPMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMD
    MLNTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRT
    KSYRHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSV
    FYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYA
    PCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVY
    KAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPAN
    PVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSA
    AVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKAL
    LRFTEGRPVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDM
    CRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTH
    LPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVI
    KKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPV
    MTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDR
    QDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEG
    LMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMM
    GAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTL
    RVALKGQMVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEI
    MDSEYVFA.
  • An SP6 RNA polymerase suitable for the present invention can be any enzyme having substantially the same polymerase activity as bacteriophage SP6 RNA polymerase. Thus, in some embodiments, an SP6 RNA polymerase suitable for the present invention may be modified from SEQ ID NO: 9. For example, a suitable SP6 RNA polymerase may contain one or more amino acid substitutions, deletions, or additions. In some embodiments, a suitable SP6 RNA polymerase has an amino acid sequence about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, or 60% identical or homologous to SEQ ID NO: 9. In some embodiments, a suitable SP6 RNA polymerase may be a truncated protein (from N-terminus, C-terminus, or internally) but retain the polymerase activity. In some embodiments, a suitable SP6 RNA polymerase is a fusion protein.
  • An SP6 RNA polymerase suitable for the invention may be a commercially-available product, e.g., from Aldevron, Ambion, New England Biolabs (NEB), Promega, and Roche. The SP6 may be ordered and/or custom designed from a commercial source or a non-commercial source according to the amino acid sequence of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 as described herein. The SP6 may be a standard-fidelity polymerase or may be a high-fidelity/high-efficiency/high-capacity which has been modified to promote RNA polymerase activities, e.g., mutations in the SP6 RNA polymerase gene or post-translational modifications of the SP6 RNA polymerase itself. Examples of such modified SP6 include SP6 RNA Polymerase-Plus™ from Ambion, HiScribe SP6 from NEB, and RiboMAX™ and Riboprobe® Systems from Promega.
  • In some embodiments, a suitable SP6 RNA polymerase is a fusion protein. For example, an SP6 RNA polymerase may include one or more tags to promote isolation, purification, or solubility of the enzyme. A suitable tag may be located at the N-terminus, C-terminus, and/or internally. Non-limiting examples of a suitable tag include Calmodulin-binding protein (CBP); Fasciola hepatica 8-kDa antigen (Fh8); FLAG tag peptide; glutathione-S-transferase (GST); Histidine tag (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N-utilization substance (NusA); small ubiquitin related modifier (SUMO) fusion tag; Streptavidin binding peptide (STREP); Tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags may be used in the present invention. These and other fusion tags have been described, e.g., Costa et al. Frontiers in Microbiology 5 (2014): 63 and in PCT/US16/57044, the contents of which are incorporated herein by reference in their entireties. In certain embodiments, a His tag is located at SP6's N-terminus.
  • SP6 Promoter
  • Any promoter that can be recognized by an SP6 RNA polymerase may be used in the present invention. Typically, an SP6 promoter comprises 5′ ATTTAGGTGACACTATAG-3′ (SEQ ID NO: 10). Variants of the SP6 promoter have been discovered and/or created to optimize recognition and/or binding of SP6 to its promoter. Non-limiting variants include but are not limited to: 5′-ATTTAGGGGACACTATAGAAGAG-3′; 5′-ATTTAGGGGACACTATAGAAGG-3′; 5′-ATTTAGGGGACACTATAGAAGGG-3′; 5′-ATTTAGGTGACACTATAGAA-3′; 5′-ATTTAGGTGACACTATAGAAGA-3′; 5′-ATTTAGGTGACACTATAGAAGAG-3′; 5′-ATTTAGGTGACACTATAGAAGG-3′; 5′-ATTTAGGTGACACTATAGAAGGG-3′; 5′-ATTTAGGTGACACTATAGAAGNG-3′; and 5′-CATACGATTTAGGTGACACTATAG-3′ (SEQ ID NO: 11 to SEQ ID NO: 20).
  • In addition, a suitable SP6 promoter for the present invention may be about 95%, 90%, 85%, 80%, 75%, or 70% identical or homologous to any one of SEQ ID NO: 10 to SEQ ID NO: 20. Moreover, an SP6 promoter useful in the present invention may include one or more additional nucleotides 5′ and/or 3′ to any of the promoter sequences described herein.
  • DNA Template
  • Typically, a CFTR DNA template is either entirely double-stranded or mostly single-stranded with a double-stranded SP6 promoter sequence.
  • Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzyme and/or physical means), PCR products, and/or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with SP6, provided that they contain a double-stranded SP6 promoter upstream (and in the correct orientation) of the DNA sequence to be transcribed.
  • In some embodiments, the linearized DNA template has a blunt-end.
  • In some embodiments, the DNA sequence to be transcribed may be optimized to facilitate more efficient transcription and/or translation. For example, the DNA sequence may be optimized regarding cis-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and/or other elements relevant to transcription; the DNA sequence may be optimized regarding cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability motif, and/or other elements relevant to mRNA processing and stability; the DNA sequence may be optimized regarding codon usage bias, codon adaptability, internal chi sites, ribosomal binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and/or other elements relevant to translation; and/or the DNA sequence may be optimized regarding codon context, codon-anticodon interaction, translational pause sites, and/or other elements relevant to protein folding. Optimization methods known in the art may be used in the present invention, e.g., GeneOptimizer by ThermoFisher and OptimumGene™, which are described in US 20110081708, the contents of which are incorporated herein by reference in its entirety.
  • In some embodiments, the DNA template includes a 5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length.
  • In some embodiments, a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer.
  • Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5′ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3′ end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
  • Large-Scale mRNA Synthesis
  • The present invention relates to large-scale production of codon optimized CFTR mRNA. In some embodiments, a method according to the invention synthesizes mRNA at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more at a single batch. As used herein, the term “batch” refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing setting. A batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and/or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized at a single batch would not include mRNA synthesized at different times that are combined to achieve the desired amount. Generally, a reaction mixture includes SP6 RNA polymerase, a linear DNA template, and an RNA polymerase reaction buffer (which may include ribonucleotides or may require addition of ribonucleotides).
  • According to the present invention, 1-100 mg of SP6 polymerase is typically used per gram (g) of mRNA produced. In some embodiments, about 1-90 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg of SP6 polymerase is used per gram of mRNA produced. In some embodiments, about 5-20 mg of SP6 polymerase is used to produce about 1 gram of mRNA. In some embodiments, about 0.5 to 2 grams of SP6 polymerase is used to produce about 100 grams of mRNA. In some embodiments, about 5 to 20 grams of SP6 polymerase is used to about 1 kilogram of mRNA. In some embodiments, at least 5 mg of SP6 polymerase is used to produce at least 1 gram of mRNA. In some embodiments, at least 500 mg of SP6 polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of SP6 polymerase is used to produce at least 1 kilogram of mRNA. In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA is used per gram of mRNA produced. In some embodiments, about 10-30 mg of plasmid DNA is used to produce about 1 gram of mRNA. In some embodiments, about 1 to 3 grams of plasmid DNA is used to produce about 100 grams of mRNA. In some embodiments, about 10 to 30 grams of plasmid DNA is used to about 1 kilogram of mRNA. In some embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.
  • In some embodiments, the concentration of the SP6 RNA polymerase in the reaction mixture may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. In certain embodiments, the concentration of the SP6 RNA polymerase is from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. A concentration of 100 to 10000 Units/ml of the SP6 RNA polymerase may be used, as examples, concentrations of 100 to 9000 Units/ml, 100 to 8000 Units/ml, 100 to 7000 Units/ml, 100 to 6000 Units/ml, 100 to 5000 Units/ml, 100 to 1000 Units/ml, 200 to 2000 Units/ml, 500 to 1000 Units/ml, 500 to 2000 Units/ml, 500 to 3000 Units/ml, 500 to 4000 Units/ml, 500 to 5000 Units/ml, 500 to 6000 Units/ml, 1000 to 7500 Units/ml, and 2500 to 5000 Units/ml may be used.
  • The concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in a reaction mixture is between about 0.1 mM and about 10 mM, e.g., between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about 10 mM, between about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, between about 3 mM and about 8 mM, between about 3 mM and about 6 mM, between about 4 mM and about 5 mM. In some embodiments, each ribonucleotide is at about 5 mM in a reaction mixture. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP and UTPs combined) used in the reaction range between 1 mM and 40 mM. In some embodiments, the total concentration of rNTPs (for example, ATP, GTP, CTP and UTPs combined) used in the reaction range between 1 mM and 30 mM, or between 1 mM and 28 mM, or between 1 mM to 25 mM, or between 1 mM and 20 mM. In some embodiments, the total rNTPs concentration is less than 30 mM. In some embodiments, the total rNTPs concentration is less than 25 mM. In some embodiments, the total rNTPs concentration is less than 20 mM. In some embodiments, the total rNTPs concentration is less than 15 mM. In some embodiments, the total rNTPs concentration is less than 10 mM.
  • The RNA polymerase reaction buffer typically includes a salt/buffering agent, e.g., Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate sodium phosphate, sodium chloride, and magnesium chloride.
  • The pH of the reaction mixture may be between about 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, and in some embodiments, the pH is 7.5.
  • Linear or linearized DNA template (e.g., as described above and in an amount/concentration sufficient to provide a desired amount of RNA), the RNA polymerase reaction buffer, and SP6 RNA polymerase are combined to form the reaction mixture. The reaction mixture is incubated at between about 37° C. and about 42° C. for thirty minutes to six hours, e.g., about sixty to about ninety minutes.
  • In some embodiments, about 5 mM NTPs, about 0.05 mg/mL SP6 polymerase, and about 0.1 mg/ml DNA template in a suitable RNA polymerase reaction buffer (final reaction mixture pH of about 7.5) is incubated at about 37° C. to about 42° C. for sixty to ninety minutes.
  • In some embodiments, a reaction mixture contains linearized double stranded DNA template with an SP6 polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT and a reaction buffer (when at 10× is 800 mM HEPES, 20 mM spermidine, 250 mM MgCl2, pH 7.7) and quantity sufficient (QS) to a desired reaction volume with RNase-free water; this reaction mixture is then incubated at 37° C. for 60 minutes. The polymerase reaction is then quenched by addition of DNase I and a DNase I buffer (when at 10× is 100 mM Tris-HCl, 5 mM MgCl2 and 25 mM CaCl2, pH 7.6) to facilitate digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.
  • In some embodiments, a reaction mixture includes NTPs at a concentration ranging from 1-10 mM, DNA template at a concentration ranging from 0.01-0.5 mg/ml, and SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg/ml, e.g., the reaction mixture comprises NTPs at a concentration of 5 mM, the DNA template at a concentration of 0.1 mg/ml, and the SP6 RNA polymerase at a concentration of 0.05 mg/ml.
  • Nucleotides
  • Various naturally-occurring or modified nucleosides may be used to product mRNA according to the present invention. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
  • In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5 mC”), pseudouridine (“ψU”), and/or 2-thio-uridine (“2sU”). See, e.g., U.S. Pat. No. 8,278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA. The mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed US Patent Publication US20120195936 and international publication WO2011012316, both of which are hereby incorporated by reference in their entirety. The presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2′-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and/or peptide polynucleotides (PNA). In some embodiments where the sugar modification is a 2′-O-alkyl modification, such modification may include, but are not limited to a 2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a 2′-O-methoxyethyl modification and a 2′-deoxy modification. In some embodiments, any of these modifications may be present in 0-100% of the nucleotides—for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination. Post-synthesis processing
  • Typically, a 5′ cap and/or a 3′ tail may be added after the synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. Additional cap structures are described in published US Application No. US 2016/0032356 and U.S. Provisional Application 62/464,327, filed Feb. 27, 2017, which are incorporated herein by reference.
  • Typically, a tail structure includes a poly(A) and/or poly(C) tail. A poly-A or poly-C tail on the 3′ terminus of mRNA typically includes at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb adenosine or cytosine nucleotides, respectively. In some embodiments, a poly A or poly C tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to 600 adenosine or cytosine nucleotides, about 300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosine or cytosine nucleotides, about 400 to 600 adenosine or cytosine nucleotides, about 450 to 600 adenosine or cytosine nucleotides, about 500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosine or cytosine nucleotides, about 10 to 100 adenosine or cytosine nucleotides, about 20 to 70 adenosine or cytosine nucleotides, or about 20 to 60 adenosine or cytosine nucleotides) respectively. In some embodiments, a tail structure includes is a combination of poly (A) and poly (C) tails with various lengths described herein. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.
  • As described herein, the addition of the 5′ cap and/or the 3′ tail facilitates the detection of abortive transcripts generated during in vitro synthesis because without capping and/or tailing, the size of those prematurely aborted mRNA transcripts can be too small to be detected. Thus, in some embodiments, the 5′ cap and/or the 3′ tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, the 5′ cap and/or the 3′ tail are added to the synthesized mRNA before the mRNA is purified as described herein. In other embodiments, the 5′ cap and/or the 3′ tail are added to the synthesized mRNA after the mRNA is purified as described herein.
  • mRNA synthesized according to the present invention may be used without further purification. In particular, mRNA synthesized according to the present invention may be used without a step of removing shortmers. In some embodiments, mRNA synthesized according to the present invention may be further purified. Various methods may be used to purify mRNA synthesized according to the present invention. For example, purification of mRNA can be performed using centrifugation, filtration and/or chromatographic methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol: chloroform: isoamyl alcohol solution, well known to one of skill in the art. In some embodiments, the mRNA is purified using Tangential Flow Filtration. Suitable purification methods include those described in US 2016/0040154, US 2015/0376220, PCT application PCT/US18/19954 entitled “METHODS FOR PURIFICATION OF MESSENGER RNA” filed on Feb. 27, 2018, and PCT application PCT/US18/19978 entitled “METHODS FOR PURIFICATION OF MESSENGER RNA” filed on Feb. 27, 2018, all of which are incorporated by reference herein and may be used to practice the present invention.
  • In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.
  • In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation.
  • In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by filtration.
  • In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF).
  • In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing by chromatography.
  • Characterization of mRNA
  • Full-length or abortive transcripts of mRNA may be detected and quantified using any methods available in the art. In some embodiments, the synthesized mRNA molecules are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver stain, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. Other detection methods known in the art are included in the present invention. In some embodiments, the synthesized mRNA molecules are detected using UV absorption spectroscopy with separation by capillary electrophoresis. In some embodiments, mRNA is first denatured by a Glyoxal dye before gel electrophoresis (“Glyoxal gel electrophoresis”). In some embodiments, synthesized mRNA is characterized before capping or tailing. In some embodiments, synthesized mRNA is characterized after capping and tailing.
  • In some embodiments, mRNA generated by the method disclosed herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% impurities other than full length mRNA. The impurities include IVT contaminants, e.g., proteins, enzymes, free nucleotides and/or shortmers.
  • In some embodiments, mRNA produced according to the invention is substantially free of shortmers or abortive transcripts. In particular, mRNA produced according to the invention contains undetectable level of shortmers or abortive transcripts by capillary electrophoresis or Glyoxal gel electrophoresis. As used herein, the term “shortmers” or “abortive transcripts” refers to any transcripts that are less than full-length. In some embodiments, “shortmers” or “abortive transcripts” are less than 100 nucleotides in length, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. In some embodiments, shortmers are detected or quantified after adding a 5′-cap, and/or a 3′-poly A tail.
  • mRNA Solution
  • In some embodiments, mRNA may be provided in a solution to be mixed with a lipid solution such that the mRNA may be encapsulated in lipid nanoparticles. A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain an mRNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable mRNA solution may contain an mRNA at a concentration ranging from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml. In some embodiments, a suitable mRNA solution may contain an mRNA at a concentration up to about 5.0 mg/ml, 4.0 mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07 mg/ml, 0.06 mg/ml, or 0.05 mg/ml.
  • Typically, a suitable mRNA solution may also contain a buffering agent and/or salt. Generally, buffering agents can include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. In some embodiments, suitable concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some embodiments, suitable concentration of the buffering agent is or greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.
  • Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. Salt concentration in a suitable mRNA solution is or greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.
  • In some embodiments, a suitable mRNA solution may have a pH ranging from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, a suitable mRNA solution may have a pH of or no greater than about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5.
  • Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffer solution described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.
  • In some embodiments, an mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps.
  • Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20× greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In some embodiments, a buffer solution is mixed at a flow rate of or greater than about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute.
  • In some embodiments, an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute). In some embodiments, an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute.
    • Delivery Vehicles
  • According to the present invention, mRNA encoding a CFTR protein (e.g., a full length, fragment, or portion of a CFTR protein) as described herein may be delivered as naked RNA (unpackaged) or via delivery vehicles. As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably.
  • Delivery vehicles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid to a target cell.
  • In some embodiments, a delivery vehicle comprising CFTR mRNA is administered by pulmonary delivery, e.g., comprising nebulization. In these embodiments, the delivery vehicle may be in an aerosolized composition which can be inhaled. In some embodiments, the mRNA is expressed in the tissue in which the delivery vehicle was administered, e.g., nasal cavity, trachea, bronchi, bronchioles, and/or other pulmonary system-related cell or tissue. Additional teaching of pulmonary delivery and nebulization are described in the related international application PCT/US17/61100 filed Nov. 10, 2017 by Applicant entitled “NOVEL ICE-BASED LIPID NANOPARTICLE FORMULATION FOR DELIVERY OF MRNA”, and the U.S. Ser. No. 62/507,061, each of which is incorporated by reference in its entirety.
  • In some embodiments, mRNAs encoding a CFTR protein may be delivered via a single delivery vehicle. In some embodiments, mRNAs encoding a CFTR protein may be delivered via one or more delivery vehicles each of a different composition. According to various embodiments, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable transfer vehicle. (Hum. Gene Ther. 2008 September; 19(9):887-95).
  • A delivery vehicle comprising CFTR mRNA may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration (e.g., local and systemic, including oral, pulmonary, and via injection), the scheduling of administration, the subject's age, sex, body weight, and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein production.
  • In some embodiments, delivery vehicles are formulated such that they are suitable for extended-release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • Liposomal Delivery Vehicles
  • In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, e.g., a lipid nanoparticle. As used herein, liposomal delivery vehicles, e.g., lipid nanoparticles, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue. In some embodiments, a nanoparticle delivery vehicle is a liposome. In some embodiments, a liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In some embodiments, a liposome comprises no more than three distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid.
  • Cationic Lipids
  • In some embodiments, liposomes may comprise one or more cationic lipids. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. An example of suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (for example, CI 2-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-1-yl)tetracosa-5, 15, 18-trien-1-amine (HGT5002).
  • In some embodiments, provided liposomes include a cationic lipid described in WO 2013/063468 and in U.S. provisional application entitled “Lipid Formulations for Delivery of Messenger RNA” filed concurrently with the present application on even date, both of which are incorporated by reference herein.
  • In some embodiments, a cationic lipid comprises a compound of formula I-c1-a:
  • Figure US20220193247A1-20220623-C00001
  • or a pharmaceutically acceptable salt thereof, wherein:
  • each R2 independently is hydrogen or C1-3 alkyl;
  • each q independently is 2 to 6;
  • each R′ independently is hydrogen or C1-3 alkyl;
  • and each RL independently is C8-12 alkyl.
  • In some embodiments, each R2 independently is hydrogen, methyl or ethyl. In some embodiments, each R2 independently is hydrogen or methyl. In some embodiments, each R2 is hydrogen.
  • In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.
  • In some embodiments, each R′ independently is hydrogen, methyl or ethyl. In some embodiments, each R′ independently is hydrogen or methyl. In some embodiments, each R′ independently is hydrogen.
  • In some embodiments, each RL independently is C8-12 alkyl. In some embodiments, each RL independently is n-C8-12alkyl. In some embodiments, each RL independently is C9-11 alkyl. In some embodiments, each RL independently is n-C9-11 alkyl. In some embodiments, each RL independently is C10 alkyl. In some embodiments, each RL independently is n-C10 alkyl.
  • In some embodiments, each R2 independently is hydrogen or methyl; each q independently is 3 to 5; each R′ independently is hydrogen or methyl; and each RL independently is C8-12 alkyl.
  • In some embodiments, each R2 is hydrogen; each q independently is 3 to 5; each R′ is hydrogen; and each RL independently is C8-12 alkyl.
  • In some embodiments, each R2 is hydrogen; each q is 4; each R′ is hydrogen; and each RL independently is C8-12 alkyl.
  • In some embodiments, a cationic lipid comprises a compound of formula I-g:
  • Figure US20220193247A1-20220623-C00002
  • or a pharmaceutically acceptable salt thereof, wherein each RL independently is C8-12 alkyl. In some embodiments, each RL independently is n-C8-12 alkyl. In some embodiments, each RL independently is C9-11 alkyl. In some embodiments, each RL independently is n-C9-11 alkyl. In some embodiments, each RL independently is C10 alkyl. In some embodiments, each RL is n-C10 alkyl.
  • In particular embodiments, provided liposomes include a cationic lipid cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione). The structure of cKK-E12 is shown below:
  • Figure US20220193247A1-20220623-C00003
  • Additional exemplary cationic lipids include those of formula I:
  • Figure US20220193247A1-20220623-C00004
  • and pharmaceutically acceptable salts thereof,
    wherein,
  • R is
  • Figure US20220193247A1-20220623-C00005
  • R is
  • Figure US20220193247A1-20220623-C00006
  • R is
  • Figure US20220193247A1-20220623-C00007
  • or
  • R is
  • Figure US20220193247A1-20220623-C00008
  • (see, e.g., Fenton, Owen S., et al. “Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.” Advanced materials (2016)).
  • In some embodiments, the one or more cationic lipids may be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA” (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc. Nat.′1 Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”,1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.
  • Additional exemplary cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”, 1, 2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”, N-dioleyl-N,N-dimethylammonium chloride or “DODAC”, N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane or “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy 1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”, 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”, 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”, 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin- -DMA”, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or “DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z,12Z)-octadeca-9,1 2-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • In some embodiments, the one or more cationic lipids may be chosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), ICE (WO 2011/068810, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001 (cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al. “Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech. 2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materials for low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).
  • Sterol Cationic Lipids
  • In some embodiments, sterol-based cationic lipids are dialkylamino-, imidazole-, and guanidinium-containing sterol-based cationic lipids. For example, certain embodiments are directed to a composition comprising one or more sterol-based cationic lipids comprising an imidazole, for example, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (II) below. In certain embodiments, a lipid nanoparticle for delivery of RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (II).
  • Figure US20220193247A1-20220623-C00009
  • In some embodiments, the percentage of cationic lipid in a liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some embodiments, cationic lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight. In some embodiments, the cationic lipid (e.g., ICE lipid) constitutes about 30%, about 35%, about 40%, about 45%, or about 50% of the liposome by molar ratio.
  • Non-Cationic/Helper Lipids
  • In some embodiments, provided liposomes contain one or more non-cationic (“helper”) lipids. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.
  • In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other lipids, for example, cationic lipids. In some embodiments, the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipid present in a liposome. In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • Cholesterol-Based Lipids
  • In some embodiments, provided liposomes comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • PEG-Modified Lipids
  • The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention, either alone or preferably in combination with other lipid formulations together which comprise the transfer vehicle (e.g., a lipid nanoparticle). Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to S kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.
  • According to various embodiments, the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the MCNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.
  • Polymers
  • In some embodiments, a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein. Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass nanoparticles comprising polymers. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).
  • A suitable liposome for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids and/or polymers described herein at various ratios. As non-limiting examples, a suitable liposome formulation may include a combination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, and DMG-PEG2K.
  • In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) is or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome by molar ratio.
  • In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.
  • Ratio of Distinct Lipid Components
  • In embodiments where a lipid nanoparticle comprises three and no more than three distinct components of lipids, the ratio of total lipid content (i.e., the ratio of lipid component (1):lipid component (2):lipid component (3)) can be represented as x:y:z, wherein

  • (y+z)=100−x.
  • In some embodiments, each of “x,” “y,” and “z” represents molar percentages of the three distinct components of lipids, and the ratio is a molar ratio.
  • In some embodiments, each of “x,” “y,” and “z” represents weight percentages of the three distinct components of lipids, and the ratio is a weight ratio.
  • In some embodiments, lipid component (1), represented by variable “x,” is a sterol-based cationic lipid.
  • In some embodiments, lipid component (2), represented by variable “y,” is a helper lipid.
  • In some embodiments, lipid component (3), represented by variable “z” is a PEG lipid.
  • In some embodiments, variable “x,” representing the molar percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • In some embodiments, variable “x,” representing the molar percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%.
  • In some embodiments, variable “x,” representing the molar percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%.
  • In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.
  • In some embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.
  • In some embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.
  • For compositions having three and only three distinct lipid components, variables “x,” “y,” and “z” may be in any combination so long as the total of the three variables sums to 100% of the total lipid content.
  • Formation of Liposomes Encapsulating mRNA
  • The liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques which are presently known in the art. The liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.
  • In certain embodiments, provided compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions.
  • In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in a liposome. In some embodiments, the one or more mRNA species may be encapsulated in the same liposome. In some embodiments, the one or more mRNA species may be encapsulated in different liposomes. In some embodiments, the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands and/or combinations thereof. In some embodiments, the one or more liposome may have a different composition of sterol-based cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof. In some embodiments the one or more liposomes may have a different molar ratio of cholesterol-based cationic lipid, neutral lipid, and PEG-modified lipid used to create the liposome.
  • The process of incorporation of a desired mRNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating an mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA to the target cell or tissue.
  • Suitable liposomes in accordance with the present invention may be made in various sizes. In some embodiments, provided liposomes may be made smaller than previously known mRNA encapsulating liposomes. In some embodiments, decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.
  • In some embodiments, an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA. In some embodiments, it may be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • Alternatively or additionally, a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • A variety of alternative methods known in the art are available for sizing of a population of liposomes. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
    • EXAMPLES
  • While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.
    • Example 1. Synthesis and Comparison of hCFTR mRNA Constructs
  • Codon-optimized Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) messenger RNA was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides in length as determined by gel electrophoresis. 5′ and 3′ untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated (vide infra).
  • Exemplary Codon-Optimized Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNAs
  • Construct design:
    • X-SEQ ID NO: 1-Y
  • 5′ and 3′ UTR Sequences:
  • X (5′ UTR Sequence) =
    (SEQ ID NO: 4)
    GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGA
    AGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGA
    ACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG
    Y (3′ UTR Sequence) =
    (SEQ ID NO: 5)
    CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGA
    AGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUG
    CAUCAAGCU
    OR
    (SEQ ID NO: 6)
    GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAA
    GUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGC
    AUCAAAGCU
  • An exemplary codon-optimized human CFTR mRNA sequence includes SEQ ID NO: 1 as described in the detailed description section.
  • An exemplary full-length codon-optimized human CFTR mRNA sequence is shown below:
  • (SEQ ID NO: 7)
    GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACC
    GAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG
    UGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAACGCUCUCCUCUUGAAAAGG
    CCUCGGUGGUGUCCAAGCUCUUCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGG
    GUACAGACAGCGCUUGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCC
    GCGGACAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUCAA
    AGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCA
    UGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCU
    GUUGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGC
    AUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCU
    UGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUG
    CCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGA
    CAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUC
    GACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGC
    UUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUG
    GAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGA
    AGUACAGGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGG
    AAAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGG
    AAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCCGCAAGGCCGC
    UUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUUCUUCGUGGUG
    UUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCU
    UCACCACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCC
    AUGGGCCGUGCAGACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGAC
    UUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGG
    UCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCG
    AGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCU
    CUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUC
    AAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAG
    ACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUC
    AAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAA
    CCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUC
    CGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGA
    UAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAG
    AAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCC
    CCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGU
    GCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCU
    GAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCCUACUUUUACGG
    CACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAUCGAAGCUGAUGGGU
    UGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAA
    CCUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAA
    GAAGCAGAGCUUCAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAU
    CUUGAACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUG
    CAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCC
    UGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUC
    CACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACC
    CACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGACUACCGCAUCCACCCGGA
    AAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAG
    ACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAUCU
    GAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAA
    CACUUAUCUGCGGUACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUG
    GUGCCUGGUGAUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUG
    UUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAACA
    GCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGU
    CGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUC
    CACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGC
    UGCAGGCCCCCAUGUCCACCCUCAACACUCUGAAGGCCGGAGGCAUUCUGAACAG
    AUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUCUUUGAC
    UUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUG
    CAGCCUUACAUUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGC
    GGGCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACG
    AUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACCCUCCGG
    GCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCC
    ACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAU
    CGAGAUGAUUUUCGUCAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACU
    ACCGGAGAGGGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUU
    AUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGC
    GAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCAC
    UAAGUCCACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAA
    AACUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGA
    AGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAG
    CUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGG
    AAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCC
    AAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCU
    UCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCUCGGGUACCUUCCGGAAGAACCU
    GGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGU
    CGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUC
    GACGGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGU
    CCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGA
    UCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGCCUUUGCCGAUUGC
    ACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAGCAGUUCC
    UGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAA
    CGAGCGGUCGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUC
    CCGCAUCGGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGG
    AAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAACGGGUGGCAUCCCUGU
    GACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCA
    GCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU
  • In another example, a full length codon-optimized human CFTR mRNA sequence is shown below:
  • (SEQ ID NO: 8)
    GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACC
    GAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG
    UGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAACGCUCUCCUCUUGAAAAGG
    CCUCGGUGGUGUCCAAGCUCUUCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGG
    GUACAGACAGCGCUUGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCC
    GCGGACAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUCAA
    AGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCA
    UGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCU
    GUUGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGC
    AUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCU
    UGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUG
    CCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGA
    CAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUC
    GACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGC
    UUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUG
    GAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGA
    AGUACAGGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGG
    AAAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGG
    AAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCCGCAAGGCCGC
    UUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUUCUUCGUGGUG
    UUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCU
    UCACCACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCC
    AUGGGCCGUGCAGACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGAC
    UUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGG
    UCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCG
    AGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCU
    CUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUC
    AAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAG
    ACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUC
    AAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAA
    CCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUC
    CGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGA
    UAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAG
    AAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCC
    CCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGU
    GCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCU
    GAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCCUACUUUUACGG
    CACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAUCGAAGCUGAUGGGU
    UGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAA
    CCUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAA
    GAAGCAGAGCUUCAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAU
    CUUGAACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUG
    CAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCC
    UGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUC
    CACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACC
    CACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGACUACCGCAUCCACCCGGA
    AAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAG
    ACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAUCU
    GAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAA
    CACUUAUCUGCGGUACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUG
    GUGCCUGGUGAUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUG
    UUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAACA
    GCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGU
    CGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUC
    CACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGC
    UGCAGGCCCCCAUGUCCACCCUCAACACUCUGAAGGCCGGAGGCAUUCUGAACAG
    AUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUCUUUGAC
    UUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUG
    CAGCCUUACAUUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGC
    GGGCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACG
    AUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACCCUCCGG
    GCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCC
    ACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAU
    CGAGAUGAUUUUCGUCAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACU
    ACCGGAGAGGGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUU
    AUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGC
    GAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCAC
    UAAGUCCACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAA
    AACUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGA
    AGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAG
    CUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGG
    AAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCC
    AAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCU
    UCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCUCGGGUACCUUCCGGAAGAACCU
    GGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGU
    CGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUC
    GACGGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGU
    CCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGA
    UCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGCCUUUGCCGAUUGC
    ACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAGCAGUUCC
    UGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAA
    CGAGCGGUCGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUC
    CCGCAUCGGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGG
    AAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAAGGGUGGCAUCCCUGUG
    ACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGC
    CUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

    Comparison of hCFTR mRNA Constructs
  • A previous hCFTR sequence (SEQ ID NO: 2) was codon-optimized using a T7 promoter. Upon changing the promoter used to synthesize the hCFTR mRNA to SP6, “cleaner” mRNA was synthesized with respect to pre-aborted sequences, but a second species of approximately 1800 nt (“longmer”) was being produced in low quantities. This was visualized by gel electrophoresis as depicted in FIG. 1. In FIG. 1, lane 1 contains an RNA ladder, lane 2 contains mRNA of SEQ ID NO: 1 and lane 3 contains mRNA of SEQ ID NO: 2. As indicated by the arrow, a secondary polynucleotide species approximately 1800 nucleotides in length is present in lane 3. Several new sequences (relative to SEQ ID NO: 2) were designed with site mutations to remove suspected cryptic promoters, but that did not result in the disappearance of the ˜1800 nt secondary species. Complete codon-re-optimization was performed to create SEQ ID NO: 1, which successfully led to an mRNA product without the additional production of the second species at ˜1800 nt (lane 1).
  • Thus, SEQ ID NO: 1 is particularly useful in a homogenous, safe and efficacious pharmaceutical composition.
    • Example 2. Additional Exemplary Codon Optimized CFTR Sequences
  • The following additional exemplary codon optimized sequences are used for synthesis of CFTR mRNA for safe and efficacious clinical use:
  • (SEQ ID NO: 21)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 22)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCTGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATTTCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTTGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 23)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTTGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACACACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTTGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 24)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCAACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 25)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCTATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTTGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 26)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATTTCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCAACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 27)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCTGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCCGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTTGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAGCCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 28)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCTATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTTGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACACACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 29)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCCGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAGTCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 30)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCTGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATTTCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCTATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACACACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    • Example 3. Additional Exemplary Codon Optimized CFTR Sequences
  • The following additional exemplary codon optimized sequences are used for generating human CFTR mRNA for safe and efficacious clinical use:
  • (SEQ ID NO: 31)
    ATGCAGAGAAGCCCCCTGGAGAAGGCCTCTGTGGTGAGCAAGCTGTTCTTCAGCTG
    GACCAGACCCATCCTGAGAAAGGGCTACAGACAGAGACTGGAGCTGTCTGACATCT
    ACCAGATCCCCTCTGTGGACTCTGCCGACAACCTGTCTGAGAAGCTGGAGAGAGAG
    TGGGACAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAATGCCCTGAGAA
    GATGCTTCTTCTGGAGATTCATGTTCTATGGCATCTTCCTGTACCTGGGAGAGGTGAC
    CAAGGCCGTGCAGCCCCTGCTGCTGGGCAGGATCATTGCCAGCTATGACCCTGACA
    ACAAGGAGGAGAGAAGCATTGCCATCTACCTGGGCATTGGCCTGTGCCTGCTGTTCA
    TTGTGAGAACCCTGCTGCTGCACCCTGCCATCTTTGGCCTGCACCACATTGGCATGC
    AGATGAGAATTGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGCAGC
    AGAGTGCTGGACAAGATCAGCATTGGCCAGCTGGTGAGCCTGCTGAGCAACAACCT
    GAACAAGTTTGATGAGGGCCTGGCCCTGGCCCACTTTGTGTGGATTGCCCCCCTGCA
    GGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCCTCTGCCTTCTGTGG
    CCTGGGCTTCCTGATTGTGCTGGCCCTGTTCCAGGCCGGCCTGGGCAGAATGATGAT
    GAAGTACAGAGACCAGAGAGCCGGCAAGATCTCTGAGAGACTGGTGATCACCTCTG
    AGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGCTGGGAGGAGGCCATGGAG
    AAGATGATTGAGAACCTGAGACAGACAGAGCTGAAGCTGACCAGGAAGGCCGCCTA
    TGTGAGATACTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGTGGTGTTCCTGT
    CTGTGCTGCCCTATGCCCTGATCAAGGGCATCATCCTGAGGAAGATCTTCACCACCA
    TCAGCTTCTGCATTGTGCTGAGGATGGCCGTGACCAGGCAGTTCCCCTGGGCCGTGC
    AGACCTGGTATGACAGCCTGGGGGCCATCAACAAGATCCAGGACTTCCTGCAGAAG
    CAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACAGAGGTGGTGATGGAGAA
    TGTGACAGCCTTCTGGGAGGAGGGCTTTGGAGAGCTGTTTGAGAAGGCCAAGCAGA
    ACAACAACAACAGAAAGACCAGCAATGGAGATGACAGCCTGTTCTTCAGCAACTTC
    AGCCTGCTGGGCACCCCTGTGCTGAAGGACATCAACTTCAAGATTGAGAGGGGCCA
    GCTGCTGGCCGTGGCCGGCAGCACAGGAGCCGGCAAGACCAGCCTGCTGATGGTGA
    TCATGGGAGAGCTGGAGCCCTCTGAGGGCAAGATCAAGCACTCTGGCAGAATCAGC
    TTCTGCAGCCAGTTCAGCTGGATCATGCCTGGCACCATCAAGGAGAACATCATCTTT
    GGGGTGAGCTATGATGAGTACAGGTACAGATCTGTGATCAAGGCCTGCCAGCTGGA
    GGAGGACATCTCCAAGTTTGCCGAGAAGGACAACATTGTGCTGGGGGAGGGAGGCA
    TCACCCTGTCTGGGGGCCAGAGAGCCAGAATCAGCCTGGCCAGAGCCGTGTACAAG
    GATGCCGACCTGTACCTGCTGGACAGCCCCTTTGGCTACCTGGATGTGCTGACAGAG
    AAGGAGATCTTTGAGAGCTGTGTGTGCAAGCTGATGGCCAACAAGACCAGGATCCT
    GGTGACCAGCAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGCATG
    AGGGCAGCAGCTACTTCTATGGCACCTTCTCTGAGCTGCAGAACCTGCAGCCTGACT
    TCAGCAGCAAGCTGATGGGCTGTGACAGCTTTGACCAGTTCTCTGCTGAGAGAAGA
    AACAGCATCCTGACAGAGACCCTGCACAGGTTCAGCCTGGAGGGGGATGCCCCTGT
    GAGCTGGACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGAGAGTTTGGGGAG
    AAGAGGAAGAACAGCATCCTGAACCCCATCAACAGCATCAGGAAGTTCAGCATTGT
    GCAGAAGACCCCCCTGCAGATGAATGGCATTGAGGAGGACTCTGATGAGCCCCTGG
    AGAGAAGACTGAGCCTGGTGCCAGACTCTGAGCAGGGAGAGGCCATCCTGCCCAGG
    ATCTCTGTGATCAGCACAGGCCCCACCCTGCAGGCCAGAAGAAGACAGTCTGTGCT
    GAACCTGATGACCCACTCTGTGAACCAGGGCCAGAATATCCACAGAAAGACCACAG
    CCAGCACCAGAAAGGTGAGCCTGGCCCCCCAGGCCAACCTGACAGAGCTGGACATC
    TACAGCAGAAGGCTGAGCCAGGAGACAGGCCTGGAGATCTCTGAGGAGATCAATGA
    GGAGGACCTGAAGGAGTGCTTCTTTGATGACATGGAGAGCATCCCTGCCGTGACCA
    CCTGGAACACCTACCTGAGATACATCACAGTGCACAAGAGCCTGATCTTTGTGCTGA
    TCTGGTGCCTGGTGATCTTCCTGGCCGAGGTGGCCGCCAGCCTGGTGGTGCTGTGGC
    TGCTGGGCAACACCCCCCTGCAGGACAAGGGCAACAGCACCCACAGCAGAAACAAC
    AGCTATGCTGTGATCATCACCAGCACCAGCAGCTACTATGTGTTCTACATCTATGTG
    GGAGTGGCTGACACCCTGCTGGCCATGGGCTTCTTCAGAGGCCTGCCCCTGGTGCAC
    ACCCTGATCACAGTGAGCAAGATCCTGCACCACAAGATGCTGCACTCTGTGCTGCAG
    GCCCCCATGAGCACCCTGAACACCCTGAAGGCTGGAGGCATCCTGAACAGATTCAG
    CAAGGACATTGCCATCCTGGATGACCTGCTGCCCCTGACCATCTTTGACTTCATCCA
    GCTGCTGCTGATTGTGATTGGAGCCATTGCCGTGGTGGCCGTGCTGCAGCCCTACAT
    CTTTGTGGCCACAGTGCCTGTGATTGTGGCCTTCATCATGCTGAGGGCCTACTTCCTG
    CAGACCAGCCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATCTTCAC
    CCACCTGGTGACCAGCCTGAAGGGCCTGTGGACCCTGAGGGCCTTTGGCAGACAGC
    CCTACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAACTGGTTCC
    TGTACCTGAGCACCCTGAGATGGTTCCAGATGAGGATTGAGATGATCTTTGTGATCT
    TCTTCATTGCCGTGACCTTCATCAGCATCCTGACCACAGGGGAGGGCGAGGGCAGA
    GTGGGCATCATCCTGACCCTGGCCATGAACATCATGAGCACCCTGCAGTGGGCCGTG
    AACAGCAGCATTGATGTGGACAGCCTGATGAGATCTGTGAGCAGAGTGTTCAAGTT
    CATTGACATGCCCACAGAGGGCAAGCCCACCAAGAGCACCAAGCCCTACAAGAATG
    GCCAGCTGAGCAAGGTGATGATCATTGAGAACAGCCATGTGAAGAAGGATGACATC
    TGGCCCTCTGGAGGCCAGATGACAGTGAAGGACCTGACAGCCAAGTACACAGAGGG
    GGGCAATGCCATCCTGGAGAACATCAGCTTCAGCATCAGCCCTGGCCAGAGGGTGG
    GCCTGCTGGGCAGAACAGGCTCTGGCAAGAGCACCCTGCTGTCTGCCTTCCTGAGGC
    TGCTGAACACAGAGGGAGAGATCCAGATTGATGGGGTGAGCTGGGACAGCATCACC
    CTGCAGCAGTGGAGGAAGGCCTTTGGGGTGATCCCCCAGAAGGTGTTCATCTTCTCT
    GGCACCTTCAGGAAGAACCTGGACCCCTATGAGCAGTGGTCTGACCAGGAGATCTG
    GAAGGTGGCCGATGAGGTGGGCCTGAGATCTGTGATTGAGCAGTTCCCTGGCAAGC
    TGGACTTTGTGCTGGTGGATGGAGGCTGTGTGCTGAGCCATGGCCACAAGCAGCTGA
    TGTGCCTGGCCAGATCTGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGATGAGCCCT
    CTGCCCACCTGGACCCTGTGACCTACCAGATCATCAGAAGAACCCTGAAGCAGGCC
    TTTGCCGACTGCACAGTGATCCTGTGTGAGCACAGAATTGAGGCCATGCTGGAGTGC
    CAGCAGTTCCTGGTGATTGAGGAGAACAAGGTGAGGCAGTATGACAGCATCCAGAA
    GCTGCTGAATGAGAGAAGCCTGTTCAGACAGGCCATCAGCCCCTCTGACAGAGTGA
    AGCTGTTCCCCCACAGGAACAGCAGCAAGTGCAAGAGCAAGCCCCAGATTGCCGCC
    CTGAAGGAGGAGACAGAGGAGGAGGTGCAGGACACCAGACTGTGA
    (SEQ ID NO: 32)
    ATGCAGAGGAGCCCCCTGGAGAAGGCCAGCGTGGTGAGCAAGCTGTTCTTCAGCTG
    GACCAGGCCCATCCTGAGGAAGGGCTACAGGCAGAGGCTGGAGCTGAGCGACATCT
    ACCAGATCCCCAGCGTGGACAGCGCCGACAACCTGAGCGAGAAGCTGGAGAGGGA
    GTGGGACAGGGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAACGCCCTGAGG
    AGGTGCTTCTTCTGGAGGTTCATGTTCTACGGCATCTTCCTGTACCTGGGCGAGGTG
    ACCAAGGCCGTGCAGCCCCTGCTGCTGGGCAGGATCATCGCCAGCTACGACCCCGA
    CAACAAGGAGGAGAGGAGCATCGCCATCTACCTGGGCATCGGCCTGTGCCTGCTGT
    TCATCGTGAGGACCCTGCTGCTGCACCCCGCCATCTTCGGCCTGCACCACATCGGCA
    TGCAGATGAGGATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGC
    AGCAGGGTGCTGGACAAGATCAGCATCGGCCAGCTGGTGAGCCTGCTGAGCAACAA
    CCTGAACAAGTTCGACGAGGGCCTGGCCCTGGCCCACTTCGTGTGGATCGCCCCCCT
    GCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCCAGCGCCTTCTG
    CGGCCTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCCGGCCTGGGCAGGATGAT
    GATGAAGTACAGGGACCAGAGGGCCGGCAAGATCAGCGAGAGGCTGGTGATCACC
    AGCGAGATGATCGAGAACATCCAGAGCGTGAAGGCCTACTGCTGGGAGGAGGCCAT
    GGAGAAGATGATCGAGAACCTGAGGCAGACCGAGCTGAAGCTGACCAGGAAGGCC
    GCCTACGTGAGGTACTTCAACAGCAGCGCCTTCTTCTTCAGCGGCTTCTTCGTGGTGT
    TCCTGAGCGTGCTGCCCTACGCCCTGATCAAGGGCATCATCCTGAGGAAGATCTTCA
    CCACCATCAGCTTCTGCATCGTGCTGAGGATGGCCGTGACCAGGCAGTTCCCCTGGG
    CCGTGCAGACCTGGTACGACAGCCTGGGCGCCATCAACAAGATCCAGGACTTCCTG
    CAGAAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACCGAGGTGGTGAT
    GGAGAACGTGACCGCCTTCTGGGAGGAGGGCTTCGGCGAGCTGTTCGAGAAGGCCA
    AGCAGAACAACAACAACAGGAAGACCAGCAACGGCGACGACAGCCTGTTCTTCAGC
    AACTTCAGCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAG
    GGGCCAGCTGCTGGCCGTGGCCGGCAGCACCGGCGCCGGCAAGACCAGCCTGCTGA
    TGGTGATCATGGGCGAGCTGGAGCCCAGCGAGGGCAAGATCAAGCACAGCGGCAG
    GATCAGCTTCTGCAGCCAGTTCAGCTGGATCATGCCCGGCACCATCAAGGAGAACA
    TCATCTTCGGCGTGAGCTACGACGAGTACAGGTACAGGAGCGTGATCAAGGCCTGC
    CAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGACAACATCGTGCTGGGCGA
    GGGCGGCATCACCCTGAGCGGCGGCCAGAGGGCCAGGATCAGCCTGGCCAGGGCCG
    TGTACAAGGACGCCGACCTGTACCTGCTGGACAGCCCCTTCGGCTACCTGGACGTGC
    TGACCGAGAAGGAGATCTTCGAGAGCTGCGTGTGCAAGCTGATGGCCAACAAGACC
    AGGATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGAT
    CCTGCACGAGGGCAGCAGCTACTTCTACGGCACCTTCAGCGAGCTGCAGAACCTGC
    AGCCCGACTTCAGCAGCAAGCTGATGGGCTGCGACAGCTTCGACCAGTTCAGCGCC
    GAGAGGAGGAACAGCATCCTGACCGAGACCCTGCACAGGTTCAGCCTGGAGGGCGA
    CGCCCCCGTGAGCTGGACCGAGACCAAGAAGCAGAGCTTCAAGCAGACCGGCGAGT
    TCGGCGAGAAGAGGAAGAACAGCATCCTGAACCCCATCAACAGCATCAGGAAGTTC
    AGCATCGTGCAGAAGACCCCCCTGCAGATGAACGGCATCGAGGAGGACAGCGACG
    AGCCCCTGGAGAGGAGGCTGAGCCTGGTGCCCGACAGCGAGCAGGGCGAGGCCATC
    CTGCCCAGGATCAGCGTGATCAGCACCGGCCCCACCCTGCAGGCCAGGAGGAGGCA
    GAGCGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAACATCCACAGGA
    AGACCACCGCCAGCACCAGGAAGGTGAGCCTGGCCCCCCAGGCCAACCTGACCGAG
    CTGGACATCTACAGCAGGAGGCTGAGCCAGGAGACCGGCCTGGAGATCAGCGAGG
    AGATCAACGAGGAGGACCTGAAGGAGTGCTTCTTCGACGACATGGAGAGCATCCCC
    GCCGTGACCACCTGGAACACCTACCTGAGGTACATCACCGTGCACAAGAGCCTGAT
    CTTCGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCCGAGGTGGCCGCCAGCCTGGT
    GGTGCTGTGGCTGCTGGGCAACACCCCCCTGCAGGACAAGGGCAACAGCACCCACA
    GCAGGAACAACAGCTACGCCGTGATCATCACCAGCACCAGCAGCTACTACGTGTTC
    TACATCTACGTGGGCGTGGCCGACACCCTGCTGGCCATGGGCTTCTTCAGGGGCCTG
    CCCCTGGTGCACACCCTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCAC
    AGCGTGCTGCAGGCCCCCATGAGCACCCTGAACACCCTGAAGGCCGGCGGCATCCT
    GAACAGGTTCAGCAAGGACATCGCCATCCTGGACGACCTGCTGCCCCTGACCATCTT
    CGACTTCATCCAGCTGCTGCTGATCGTGATCGGCGCCATCGCCGTGGTGGCCGTGCT
    GCAGCCCTACATCTTCGTGGCCACCGTGCCCGTGATCGTGGCCTTCATCATGCTGAG
    GGCCTACTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCAGGA
    GCCCCATCTTCACCCACCTGGTGACCAGCCTGAAGGGCCTGTGGACCCTGAGGGCCT
    TCGGCAGGCAGCCCTACTTCGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACCG
    CCAACTGGTTCCTGTACCTGAGCACCCTGAGGTGGTTCCAGATGAGGATCGAGATGA
    TCTTCGTGATCTTCTTCATCGCCGTGACCTTCATCAGCATCCTGACCACCGGCGAGG
    GCGAGGGCAGGGTGGGCATCATCCTGACCCTGGCCATGAACATCATGAGCACCCTG
    CAGTGGGCCGTGAACAGCAGCATCGACGTGGACAGCCTGATGAGGAGCGTGAGCAG
    GGTGTTCAAGTTCATCGACATGCCCACCGAGGGCAAGCCCACCAAGAGCACCAAGC
    CCTACAAGAACGGCCAGCTGAGCAAGGTGATGATCATCGAGAACAGCCACGTGAAG
    AAGGACGACATCTGGCCCAGCGGCGGCCAGATGACCGTGAAGGACCTGACCGCCAA
    GTACACCGAGGGCGGCAACGCCATCCTGGAGAACATCAGCTTCAGCATCAGCCCCG
    GCCAGAGGGTGGGCCTGCTGGGCAGGACCGGCAGCGGCAAGAGCACCCTGCTGAGC
    GCCTTCCTGAGGCTGCTGAACACCGAGGGCGAGATCCAGATCGACGGCGTGAGCTG
    GGACAGCATCACCCTGCAGCAGTGGAGGAAGGCCTTCGGCGTGATCCCCCAGAAGG
    TGTTCATCTTCAGCGGCACCTTCAGGAAGAACCTGGACCCCTACGAGCAGTGGAGC
    GACCAGGAGATCTGGAAGGTGGCCGACGAGGTGGGCCTGAGGAGCGTGATCGAGC
    AGTTCCCCGGCAAGCTGGACTTCGTGCTGGTGGACGGCGGCTGCGTGCTGAGCCAC
    GGCCACAAGCAGCTGATGTGCCTGGCCAGGAGCGTGCTGAGCAAGGCCAAGATCCT
    GCTGCTGGACGAGCCCAGCGCCCACCTGGACCCCGTGACCTACCAGATCATCAGGA
    GGACCCTGAAGCAGGCCTTCGCCGACTGCACCGTGATCCTGTGCGAGCACAGGATC
    GAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATCGAGGAGAACAAGGTGAGGCA
    GTACGACAGCATCCAGAAGCTGCTGAACGAGAGGAGCCTGTTCAGGCAGGCCATCA
    GCCCCAGCGACAGGGTGAAGCTGTTCCCCCACAGGAACAGCAGCAAGTGCAAGAGC
    AAGCCCCAGATCGCCGCCCTGAAGGAGGAGACCGAGGAGGAGGTGCAGGACACCA
    GGCTGTGA
    (SEQ ID NO: 33)
    ATGCAGAGATCCCCTCTGGAGAAGGCCTCAGTGGTGTCCAAGCTTTTCTTCTCCTGG
    ACCAGGCCCATTTTAAGAAAGGGCTACAGGCAGAGACTTGAGCTGTCTGACATCTAT
    CAGATCCCTTCTGTGGATTCTGCTGACAATCTTAGTGAAAAATTGGAAAGGGAGTGG
    GACAGAGAGCTGGCAAGTAAAAAGAACCCCAAGCTGATTAATGCCCTGAGGCGCTG
    CTTTTTTTGGAGATTCATGTTCTATGGCATATTCCTCTACCTTGGAGAAGTAACCAAA
    GCTGTACAGCCTCTCCTCCTTGGCAGAATCATTGCCTCCTATGATCCTGATAACAAG
    GAGGAGAGAAGCATAGCCATCTACCTGGGCATTGGGCTGTGCCTCTTGTTTATTGTG
    AGGACCCTTCTCTTGCACCCTGCCATCTTTGGCCTTCATCACATTGGCATGCAAATGA
    GAATAGCAATGTTTAGTCTTATTTACAAAAAAACATTAAAACTCTCTTCCAGGGTGT
    TGGACAAGATCAGTATTGGACAACTGGTCAGCCTGCTGAGCAACAACCTGAACAAG
    TTTGATGAAGGACTGGCCCTGGCCCACTTTGTCTGGATTGCCCCCCTTCAGGTGGCTC
    TTTTGATGGGCCTGATCTGGGAACTCCTGCAGGCCTCTGCCTTCTGTGGGTTAGGCTT
    CCTGATAGTGCTAGCTCTCTTTCAGGCAGGGTTGGGTAGAATGATGATGAAGTACAG
    AGACCAGAGGGCTGGGAAGATATCTGAGAGGCTGGTCATTACTTCTGAAATGATAG
    AAAACATCCAGTCTGTTAAAGCTTACTGCTGGGAGGAGGCTATGGAAAAGATGATT
    GAGAACTTGAGGCAAACAGAGCTCAAGCTGACTAGGAAGGCAGCCTATGTCAGGTA
    TTTCAACAGCAGTGCTTTCTTCTTCTCAGGCTTTTTCGTGGTCTTCTTGAGTGTTCTGC
    CCTATGCCCTCATCAAGGGGATAATTTTGAGAAAGATTTTCACCACTATTTCCTTTTG
    CATTGTCCTGAGGATGGCTGTCACCAGGCAATTCCCCTGGGCTGTGCAGACATGGTA
    TGACTCTCTGGGGGCCATCAACAAAATCCAAGATTTCCTGCAGAAGCAGGAGTACA
    AGACCCTGGAATACAACCTCACCACCACAGAAGTTGTGATGGAGAATGTGACTGCA
    TTCTGGGAGGAAGGATTTGGGGAGCTGTTTGAGAAAGCAAAACAAAACAATAATAA
    CAGGAAAACCAGCAATGGAGATGACTCCCTGTTCTTTTCCAACTTCTCTTTGTTGGG
    CACCCCTGTCCTGAAAGATATAAACTTTAAAATTGAAAGAGGGCAGCTGTTGGCAGT
    TGCTGGCTCCACAGGAGCTGGAAAAACTTCACTACTGATGGTGATCATGGGGGAGTT
    AGAACCCTCTGAAGGGAAAATAAAACATTCTGGGAGGATTAGTTTCTGCAGCCAGT
    TCAGCTGGATCATGCCTGGGACCATTAAAGAAAATATTATATTTGGAGTGAGCTATG
    ATGAATATAGATATAGGAGTGTCATCAAAGCCTGTCAGTTGGAGGAAGACATCAGC
    AAATTTGCAGAGAAAGACAACATTGTTCTGGGTGAAGGTGGCATCACCCTGTCAGG
    AGGGCAAAGGGCCAGGATCAGCTTGGCCAGAGCAGTCTATAAAGATGCTGATCTGT
    ACCTCCTGGATAGCCCTTTTGGCTATCTGGATGTTTTGACAGAGAAGGAAATTTTTG
    AGTCCTGTGTCTGCAAGTTAATGGCAAATAAAACAAGGATACTTGTGACCTCAAAA
    ATGGAACACCTGAAGAAGGCTGACAAAATTCTGATCCTGCATGAGGGCAGCAGCTA
    CTTTTATGGAACATTTTCTGAACTGCAGAATTTGCAACCAGACTTTTCATCAAAGCTC
    ATGGGATGTGACAGTTTTGATCAGTTTTCTGCAGAAAGGAGAAACTCCATTTTGACT
    GAGACCCTGCACAGGTTCAGTCTGGAGGGGGATGCCCCAGTGAGTTGGACTGAGAC
    AAAGAAACAGAGCTTCAAGCAGACTGGAGAGTTTGGAGAAAAGAGGAAAAACTCA
    ATTCTCAATCCCATCAATAGCATCAGGAAGTTCAGCATAGTTCAGAAGACTCCTTTG
    CAGATGAATGGGATTGAAGAGGACTCAGATGAGCCCCTGGAAAGGAGACTCTCCTT
    GGTGCCAGATTCAGAGCAGGGGGAAGCCATACTGCCAAGGATCTCTGTGATTTCTAC
    AGGGCCCACCCTCCAAGCAAGAAGGAGACAGTCAGTTTTAAACCTGATGACCCACT
    CTGTCAACCAGGGACAGAACATTCATAGAAAGACAACAGCATCTACAAGAAAAGTT
    TCACTGGCCCCTCAAGCCAATTTAACTGAACTAGATATCTACAGCAGGAGGCTCAGC
    CAAGAAACAGGCCTGGAGATCTCAGAAGAAATAAATGAGGAGGATTTGAAGGAAT
    GCTTCTTTGATGATATGGAGAGCATCCCAGCTGTCACAACCTGGAACACCTACCTGA
    GATACATCACAGTGCACAAATCCCTCATCTTTGTACTTATATGGTGCCTTGTCATCTT
    CTTAGCTGAGGTGGCTGCTTCCCTGGTGGTGCTGTGGCTGCTGGGAAACACACCCCT
    CCAGGATAAAGGGAACTCTACTCACAGCAGGAACAACAGTTATGCTGTGATCATCA
    CCAGTACCTCCTCCTACTATGTGTTCTACATTTATGTTGGAGTTGCAGACACATTGCT
    TGCCATGGGTTTTTTTAGAGGACTCCCCCTGGTGCATACTCTCATCACTGTTTCCAAA
    ATCCTTCACCACAAGATGCTGCACAGTGTACTACAGGCTCCCATGAGCACCCTCAAC
    ACTCTTAAAGCAGGAGGAATCTTGAACAGATTTAGCAAGGACATTGCAATTCTTGAT
    GACCTGCTTCCACTGACCATCTTTGACTTCATCCAGCTTCTGCTCATTGTAATTGGTG
    CCATTGCTGTGGTAGCAGTGCTCCAGCCATATATTTTTGTGGCCACTGTGCCTGTTAT
    TGTGGCCTTCATTATGTTGAGAGCCTACTTCCTGCAGACCTCTCAGCAGCTCAAGCA
    ACTTGAAAGTGAGGGCAGGAGCCCCATATTTACACACTTGGTCACTTCCCTCAAAGG
    CCTCTGGACACTCAGAGCTTTTGGAAGACAACCTTATTTTGAAACTCTCTTCCACAA
    GGCTCTGAATCTCCACACAGCCAACTGGTTTCTGTATCTTTCAACACTGCGCTGGTTC
    CAGATGAGGATTGAGATGATCTTTGTTATCTTCTTCATAGCTGTTACCTTCATCTCTA
    TTCTGACAACTGGTGAGGGGGAAGGGAGAGTAGGCATCATCCTCACACTAGCCATG
    AACATAATGTCTACCTTACAATGGGCCGTGAACAGCTCCATAGATGTGGACAGCCTC
    ATGAGAAGTGTGTCAAGAGTTTTCAAATTCATTGACATGCCCACAGAAGGCAAACC
    AACCAAGAGCACAAAACCCTACAAGAATGGCCAGCTGAGTAAGGTCATGATCATTG
    AAAATTCTCATGTGAAGAAGGATGATATTTGGCCCAGTGGGGGCCAGATGACAGTC
    AAGGACCTCACTGCCAAATACACAGAGGGTGGAAATGCTATCCTAGAGAACATCTC
    CTTCTCCATCTCCCCAGGCCAAAGAGTTGGCTTGCTGGGCAGGACTGGCAGTGGCAA
    GTCCACCTTGCTCTCAGCATTTCTCAGGCTTTTAAATACAGAGGGAGAGATTCAAAT
    TGATGGGGTGTCTTGGGATAGTATAACACTTCAACAGTGGAGGAAAGCCTTTGGTGT
    GATTCCTCAGAAAGTGTTTATCTTCTCTGGCACTTTCAGAAAAAATCTGGACCCCTAT
    GAACAGTGGAGTGACCAGGAAATCTGGAAGGTGGCAGATGAAGTGGGCCTAAGATC
    AGTCATAGAGCAGTTTCCTGGAAAGTTGGATTTTGTGCTTGTAGATGGAGGCTGTGT
    GCTGTCCCATGGCCATAAACAGCTAATGTGCCTGGCTAGGTCAGTGCTGAGCAAGGC
    CAAGATCCTGCTGTTAGATGAGCCTTCAGCCCATCTGGACCCTGTGACATACCAGAT
    TATCAGAAGAACTCTGAAGCAGGCCTTTGCTGACTGCACTGTCATCCTGTGTGAGCA
    CAGAATTGAGGCCATGCTGGAGTGCCAGCAGTTCCTTGTTATAGAAGAGAATAAGG
    TTAGGCAGTATGACAGCATTCAGAAACTGCTAAATGAAAGATCTCTCTTCAGGCAAG
    CTATTTCACCATCTGATAGAGTGAAACTTTTTCCCCACAGAAATTCCTCTAAATGTAA
    ATCTAAGCCCCAGATAGCTGCCTTGAAAGAGGAGACTGAAGAAGAAGTCCAGGACA
    CCAGACTGTGA
    (SEQ ID NO: 34)
    ATGCAGAGATCCCCGCTGGAGAAGGCATCTGTGGTGTCAAAACTGTTCTTTAGCTGG
    ACAAGGCCCATCCTTAGGAAAGGGTACAGACAGAGGTTGGAGCTGTCAGACATATA
    TCAGATCCCTTCAGTGGACTCTGCAGACAACCTCTCTGAAAAGCTGGAGAGGGAAT
    GGGACAGGGAACTGGCCAGCAAAAAAAACCCTAAACTGATTAATGCCCTGAGGAGG
    TGCTTCTTTTGGAGATTCATGTTCTATGGGATCTTCCTTTACCTGGGGGAGGTGACTA
    AAGCTGTTCAGCCTCTTCTTCTGGGGAGGATTATTGCCTCCTATGACCCAGACAACA
    AAGAAGAAAGAAGCATAGCCATTTACTTAGGCATAGGCCTCTGCTTGCTCTTCATAG
    TTAGAACCCTCCTACTCCACCCAGCCATCTTTGGTCTCCACCACATAGGTATGCAGA
    TGAGAATAGCAATGTTCTCCTTGATCTACAAGAAGACCCTCAAGCTGTCCAGCAGGG
    TGCTGGACAAGATCTCCATAGGCCAGTTAGTCAGTCTACTGTCCAATAACTTAAATA
    AGTTTGATGAGGGACTGGCACTGGCACATTTTGTGTGGATTGCCCCCCTCCAAGTGG
    CCCTTCTTATGGGCCTTATCTGGGAGCTGTTGCAGGCCTCTGCTTTCTGTGGCCTGGG
    TTTCCTCATAGTCCTAGCCTTATTCCAGGCTGGACTGGGCAGAATGATGATGAAGTA
    TAGGGACCAAAGAGCAGGGAAGATTTCTGAAAGGCTGGTTATAACTTCTGAGATGA
    TTGAGAACATTCAGTCAGTGAAAGCTTACTGCTGGGAAGAAGCTATGGAAAAAATG
    ATTGAAAATCTCAGACAGACTGAATTAAAGTTGACCAGGAAAGCTGCTTATGTCAG
    ATACTTCAACTCCTCAGCCTTCTTTTTTTCTGGCTTCTTTGTTGTATTCCTTTCAGTCC
    TCCCCTATGCCCTGATTAAGGGCATTATCTTGAGGAAAATTTTCACAACCATCTCCTT
    TTGTATTGTCCTCAGGATGGCTGTTACAAGGCAATTTCCTTGGGCTGTGCAAACTTG
    GTATGATAGCCTTGGAGCAATCAACAAGATCCAGGATTTCCTGCAAAAGCAGGAGT
    ACAAGACATTGGAATACAACCTTACCACCACTGAGGTGGTGATGGAAAATGTGACT
    GCCTTCTGGGAGGAGGGGTTTGGAGAGCTGTTTGAGAAAGCCAAACAGAACAACAA
    CAATAGAAAGACCTCTAATGGTGATGATTCCCTGTTCTTTTCTAACTTTAGTCTTCTG
    GGGACCCCAGTTCTGAAAGATATTAACTTTAAAATTGAAAGGGGACAGTTGCTGGCT
    GTGGCTGGGTCCACTGGGGCTGGGAAGACAAGCCTGCTCATGGTGATCATGGGAGA
    GCTGGAACCCAGTGAAGGAAAGATCAAACACTCAGGCAGGATCTCCTTCTGCAGCC
    AGTTCTCATGGATTATGCCAGGCACTATTAAAGAAAATATCATCTTTGGTGTAAGCT
    ATGATGAGTACAGGTATAGATCTGTAATTAAAGCCTGCCAGCTGGAGGAAGACATC
    TCTAAGTTTGCTGAGAAGGATAACATTGTGTTGGGGGAAGGGGGCATCACCCTTTCT
    GGTGGGCAGAGGGCTAGGATCTCCCTTGCTAGGGCAGTATACAAGGATGCTGACTT
    GTACCTCTTGGATAGTCCTTTTGGCTACCTAGATGTGCTGACAGAGAAAGAAATATT
    TGAAAGCTGTGTGTGTAAGCTCATGGCTAACAAGACCAGGATCCTGGTCACCAGTA
    AAATGGAACACCTCAAAAAAGCAGACAAGATCCTTATTCTCCATGAGGGCTCCTCCT
    ACTTCTATGGGACCTTCAGTGAGCTGCAGAATCTGCAGCCAGACTTCTCCTCAAAAC
    TTATGGGCTGTGACTCCTTTGACCAATTCTCTGCAGAAAGAAGGAATAGCATACTGA
    CAGAAACACTGCATAGATTCTCCCTGGAAGGAGATGCCCCAGTGAGTTGGACAGAA
    ACCAAAAAGCAGAGCTTCAAGCAGACTGGTGAGTTTGGTGAAAAGAGGAAGAATTC
    TATCCTGAACCCCATCAATAGCATCAGGAAATTTAGCATAGTCCAAAAGACCCCCCT
    CCAGATGAATGGAATAGAGGAGGATAGTGATGAGCCTCTTGAGAGAAGGCTGTCCC
    TGGTTCCAGACAGTGAACAGGGTGAAGCCATTCTTCCGAGGATCAGTGTCATCTCCA
    CTGGGCCCACATTGCAGGCCAGAAGAAGACAGTCTGTTCTGAATTTGATGACACATT
    CTGTGAATCAAGGCCAGAATATCCATAGAAAAACCACTGCCAGCACCAGAAAAGTT
    TCTCTAGCCCCCCAGGCTAACCTGACTGAGTTAGACATCTACAGCAGAAGGCTGAGC
    CAAGAGACTGGCTTGGAAATATCTGAGGAGATCAATGAGGAGGACCTCAAGGAGTG
    CTTCTTTGATGACATGGAGTCAATCCCTGCAGTCACTACATGGAACACTTACCTAAG
    GTACATCACAGTTCATAAGAGCCTCATCTTTGTCCTCATATGGTGTCTGGTCATCTTT
    TTAGCAGAAGTGGCTGCCAGCCTAGTTGTGCTGTGGTTACTGGGCAATACACCTCTT
    CAGGACAAAGGCAATAGCACACACAGCAGAAACAACTCCTATGCAGTGATCATCAC
    CTCTACAAGCTCTTACTATGTATTCTATATATATGTGGGAGTGGCAGATACTCTCCTG
    GCCATGGGATTCTTCAGGGGATTACCTCTAGTTCACACATTGATCACAGTGTCAAAA
    ATTCTCCACCACAAGATGTTACACAGTGTCCTGCAAGCCCCAATGTCTACTCTGAAC
    ACACTTAAGGCAGGTGGAATTTTGAATAGGTTTAGCAAGGACATAGCTATCCTGGAT
    GATCTCCTCCCTCTGACCATCTTTGACTTCATCCAGTTACTGCTCATTGTAATTGGAG
    CCATTGCAGTGGTAGCAGTCCTACAGCCTTACATTTTTGTGGCTACTGTTCCTGTTAT
    TGTGGCCTTCATTATGCTAAGAGCTTACTTCCTGCAAACAAGCCAACAGTTGAAACA
    GCTAGAAAGTGAGGGAAGGTCCCCCATCTTCACCCACCTGGTGACATCACTCAAGG
    GGCTATGGACTCTTAGGGCTTTTGGGAGACAGCCGTACTTTGAGACCTTATTCCATA
    AGGCCCTTAACCTCCATACAGCAAACTGGTTCTTATACCTGAGTACTCTGAGGTGGT
    TTCAAATGAGGATTGAAATGATTTTTGTGATCTTCTTCATTGCTGTGACCTTCATCTC
    AATCTTGACCACAGGAGAGGGGGAGGGCAGGGTGGGCATCATACTGACCTTGGCCA
    TGAACATTATGTCAACCCTGCAGTGGGCTGTCAATAGCTCCATTGATGTGGACAGTC
    TGATGAGGAGTGTCTCCAGGGTCTTCAAGTTTATTGACATGCCAACTGAGGGCAAAC
    CCACCAAAAGCACTAAGCCATATAAAAATGGCCAACTGTCCAAAGTGATGATCATT
    GAAAATTCACATGTAAAGAAGGATGATATCTGGCCCTCTGGAGGACAGATGACAGT
    GAAAGACCTGACTGCCAAGTACACAGAGGGTGGTAATGCCATTCTTGAGAACATTA
    GTTTCAGTATTTCCCCGGGGCAAAGGGTGGGCCTCCTTGGCAGAACAGGCTCTGGCA
    AGAGTACCCTGCTGTCAGCCTTTTTAAGACTGTTGAACACTGAGGGAGAAATTCAGA
    TTGATGGTGTCTCCTGGGATAGCATCACCCTCCAGCAGTGGAGAAAAGCTTTTGGAG
    TGATCCCGCAAAAGGTTTTCATCTTTTCAGGCACCTTCCGGAAGAACCTGGACCCCT
    ATGAGCAGTGGTCTGACCAGGAAATATGGAAGGTAGCTGATGAAGTTGGGCTTAGG
    TCAGTCATAGAGCAGTTCCCAGGCAAACTGGACTTTGTCCTGGTGGATGGTGGATGT
    GTACTGAGTCATGGGCACAAACAGCTGATGTGCCTAGCCAGGTCTGTGCTCAGCAA
    GGCAAAGATATTGCTGCTTGATGAACCCAGTGCCCATCTGGACCCAGTCACATATCA
    GATCATCAGAAGAACATTGAAGCAGGCCTTTGCTGATTGCACAGTTATCCTCTGTGA
    GCACAGGATTGAGGCCATGCTGGAGTGCCAGCAGTTTCTGGTGATTGAGGAGAATA
    AAGTAAGGCAGTATGACTCCATCCAGAAGCTGCTCAATGAAAGAAGCCTCTTTAGA
    CAAGCTATCTCCCCCTCAGACAGGGTCAAATTGTTCCCTCACAGAAACAGCAGCAA
    GTGCAAGAGCAAGCCCCAAATTGCAGCCTTGAAAGAGGAGACAGAGGAAGAGGTG
    CAGGACACCAGACTCTGA
    (SEQ ID NO: 35)
    ATGCAGAGAAGCCCCCTGGAGAAGGCCAGCGTGGTGAGCAAGCTGTTCTTCAGCTG
    GACCAGACCCATCCTGAGAAAGGGCTACAGACAGAGACTGGAGCTGAGCGACATCT
    ACCAGATCCCCAGCGTGGACAGCGCCGACAACCTGAGCGAGAAGCTGGAGAGAGA
    GTGGGACAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAACGCCCTGAGA
    AGATGCTTCTTCTGGAGATTCATGTTCTACGGCATCTTCCTGTACCTGGGCGAGGTG
    ACCAAGGCCGTGCAGCCCCTGCTGCTGGGCAGAATCATCGCCAGCTACGACCCCGA
    CAACAAGGAGGAGAGAAGCATCGCCATCTACCTGGGCATCGGCCTGTGCCTGCTGT
    TCATCGTGAGAACCCTGCTGCTGCACCCCGCCATCTTCGGCCTGCACCACATCGGCA
    TGCAGATGAGAATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGC
    AGCAGAGTGCTGGACAAGATCAGCATCGGCCAGCTGGTGAGCCTGCTGAGCAACAA
    CCTGAACAAGTTCGACGAGGGCCTGGCCCTGGCCCACTTCGTGTGGATCGCCCCCCT
    GCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCCAGCGCCTTCTG
    CGGCCTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCCGGCCTGGGCAGAATGAT
    GATGAAGTACAGAGACCAGAGAGCCGGCAAGATCAGCGAGAGACTGGTGATCACC
    AGCGAGATGATCGAGAACATCCAGAGCGTGAAGGCCTACTGCTGGGAGGAGGCCAT
    GGAGAAGATGATCGAGAACCTGAGACAGACCGAGCTGAAGCTGACCAGAAAGGCC
    GCCTACGTGAGATACTTCAACAGCAGCGCCTTCTTCTTCAGCGGCTTCTTCGTGGTGT
    TCCTGAGCGTGCTGCCCTACGCCCTGATCAAGGGCATCATCCTGAGAAAGATCTTCA
    CCACCATCAGCTTCTGCATCGTGCTGAGAATGGCCGTGACCAGACAGTTCCCCTGGG
    CCGTGCAGACCTGGTACGACAGCCTGGGCGCCATCAACAAGATCCAGGACTTCCTG
    CAGAAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACCGAGGTGGTGAT
    GGAGAACGTGACCGCCTTCTGGGAGGAGGGCTTCGGCGAGCTGTTCGAGAAGGCCA
    AGCAGAACAACAACAACAGAAAGACCAGCAACGGCGACGACAGCCTGTTCTTCAGC
    AACTTCAGCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAG
    AGGCCAGCTGCTGGCCGTGGCCGGCAGCACCGGCGCCGGCAAGACCAGCCTGCTGA
    TGGTGATCATGGGCGAGCTGGAGCCCAGCGAGGGCAAGATCAAGCACAGCGGCAG
    AATCAGCTTCTGCAGCCAGTTCAGCTGGATCATGCCCGGCACCATCAAGGAGAACA
    TCATCTTCGGCGTGAGCTACGACGAGTACAGATACAGAAGCGTGATCAAGGCCTGC
    CAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGACAACATCGTGCTGGGCGA
    GGGCGGCATCACCCTGAGCGGCGGCCAGAGAGCCAGAATCAGCCTGGCCAGAGCCG
    TGTACAAGGACGCCGACCTGTACCTGCTGGACAGCCCCTTCGGCTACCTGGACGTGC
    TGACCGAGAAGGAGATCTTCGAGAGCTGCGTGTGCAAGCTGATGGCCAACAAGACC
    AGAATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGAT
    CCTGCACGAGGGCAGCAGCTACTTCTACGGCACCTTCAGCGAGCTGCAGAACCTGC
    AGCCCGACTTCAGCAGCAAGCTGATGGGCTGCGACAGCTTCGACCAGTTCAGCGCC
    GAGAGAAGAAACAGCATCCTGACCGAGACCCTGCACAGATTCAGCCTGGAGGGCGA
    CGCCCCCGTGAGCTGGACCGAGACCAAGAAGCAGAGCTTCAAGCAGACCGGCGAGT
    TCGGCGAGAAGAGAAAGAACAGCATCCTGAACCCCATCAACAGCATCAGAAAGTTC
    AGCATCGTGCAGAAGACCCCCCTGCAGATGAACGGCATCGAGGAGGACAGCGACG
    AGCCCCTGGAGAGAAGACTGAGCCTGGTGCCCGACAGCGAGCAGGGCGAGGCCATC
    CTGCCCAGAATCAGCGTGATCAGCACCGGCCCCACCCTGCAGGCCAGAAGAAGACA
    GAGCGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAACATCCACAGAA
    AGACCACCGCCAGCACCAGAAAGGTGAGCCTGGCCCCCCAGGCCAACCTGACCGAG
    CTGGACATCTACAGCAGAAGACTGAGCCAGGAGACCGGCCTGGAGATCAGCGAGG
    AGATCAACGAGGAGGACCTGAAGGAGTGCTTCTTCGACGACATGGAGAGCATCCCC
    GCCGTGACCACCTGGAACACCTACCTGAGATACATCACCGTGCACAAGAGCCTGAT
    CTTCGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCCGAGGTGGCCGCCAGCCTGGT
    GGTGCTGTGGCTGCTGGGCAACACCCCCCTGCAGGACAAGGGCAACAGCACCCACA
    GCAGAAACAACAGCTACGCCGTGATCATCACCAGCACCAGCAGCTACTACGTGTTC
    TACATCTACGTGGGCGTGGCCGACACCCTGCTGGCCATGGGCTTCTTCAGAGGCCTG
    CCCCTGGTGCACACCCTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCAC
    AGCGTGCTGCAGGCCCCCATGAGCACCCTGAACACCCTGAAGGCCGGCGGCATCCT
    GAACAGATTCAGCAAGGACATCGCCATCCTGGACGACCTGCTGCCCCTGACCATCTT
    CGACTTCATCCAGCTGCTGCTGATCGTGATCGGCGCCATCGCCGTGGTGGCCGTGCT
    GCAGCCCTACATCTTCGTGGCCACCGTGCCCGTGATCGTGGCCTTCATCATGCTGAG
    AGCCTACTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCAGAA
    GCCCCATCTTCACCCACCTGGTGACCAGCCTGAAGGGCCTGTGGACCCTGAGAGCCT
    TCGGCAGACAGCCCTACTTCGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACCG
    CCAACTGGTTCCTGTACCTGAGCACCCTGAGATGGTTCCAGATGAGAATCGAGATGA
    TCTTCGTGATCTTCTTCATCGCCGTGACCTTCATCAGCATCCTGACCACCGGCGAGG
    GCGAGGGCAGAGTGGGCATCATCCTGACCCTGGCCATGAACATCATGAGCACCCTG
    CAGTGGGCCGTGAACAGCAGCATCGACGTGGACAGCCTGATGAGAAGCGTGAGCAG
    AGTGTTCAAGTTCATCGACATGCCCACCGAGGGCAAGCCCACCAAGAGCACCAAGC
    CCTACAAGAACGGCCAGCTGAGCAAGGTGATGATCATCGAGAACAGCCACGTGAAG
    AAGGACGACATCTGGCCCAGCGGCGGCCAGATGACCGTGAAGGACCTGACCGCCAA
    GTACACCGAGGGCGGCAACGCCATCCTGGAGAACATCAGCTTCAGCATCAGCCCCG
    GCCAGAGAGTGGGCCTGCTGGGCAGAACCGGCAGCGGCAAGAGCACCCTGCTGAGC
    GCCTTCCTGAGACTGCTGAACACCGAGGGCGAGATCCAGATCGACGGCGTGAGCTG
    GGACAGCATCACCCTGCAGCAGTGGAGAAAGGCCTTCGGCGTGATCCCCCAGAAGG
    TGTTCATCTTCAGCGGCACCTTCAGAAAGAACCTGGACCCCTACGAGCAGTGGAGC
    GACCAGGAGATCTGGAAGGTGGCCGACGAGGTGGGCCTGAGAAGCGTGATCGAGC
    AGTTCCCCGGCAAGCTGGACTTCGTGCTGGTGGACGGCGGCTGCGTGCTGAGCCAC
    GGCCACAAGCAGCTGATGTGCCTGGCCAGAAGCGTGCTGAGCAAGGCCAAGATCCT
    GCTGCTGGACGAGCCCAGCGCCCACCTGGACCCCGTGACCTACCAGATCATCAGAA
    GAACCCTGAAGCAGGCCTTCGCCGACTGCACCGTGATCCTGTGCGAGCACAGAATC
    GAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATCGAGGAGAACAAGGTGAGACA
    GTACGACAGCATCCAGAAGCTGCTGAACGAGAGAAGCCTGTTCAGACAGGCCATCA
    GCCCCAGCGACAGAGTGAAGCTGTTCCCCCACAGAAACAGCAGCAAGTGCAAGAGC
    AAGCCCCAGATCGCCGCCCTGAAGGAGGAGACCGAGGAGGAGGTGCAGGACACCA
    GACTGTGA
    (SEQ ID NO: 36)
    ATGCAGCGCAGCCCCCTGGAGAAGGCCAGCGTGGTGAGCAAGCTGTTCTTCAGCTG
    GACCCGCCCCATCCTGCGCAAGGGCTACCGCCAGCGCCTGGAGCTGAGCGACATCT
    ACCAGATCCCCAGCGTGGACAGCGCCGACAACCTGAGCGAGAAGCTGGAGCGCGA
    GTGGGACCGCGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAACGCCCTGCGCC
    GCTGCTTCTTCTGGCGCTTCATGTTCTACGGCATCTTCCTGTACCTGGGCGAGGTGAC
    CAAGGCCGTGCAGCCCCTGCTGCTGGGCCGCATCATCGCCAGCTACGACCCCGACA
    ACAAGGAGGAGCGCAGCATCGCCATCTACCTGGGCATCGGCCTGTGCCTGCTGTTCA
    TCGTGCGCACCCTGCTGCTGCACCCCGCCATCTTCGGCCTGCACCACATCGGCATGC
    AGATGCGCATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGCAGC
    CGCGTGCTGGACAAGATCAGCATCGGCCAGCTGGTGAGCCTGCTGAGCAACAACCT
    GAACAAGTTCGACGAGGGCCTGGCCCTGGCCCACTTCGTGTGGATCGCCCCCCTGCA
    GGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCCAGCGCCTTCTGCGG
    CCTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCCGGCCTGGGCCGCATGATGAT
    GAAGTACCGCGACCAGCGCGCCGGCAAGATCAGCGAGCGCCTGGTGATCACCAGCG
    AGATGATCGAGAACATCCAGAGCGTGAAGGCCTACTGCTGGGAGGAGGCCATGGAG
    AAGATGATCGAGAACCTGCGCCAGACCGAGCTGAAGCTGACCCGCAAGGCCGCCTA
    CGTGCGCTACTTCAACAGCAGCGCCTTCTTCTTCAGCGGCTTCTTCGTGGTGTTCCTG
    AGCGTGCTGCCCTACGCCCTGATCAAGGGCATCATCCTGCGCAAGATCTTCACCACC
    ATCAGCTTCTGCATCGTGCTGCGCATGGCCGTGACCCGCCAGTTCCCCTGGGCCGTG
    CAGACCTGGTACGACAGCCTGGGCGCCATCAACAAGATCCAGGACTTCCTGCAGAA
    GCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACCGAGGTGGTGATGGAGA
    ACGTGACCGCCTTCTGGGAGGAGGGCTTCGGCGAGCTGTTCGAGAAGGCCAAGCAG
    AACAACAACAACCGCAAGACCAGCAACGGCGACGACAGCCTGTTCTTCAGCAACTT
    CAGCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGCGCGGCC
    AGCTGCTGGCCGTGGCCGGCAGCACCGGCGCCGGCAAGACCAGCCTGCTGATGGTG
    ATCATGGGCGAGCTGGAGCCCAGCGAGGGCAAGATCAAGCACAGCGGCCGCATCA
    GCTTCTGCAGCCAGTTCAGCTGGATCATGCCCGGCACCATCAAGGAGAACATCATCT
    TCGGCGTGAGCTACGACGAGTACCGCTACCGCAGCGTGATCAAGGCCTGCCAGCTG
    GAGGAGGACATCAGCAAGTTCGCCGAGAAGGACAACATCGTGCTGGGCGAGGGCG
    GCATCACCCTGAGCGGCGGCCAGCGCGCCCGCATCAGCCTGGCCCGCGCCGTGTAC
    AAGGACGCCGACCTGTACCTGCTGGACAGCCCCTTCGGCTACCTGGACGTGCTGACC
    GAGAAGGAGATCTTCGAGAGCTGCGTGTGCAAGCTGATGGCCAACAAGACCCGCAT
    CCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGC
    ACGAGGGCAGCAGCTACTTCTACGGCACCTTCAGCGAGCTGCAGAACCTGCAGCCC
    GACTTCAGCAGCAAGCTGATGGGCTGCGACAGCTTCGACCAGTTCAGCGCCGAGCG
    CCGCAACAGCATCCTGACCGAGACCCTGCACCGCTTCAGCCTGGAGGGCGACGCCC
    CCGTGAGCTGGACCGAGACCAAGAAGCAGAGCTTCAAGCAGACCGGCGAGTTCGGC
    GAGAAGCGCAAGAACAGCATCCTGAACCCCATCAACAGCATCCGCAAGTTCAGCAT
    CGTGCAGAAGACCCCCCTGCAGATGAACGGCATCGAGGAGGACAGCGACGAGCCCC
    TGGAGCGCCGCCTGAGCCTGGTGCCCGACAGCGAGCAGGGCGAGGCCATCCTGCCC
    CGCATCAGCGTGATCAGCACCGGCCCCACCCTGCAGGCCCGCCGCCGCCAGAGCGT
    GCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAACATCCACCGCAAGACCA
    CCGCCAGCACCCGCAAGGTGAGCCTGGCCCCCCAGGCCAACCTGACCGAGCTGGAC
    ATCTACAGCCGCCGCCTGAGCCAGGAGACCGGCCTGGAGATCAGCGAGGAGATCAA
    CGAGGAGGACCTGAAGGAGTGCTTCTTCGACGACATGGAGAGCATCCCCGCCGTGA
    CCACCTGGAACACCTACCTGCGCTACATCACCGTGCACAAGAGCCTGATCTTCGTGC
    TGATCTGGTGCCTGGTGATCTTCCTGGCCGAGGTGGCCGCCAGCCTGGTGGTGCTGT
    GGCTGCTGGGCAACACCCCCCTGCAGGACAAGGGCAACAGCACCCACAGCCGCAAC
    AACAGCTACGCCGTGATCATCACCAGCACCAGCAGCTACTACGTGTTCTACATCTAC
    GTGGGCGTGGCCGACACCCTGCTGGCCATGGGCTTCTTCCGCGGCCTGCCCCTGGTG
    CACACCCTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCACAGCGTGCT
    GCAGGCCCCCATGAGCACCCTGAACACCCTGAAGGCCGGCGGCATCCTGAACCGCT
    TCAGCAAGGACATCGCCATCCTGGACGACCTGCTGCCCCTGACCATCTTCGACTTCA
    TCCAGCTGCTGCTGATCGTGATCGGCGCCATCGCCGTGGTGGCCGTGCTGCAGCCCT
    ACATCTTCGTGGCCACCGTGCCCGTGATCGTGGCCTTCATCATGCTGCGCGCCTACTT
    CCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCCGCAGCCCCATCT
    TCACCCACCTGGTGACCAGCCTGAAGGGCCTGTGGACCCTGCGCGCCTTCGGCCGCC
    AGCCCTACTTCGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACCGCCAACTGGT
    TCCTGTACCTGAGCACCCTGCGCTGGTTCCAGATGCGCATCGAGATGATCTTCGTGA
    TCTTCTTCATCGCCGTGACCTTCATCAGCATCCTGACCACCGGCGAGGGCGAGGGCC
    GCGTGGGCATCATCCTGACCCTGGCCATGAACATCATGAGCACCCTGCAGTGGGCC
    GTGAACAGCAGCATCGACGTGGACAGCCTGATGCGCAGCGTGAGCCGCGTGTTCAA
    GTTCATCGACATGCCCACCGAGGGCAAGCCCACCAAGAGCACCAAGCCCTACAAGA
    ACGGCCAGCTGAGCAAGGTGATGATCATCGAGAACAGCCACGTGAAGAAGGACGA
    CATCTGGCCCAGCGGCGGCCAGATGACCGTGAAGGACCTGACCGCCAAGTACACCG
    AGGGCGGCAACGCCATCCTGGAGAACATCAGCTTCAGCATCAGCCCCGGCCAGCGC
    GTGGGCCTGCTGGGCCGCACCGGCAGCGGCAAGAGCACCCTGCTGAGCGCCTTCCT
    GCGCCTGCTGAACACCGAGGGCGAGATCCAGATCGACGGCGTGAGCTGGGACAGCA
    TCACCCTGCAGCAGTGGCGCAAGGCCTTCGGCGTGATCCCCCAGAAGGTGTTCATCT
    TCAGCGGCACCTTCCGCAAGAACCTGGACCCCTACGAGCAGTGGAGCGACCAGGAG
    ATCTGGAAGGTGGCCGACGAGGTGGGCCTGCGCAGCGTGATCGAGCAGTTCCCCGG
    CAAGCTGGACTTCGTGCTGGTGGACGGCGGCTGCGTGCTGAGCCACGGCCACAAGC
    AGCTGATGTGCCTGGCCCGCAGCGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGAC
    GAGCCCAGCGCCCACCTGGACCCCGTGACCTACCAGATCATCCGCCGCACCCTGAA
    GCAGGCCTTCGCCGACTGCACCGTGATCCTGTGCGAGCACCGCATCGAGGCCATGCT
    GGAGTGCCAGCAGTTCCTGGTGATCGAGGAGAACAAGGTGCGCCAGTACGACAGCA
    TCCAGAAGCTGCTGAACGAGCGCAGCCTGTTCCGCCAGGCCATCAGCCCCAGCGAC
    CGCGTGAAGCTGTTCCCCCACCGCAACAGCAGCAAGTGCAAGAGCAAGCCCCAGAT
    CGCCGCCCTGAAGGAGGAGACCGAGGAGGAGGTGCAGGACACCCGCCTGTAA
    (SEQ ID NO: 37)
    ATGCAGAGAAGCCCCCTGGAGAAGGCCAGCGTGGTGAGCAAGCTGTTCTTCAGCTG
    GACCAGACCCATCCTGAGAAAGGGCTACAGACAGAGACTGGAGCTGAGCGACATCT
    ACCAGATCCCCAGCGTGGACAGCGCCGACAACCTGAGCGAGAAGCTGGAGAGAGA
    GTGGGACAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAACGCCCTGAGA
    AGATGCTTCTTCTGGAGATTCATGTTCTACGGCATCTTCCTGTACCTGGGCGAGGTG
    ACCAAGGCCGTGCAGCCCCTGCTGCTGGGCAGAATCATCGCCAGCTACGACCCCGA
    CAACAAGGAGGAGAGAAGCATCGCCATCTACCTGGGCATCGGCCTGTGCCTGCTGT
    TCATCGTGAGAACCCTGCTGCTGCACCCCGCCATCTTCGGCCTGCACCACATCGGCA
    TGCAGATGAGAATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAGCTGAGC
    AGCAGAGTGCTGGACAAGATCAGCATCGGCCAGCTGGTGAGCCTGCTGAGCAACAA
    CCTGAACAAGTTCGACGAGGGCCTGGCCCTGGCCCACTTCGTGTGGATCGCCCCCCT
    GCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCCAGCGCCTTCTG
    CGGCCTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCCGGCCTGGGCAGAATGAT
    GATGAAGTACAGGGACCAGAGAGCCGGCAAGATCAGCGAGAGACTGGTGATCACC
    AGCGAGATGATCGAGAACATCCAGAGCGTGAAGGCCTACTGCTGGGAGGAGGCCAT
    GGAGAAGATGATCGAGAACCTGAGACAGACCGAGCTGAAGCTGACCAGAAAGGCC
    GCCTACGTGAGATACTTCAACAGCAGCGCCTTCTTCTTCAGCGGCTTCTTCGTGGTGT
    TCCTGAGCGTGCTGCCCTACGCCCTGATCAAGGGCATCATCCTGAGAAAGATCTTCA
    CCACCATCAGCTTCTGCATCGTGCTGAGAATGGCCGTGACCAGACAGTTCCCCTGGG
    CCGTGCAGACCTGGTACGACAGCCTGGGCGCCATCAACAAGATCCAGGACTTCCTG
    CAGAAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACCGAGGTGGTGAT
    GGAGAACGTGACCGCCTTCTGGGAGGAGGGCTTCGGCGAGCTGTTCGAGAAGGCCA
    AGCAGAACAACAACAACAGAAAGACCAGCAACGGCGACGACAGCCTGTTCTTCAGC
    AACTTCAGCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAG
    AGGCCAGCTGCTGGCCGTGGCCGGCAGCACCGGCGCCGGCAAGACCAGCCTGCTGA
    TGGTGATCATGGGCGAGCTGGAGCCCAGCGAGGGCAAGATCAAGCACAGCGGCAG
    AATCAGCTTCTGCAGCCAGTTCAGCTGGATCATGCCCGGCACCATCAAGGAGAACA
    TCATCTTCGGCGTGAGCTACGACGAGTACAGATACAGAAGCGTGATCAAGGCCTGC
    CAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGACAACATCGTGCTGGGCGA
    GGGCGGCATCACCCTGAGCGGCGGCCAGAGAGCCAGAATCAGCCTGGCCAGAGCCG
    TGTACAAGGACGCCGACCTGTACCTGCTGGACAGCCCCTTCGGCTACCTGGACGTGC
    TGACCGAGAAGGAGATCTTCGAGAGCTGCGTGTGCAAGCTGATGGCCAACAAGACC
    AGAATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGAT
    CCTGCACGAGGGCAGCAGCTACTTCTACGGCACCTTCAGCGAGCTGCAGAACCTGC
    AGCCCGACTTCAGCAGCAAGCTGATGGGCTGCGACAGCTTCGACCAGTTCAGCGCC
    GAGAGAAGAAACAGCATCCTGACCGAGACCCTGCACAGATTCAGCCTGGAGGGCGA
    CGCCCCCGTGAGCTGGACCGAGACCAAGAAGCAGAGCTTCAAGCAGACCGGCGAGT
    TCGGCGAGAAGAGAAAGAACAGCATCCTGAACCCCATCAACAGCATCAGAAAGTTC
    AGCATCGTGCAGAAGACCCCCCTGCAGATGAACGGCATCGAGGAGGACAGCGACG
    AGCCCCTGGAGAGAAGACTGAGCCTGGTGCCCGACAGCGAGCAGGGCGAGGCCATC
    CTGCCCAGAATCAGCGTGATCAGCACCGGCCCCACCCTGCAGGCCAGAAGAAGACA
    GAGCGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAACATCCACAGAA
    AGACCACCGCCAGCACCAGAAAGGTGAGCCTGGCCCCCCAGGCCAACCTGACCGAG
    CTGGACATCTACAGCAGAAGACTGAGCCAGGAGACCGGCCTGGAGATCAGCGAGG
    AGATCAACGAGGAGGACCTGAAGGAGTGCTTCTTCGACGACATGGAGAGCATCCCC
    GCCGTGACCACCTGGAACACCTACCTGAGATACATCACCGTGCACAAGAGCCTGAT
    CTTCGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCCGAGGTGGCCGCCAGCCTGGT
    GGTGCTGTGGCTGCTGGGCAACACCCCCCTGCAGGACAAGGGCAACAGCACCCACA
    GCAGAAACAACAGCTACGCCGTGATCATCACCAGCACCAGCAGCTACTACGTGTTC
    TACATCTACGTGGGCGTGGCCGACACCCTGCTGGCCATGGGCTTCTTCAGAGGCCTG
    CCCCTGGTGCACACCCTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCAC
    AGCGTGCTGCAGGCCCCCATGAGCACCCTGAACACCCTGAAGGCCGGCGGCATCCT
    GAACAGATTCAGCAAGGACATCGCCATCCTGGACGACCTGCTGCCCCTGACCATCTT
    CGACTTCATCCAGCTGCTGCTGATCGTGATCGGCGCCATCGCCGTGGTGGCCGTGCT
    GCAGCCCTACATCTTCGTGGCCACCGTGCCCGTGATCGTGGCCTTCATCATGCTGAG
    AGCCTACTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCAGGA
    GCCCCATCTTCACCCACCTGGTGACCAGCCTGAAGGGCCTGTGGACCCTGAGAGCCT
    TCGGCAGACAGCCCTACTTCGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACCG
    CCAACTGGTTCCTGTACCTGAGCACCCTGAGATGGTTCCAGATGAGAATCGAGATGA
    TCTTCGTGATCTTCTTCATCGCCGTGACCTTCATCAGCATCCTGACCACCGGCGAGG
    GCGAGGGCAGAGTGGGCATCATCCTGACCCTGGCCATGAACATCATGAGCACCCTG
    CAGTGGGCCGTGAACAGCAGCATCGACGTGGACAGCCTGATGAGAAGCGTGAGCAG
    AGTGTTCAAGTTCATCGACATGCCCACCGAGGGCAAGCCCACCAAGAGCACCAAGC
    CCTACAAGAACGGCCAGCTGAGCAAGGTGATGATCATCGAGAACAGCCACGTGAAG
    AAGGACGACATCTGGCCCAGCGGCGGCCAGATGACCGTGAAGGACCTGACCGCCAA
    GTACACCGAGGGCGGCAACGCCATCCTGGAGAACATCAGCTTCAGCATCAGCCCCG
    GCCAGAGAGTGGGCCTGCTGGGCAGAACCGGCAGCGGCAAGAGCACCCTGCTGAGC
    GCCTTCCTGAGACTGCTGAACACCGAGGGCGAGATCCAGATCGACGGCGTGAGCTG
    GGACAGCATCACCCTGCAGCAGTGGAGAAAGGCCTTCGGCGTGATCCCCCAGAAGG
    TGTTCATCTTCAGCGGCACCTTCAGAAAGAACCTGGACCCCTACGAGCAGTGGAGC
    GACCAGGAGATCTGGAAGGTGGCCGACGAGGTGGGCCTGAGAAGCGTGATCGAGC
    AGTTCCCCGGCAAGCTGGACTTCGTGCTGGTGGACGGCGGCTGCGTGCTGAGCCAC
    GGCCACAAGCAGCTGATGTGCCTGGCCAGAAGCGTGCTGAGCAAGGCCAAGATCCT
    GCTGCTGGACGAGCCCAGCGCCCACCTGGACCCCGTGACCTACCAGATCATCAGAA
    GAACCCTGAAGCAGGCCTTCGCCGACTGCACCGTGATCCTGTGCGAGCACAGAATC
    GAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATCGAGGAGAACAAGGTGAGACA
    GTACGACAGCATCCAGAAGCTGCTGAACGAGAGAAGCCTGTTCAGACAGGCCATCA
    GCCCCAGCGACAGAGTGAAGCTGTTCCCCCACAGAAACAGCAGCAAGTGCAAGAGC
    AAGCCCCAGATCGCCGCCCTGAAGGAGGAGACCGAGGAGGAGGTGCAGGACACCA
    GACTGTGA
    (SEQ ID NO: 38)
    ATGCAGAGGTCACCTCTGGAAAAGGCTAGCGTGGTCAGCAAGCTATTTTTTTCCTGG
    ACCCGCCCGATACTCAGGAAGGGCTACCGACAGCGGCTGGAGCTGAGTGACATTTA
    TCAGATTCCCTCCGTCGATTCCGCTGACAACCTGTCTGAGAAACTGGAGCGGGAATG
    GGATAGGGAACTGGCGTCCAAAAAAAACCCCAAACTCATCAATGCACTCCGCAGAT
    GCTTCTTCTGGCGGTTTATGTTTTATGGCATATTCCTGTATCTGGGGGAGGTGACGAA
    AGCCGTGCAGCCGCTGCTGCTTGGTCGCATTATCGCGTCATACGATCCAGATAACAA
    GGAGGAAAGAAGTATCGCTATCTATCTCGGGATAGGGCTGTGCCTGCTCTTCATTGT
    GCGGACTCTTCTCTTGCACCCCGCCATTTTCGGTCTGCATCATATAGGTATGCAGATG
    AGAATTGCGATGTTCTCATTGATTTACAAAAAAACGCTTAAGCTAAGTTCAAGGGTG
    CTAGATAAGATATCGATCGGCCAGCTGGTGTCTCTGCTTAGCAACAACCTCAATAAA
    TTCGACGAAGGCCTTGCACTGGCCCACTTCGTGTGGATCGCCCCTCTGCAGGTGGCT
    CTGCTGATGGGGTTAATATGGGAGCTGTTGCAGGCCTCCGCTTTTTGTGGCCTGGGG
    TTTCTCATCGTGTTGGCCTTGTTTCAGGCAGGGCTGGGACGTATGATGATGAAATAT
    AGGGATCAGAGGGCTGGCAAAATCTCTGAGCGCCTGGTTATTACGAGTGAAATGAT
    TGAGAACATCCAGTCAGTGAAGGCCTATTGCTGGGAGGAGGCCATGGAAAAAATGA
    TTGAGAACCTACGCCAGACTGAGCTGAAGTTAACCAGAAAAGCCGCCTATGTGCGC
    TACTTTAACAGTAGCGCATTTTTCTTCTCCGGTTTTTTCGTGGTGTTTCTTAGTGTGTT
    GCCGTATGCCTTAATCAAGGGAATAATACTCCGGAAGATTTTCACTACCATCAGCTT
    CTGTATCGTGTTGCGGATGGCCGTCACCCGGCAGTTTCCCTGGGCAGTACAGACTTG
    GTACGATTCTCTCGGAGCAATTAACAAAATCCAAGACTTTCTACAAAAGCAGGAGT
    ACAAGACCCTGGAGTACAATCTGACCACCACAGAAGTCGTAATGGAGAATGTAACT
    GCCTTCTGGGAAGAGGGCTTTGGCGAACTCTTTGAAAAGGCCAAGCAGAACAATAA
    CAACCGGAAGACCTCCAACGGGGACGACAGCTTATTTTTCAGCAATTTTTCTTTGCT
    CGGGACCCCTGTACTGAAAGATATTAACTTTAAGATCGAGCGCGGACAACTCCTGGC
    TGTCGCCGGCAGCACTGGAGCTGGAAAAACATCACTGCTTATGGTGATAATGGGAG
    AACTCGAACCAAGCGAGGGAAAAATAAAGCACTCTGGACGGATTAGTTTTTGCTCC
    CAGTTCTCGTGGATAATGCCTGGCACCATTAAGGAGAATATCATCTTTGGAGTGAGT
    TACGACGAATACCGGTACCGGTCCGTTATCAAGGCTTGTCAACTCGAGGAGGACATT
    TCTAAATTCGCCGAAAAAGATAATATAGTGCTGGGCGAAGGAGGCATTACACTGAG
    CGGGGGTCAGAGAGCTCGAATTAGCCTCGCCCGAGCAGTCTATAAAGACGCCGATC
    TTTACCTGCTGGATTCCCCTTTTGGGTATTTGGATGTTCTGACAGAGAAGGAAATCTT
    TGAATCATGTGTCTGTAAACTGATGGCCAATAAGACTAGGATTCTAGTGACTTCGAA
    AATGGAGCACCTGAAAAAAGCGGACAAAATTCTGATACTCCATGAAGGGTCTTCCT
    ACTTCTACGGCACCTTCTCAGAGTTGCAGAACTTACAACCTGATTTTTCATCTAAGCT
    TATGGGGTGCGACTCGTTTGACCAGTTCTCCGCTGAAAGACGAAACAGCATCTTAAC
    GGAAACTCTTCACAGGTTCTCATTAGAGGGAGATGCGCCGGTGTCCTGGACAGAGA
    CAAAAAAACAGTCTTTCAAACAGACAGGAGAGTTTGGCGAGAAGAGAAAAAACTC
    AATCCTCAATCCCATCAATTCTATTAGAAAGTTTAGCATCGTCCAAAAAACACCATT
    GCAGATGAATGGGATTGAGGAGGACAGTGATGAGCCTTTGGAACGAAGACTGTCCC
    TGGTACCCGATAGCGAACAGGGTGAGGCCATCCTTCCTAGGATCTCGGTCATAAGTA
    CAGGGCCCACACTGCAGGCCAGGCGACGTCAAAGTGTCCTCAATCTTATGACGCAC
    AGTGTGAATCAGGGGCAGAACATCCATCGTAAGACGACAGCTTCAACTCGAAAGGT
    CAGTCTAGCTCCACAAGCCAATCTTACAGAGCTGGACATTTATTCCCGCCGCCTCAG
    TCAGGAGACCGGATTGGAAATATCAGAGGAAATTAATGAAGAGGATCTGAAGGAAT
    GCTTCTTTGATGACATGGAATCGATCCCCGCTGTTACTACCTGGAACACATATCTGA
    GATATATTACCGTCCATAAGAGCTTAATCTTTGTACTGATATGGTGCTTGGTGATTTT
    CCTGGCAGAGGTTGCGGCGAGTTTGGTCGTGCTATGGCTCCTTGGAAACACTCCCCT
    GCAGGATAAGGGGAACTCCACTCATAGCAGGAATAACAGCTATGCCGTGATCATCA
    CCTCTACCTCCTCTTATTACGTGTTTTACATATACGTCGGTGTTGCGGATACCCTGTT
    GGCAATGGGGTTCTTTAGAGGACTACCCCTAGTTCACACCCTGATCACCGTTTCGAA
    GATCTTGCACCACAAGATGCTTCATAGCGTTCTCCAAGCTCCTATGAGCACCCTTAA
    TACACTGAAAGCAGGAGGTATCCTTAACCGCTTTTCCAAAGACATCGCTATACTCGA
    CGATTTGCTCCCATTGACCATCTTCGACTTCATTCAGCTGCTCCTCATTGTGATCGGC
    GCCATTGCCGTGGTCGCAGTGTTACAGCCATATATTTTCGTAGCCACCGTGCCCGTC
    ATCGTGGCATTTATCATGCTGCGCGCATATTTCTTACAGACATCTCAGCAACTGAAG
    CAGCTGGAATCTGAGGGCAGATCTCCTATTTTTACACACCTGGTTACCAGCCTGAAG
    GGCCTGTGGACCCTGCGTGCTTTCGGTCGCCAACCCTACTTTGAGACTCTCTTCCATA
    AGGCTCTGAATTTACATACTGCCAATTGGTTCCTATACCTTAGTACCCTTCGGTGGTT
    CCAGATGCGGATAGAAATGATCTTCGTGATTTTCTTCATCGCAGTCACTTTCATCTCT
    ATTTTGACGACCGGTGAGGGCGAGGGCAGGGTGGGCATCATTCTGACTTTGGCCATG
    AACATTATGTCAACACTCCAGTGGGCCGTTAATTCAAGCATTGATGTGGATTCCTTG
    ATGCGTTCCGTCAGCAGGGTATTTAAATTCATAGACATGCCCACCGAGGGCAAGCCA
    ACAAAATCTACCAAGCCATACAAAAATGGCCAACTAAGCAAGGTCATGATTATCGA
    GAATTCTCATGTGAAAAAGGACGACATTTGGCCTTCCGGGGGTCAAATGACTGTAA
    AGGACCTGACGGCTAAATACACTGAGGGCGGTAATGCTATCTTGGAGAACATCTCTT
    TCAGCATCTCCCCTGGCCAGAGAGTGGGACTGCTCGGGCGGACAGGCTCCGGAAAG
    TCTACGCTCCTTTCAGCATTCCTTAGACTTCTGAACACCGAAGGTGAGATTCAGATT
    GACGGGGTCTCTTGGGACTCCATCACACTTCAGCAATGGAGGAAGGCATTCGGTGTA
    ATCCCCCAAAAGGTTTTTATCTTCTCCGGAACATTTCGTAAGAATCTGGACCCGTAC
    GAGCAGTGGTCAGATCAGGAGATCTGGAAAGTAGCAGACGAGGTCGGGCTACGGA
    GCGTTATTGAACAGTTTCCTGGCAAACTGGACTTCGTTTTGGTGGACGGAGGCTGTG
    TGCTGAGTCACGGCCATAAACAACTGATGTGCTTAGCTAGGTCTGTTCTCAGCAAGG
    CAAAGATTTTACTGCTGGATGAACCAAGCGCCCACCTTGATCCAGTGACATATCAAA
    TCATCAGAAGAACTCTTAAACAGGCGTTCGCCGACTGCACAGTGATCCTGTGTGAGC
    ACAGAATAGAAGCCATGCTGGAATGTCAACAGTTTCTCGTGATTGAGGAGAACAAG
    GTGCGCCAGTACGATAGCATCCAGAAGTTACTCAATGAAAGGTCACTCTTCAGGCA
    GGCCATCTCACCCAGCGACCGCGTTAAGCTGTTTCCACACCGAAACAGTTCCAAGTG
    CAAAAGTAAGCCACAGATTGCTGCACTGAAGGAAGAGACAGAAGAAGAAGTTCAG
    GACACTCGGCTCTGA
    (SEQ ID NO: 39)
    ATGCAGAGGAGCCCACTGGAGAAAGCCTCCGTGGTGAGTAAACTCTTTTTTAGTTGG
    ACCAGACCCATCCTGCGAAAAGGATACAGGCAGCGCCTCGAGTTGTCAGATATCTA
    CCAGATTCCTTCTGTGGACTCAGCTGACAATTTGAGTGAGAAGCTGGAGCGGGAGTG
    GGATAGAGAGCTGGCGAGCAAAAAAAACCCCAAGCTTATCAATGCTCTGCGCCGCT
    GCTTTTTCTGGAGGTTCATGTTTTATGGGATCTTCCTGTACCTGGGGGAGGTCACCAA
    AGCTGTTCAGCCGCTCCTTCTTGGCCGCATCATCGCCAGCTATGACCCTGATAATAA
    AGAAGAAAGGTCTATTGCTATTTATCTGGGAATTGGCCTCTGCTTGCTCTTCATCGTC
    CGCACCCTTCTGCTGCACCCTGCCATTTTTGGCCTTCACCACATCGGCATGCAAATG
    AGAATTGCCATGTTCTCCCTCATTTACAAAAAGACCCTGAAACTTTCCTCAAGAGTG
    TTAGATAAAATATCCATTGGTCAGCTGGTCAGCCTGCTGTCCAACAATCTTAACAAA
    TTTGATGAAGGCTTGGCGCTGGCCCACTTCGTGTGGATTGCACCTCTGCAGGTGGCC
    CTGTTGATGGGACTTATATGGGAGCTGCTTCAAGCCTCTGCTTTCTGTGGGCTGGGCT
    TTTTGATTGTACTGGCACTTTTTCAGGCTGGGCTCGGAAGAATGATGATGAAATACA
    GAGATCAGCGGGCCGGGAAGATATCAGAGCGACTTGTGATCACCAGTGAAATGATT
    GAAAATATTCAGAGCGTGAAAGCCTACTGCTGGGAAGAAGCCATGGAGAAGATGAT
    TGAGAACCTGAGGCAGACAGAGCTCAAGCTCACTCGGAAGGCTGCTTATGTTCGCT
    ATTTCAACAGCAGCGCCTTCTTCTTCAGTGGCTTCTTTGTTGTCTTCCTGTCTGTTCTG
    CCATATGCACTGATAAAAGGCATTATTTTACGAAAGATCTTCACCACCATCAGTTTT
    TGCATCGTTCTCAGGATGGCCGTCACAAGACAGTTCCCCTGGGCTGTGCAGACCTGG
    TACGATTCCTTGGGGGCCATCAACAAGATTCAAGATTTCTTGCAAAAACAAGAATAT
    AAAACTTTAGAATACAACCTCACCACCACTGAAGTGGTCATGGAAAATGTGACAGC
    CTTTTGGGAGGAGGGTTTTGGAGAATTGTTCGAGAAGGCAAAGCAGAATAACAACA
    ACAGGAAGACGAGCAATGGGGACGACTCTCTCTTCTTCAGCAACTTTTCACTGCTCG
    GGACCCCTGTGTTGAAAGATATAAACTTCAAGATCGAGAGGGGCCAGCTCTTGGCT
    GTGGCAGGCTCCACTGGAGCTGGTAAAACATCTCTTCTCATGGTGATCATGGGGGAA
    CTGGAGCCTTCCGAAGGAAAAATCAAGCACAGTGGGAGAATCTCATTCTGCAGCCA
    GTTTTCCTGGATCATGCCCGGCACCATTAAGGAAAACATCATATTTGGAGTGTCCTA
    TGATGAGTACCGCTACCGGTCAGTCATCAAAGCCTGTCAGTTGGAGGAGGACATCTC
    CAAGTTTGCAGAGAAAGACAACATTGTGCTTGGAGAGGGGGGTATCACTCTTTCTGG
    AGGACAAAGAGCCAGGATCTCTTTGGCCCGGGCAGTCTACAAGGATGCAGACCTCT
    ACTTGTTGGACAGTCCCTTCGGCTACCTCGACGTGCTGACTGAAAAAGAAATTTTTG
    AAAGCTGTGTGTGCAAACTGATGGCAAACAAGACCAGGATTCTTGTCACCAGCAAG
    ATGGAACATCTGAAGAAAGCGGACAAAATTCTGATTCTGCATGAAGGGAGCTCCTA
    CTTCTATGGAACATTTAGCGAGCTTCAGAACCTACAGCCAGACTTCTCCTCCAAATT
    AATGGGCTGTGACTCCTTCGACCAGTTCTCTGCAGAAAGAAGAAACTCTATACTCAC
    AGAGACCCTCCACCGCTTCTCCCTTGAGGGAGATGCCCCAGTTTCTTGGACAGAAAC
    CAAGAAGCAGTCCTTTAAGCAGACTGGCGAGTTTGGTGAAAAGAGGAAAAATTCAA
    TTCTCAATCCAATTAACAGTATTCGCAAGTTCAGCATTGTCCAGAAGACACCCCTCC
    AGATGAATGGCATCGAAGAAGATAGTGACGAGCCGCTGGAGAGACGGCTGAGTCTG
    GTGCCAGATTCAGAACAGGGGGAGGCCATCCTGCCCCGGATCAGCGTCATTTCCAC
    AGGCCCCACATTACAAGCACGGCGCCGGCAGAGTGTTTTAAATCTCATGACCCATTC
    AGTGAACCAGGGCCAAAATATCCACAGGAAGACTACAGCTTCTACCCGGAAAGTGT
    CTCTGGCCCCTCAGGCCAATCTGACCGAGCTGGACATCTACAGCAGGAGGCTCTCCC
    AGGAAACAGGGCTGGAAATATCTGAAGAGATTAATGAAGAGGATCTTAAAGAGTGC
    TTCTTTGATGACATGGAGAGCATCCCCGCGGTGACCACATGGAACACCTACCTTAGA
    TATATTACTGTCCACAAGAGCCTCATATTTGTCCTCATCTGGTGCCTGGTTATTTTCC
    TCGCTGAGGTGGCGGCCAGTCTTGTTGTGCTCTGGCTGCTGGGCAACACTCCTCTCC
    AGGACAAGGGCAATAGTACTCACAGCAGAAATAATTCTTATGCCGTCATCATTACA
    AGCACCTCCAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCTGACACCCTCCTG
    GCCATGGGTTTCTTCCGGGGCCTGCCTTTGGTGCACACCCTCATCACAGTGTCAAAA
    ATTCTGCACCATAAAATGCTTCATTCTGTCCTGCAGGCACCCATGAGCACTTTGAAC
    ACATTGAAGGCTGGCGGCATCCTCAACAGATTTTCTAAAGATATTGCTATCCTGGAT
    GATCTCCTCCCCCTGACAATCTTTGACTTTATCCAGCTTCTGCTGATCGTGATTGGAG
    CCATAGCAGTGGTTGCTGTCCTGCAGCCCTACATTTTTGTGGCCACCGTGCCCGTGAT
    TGTTGCCTTTATTATGCTCAGAGCTTACTTCCTGCAAACTTCTCAACAGCTCAAACAG
    CTAGAATCTGAGGGCCGGAGCCCCATTTTTACCCACCTGGTGACTTCCCTGAAGGGA
    CTGTGGACTCTGAGAGCATTCGGGCGACAGCCTTACTTTGAGACACTGTTCCACAAG
    GCCCTGAACTTGCACACTGCCAACTGGTTTCTTTACCTGAGCACACTCCGCTGGTTCC
    AGATGCGGATAGAGATGATCTTCGTCATCTTTTTTATAGCTGTAACCTTCATTTCTAT
    CCTTACAACAGGAGAAGGAGAGGGCAGGGTGGGAATCATCCTCACGCTGGCTATGA
    ACATAATGTCCACCTTGCAGTGGGCCGTGAATTCCAGTATAGATGTGGATTCTCTAA
    TGAGGAGTGTCTCCCGGGTGTTTAAATTCATTGATATGCCTACTGAGGGGAAACCCA
    CCAAGTCAACAAAACCTTATAAGAATGGACAGCTGAGCAAGGTGATGATAATTGAG
    AACAGCCACGTGAAGAAGGATGACATTTGGCCCAGCGGGGGCCAGATGACTGTGAA
    GGACCTGACGGCCAAGTACACCGAAGGTGGAAATGCCATTTTGGAAAACATCAGCT
    TCTCAATCTCTCCTGGGCAGAGAGTTGGATTGCTGGGTCGCACGGGCAGCGGCAAAT
    CAACCCTGCTCAGTGCCTTCCTTCGGCTCCTGAATACAGAAGGCGAAATCCAAATTG
    ACGGGGTGAGCTGGGACAGCATCACCCTGCAGCAGTGGAGAAAAGCATTTGGGGTC
    ATTCCACAGAAAGTTTTCATCTTCTCTGGCACTTTCAGAAAGAACCTGGACCCCTAT
    GAGCAGTGGAGCGACCAGGAGATCTGGAAGGTTGCAGATGAAGTTGGCCTGCGGAG
    TGTGATAGAACAATTTCCTGGCAAGCTGGATTTTGTGCTGGTAGATGGAGGCTGCGT
    GCTGTCCCACGGCCACAAACAGCTGATGTGCCTCGCCCGCTCCGTTCTTTCAAAGGC
    CAAAATCTTGCTTTTGGATGAGCCCAGTGCTCACCTCGACCCAGTGACCTATCAGAT
    AATCCGCAGGACCTTAAAGCAAGCTTTTGCCGACTGCACCGTCATACTGTGTGAGCA
    CCGGATTGAAGCAATGCTGGAATGCCAGCAGTTTCTGGTGATCGAGGAGAATAAGG
    TCCGGCAGTACGACAGCATCCAGAAGTTGTTGAATGAGCGCAGCCTTTTCCGCCAGG
    CCATCTCCCCATCTGACAGAGTCAAGCTGTTTCCACATAGGAACTCCTCTAAGTGCA
    AGTCCAAGCCCCAGATCGCTGCCCTCAAGGAGGAAACTGAGGAAGAGGTGCAGGAT
    ACCCGCCTGTGA
    (SEQ ID NO: 40)
    ATGCAACGGAGTCCTCTGGAAAAAGCCTCTGTCGTATCTAAGCTTTTCTTCAGTTGG
    ACACGCCCGATTTTGAGAAAGGGTTATCGGCAACGCTTGGAACTTAGTGACATCTAC
    CAAATTCCAAGTGTAGACTCAGCCGATAACTTGAGCGAAAAGCTCGAACGAGAGTG
    GGATCGAGAACTGGCTAGCAAAAAAAATCCCAAACTCATAAATGCCCTGCGACGCT
    GTTTCTTTTGGCGATTTATGTTTTACGGTATTTTCCTTTATTTGGGTGAGGTCACGAA
    GGCTGTACAGCCACTGCTGCTGGGTCGCATCATTGCCTCTTACGACCCTGACAACAA
    AGAGGAGCGGTCAATAGCTATCTACCTTGGTATAGGACTTTGCTTGCTCTTCATAGT
    CCGCACGTTGCTTCTCCACCCTGCTATATTTGGTCTCCATCACATTGGGATGCAAATG
    CGGATCGCGATGTTCAGTCTTATATATAAAAAGACTCTTAAACTTTCCAGCCGGGTT
    CTGGATAAGATCTCTATTGGTCAACTGGTATCTCTTTTGTCTAACAACCTGAATAAGT
    TCGACGAGGGCCTTGCATTGGCCCATTTTGTATGGATTGCCCCTTTGCAAGTCGCCCT
    CCTGATGGGATTGATCTGGGAACTCCTGCAAGCTAGTGCTTTTTGCGGATTGGGATT
    CCTCATAGTCCTTGCGCTCTTTCAGGCGGGACTTGGACGCATGATGATGAAGTATCG
    CGACCAACGAGCTGGCAAGATCAGTGAACGGCTTGTAATAACCAGTGAAATGATAG
    AGAACATCCAGAGCGTAAAAGCTTACTGTTGGGAAGAAGCGATGGAAAAGATGATT
    GAGAACCTTCGCCAGACAGAACTTAAACTTACACGAAAGGCCGCTTATGTCCGGTA
    CTTCAACTCTTCAGCATTTTTTTTTAGTGGCTTCTTTGTAGTGTTCCTGTCCGTCCTTC
    CGTATGCACTTATCAAGGGTATAATACTTAGGAAAATCTTCACAACAATCAGTTTTT
    GCATAGTCCTTCGCATGGCAGTAACTCGCCAATTTCCCTGGGCAGTTCAGACGTGGT
    ACGACTCACTTGGCGCAATTAACAAAATTCAAGATTTCCTCCAAAAGCAAGAGTATA
    AAACCTTGGAATACAACCTTACCACCACAGAAGTTGTAATGGAAAATGTCACAGCC
    TTCTGGGAGGAAGGTTTCGGCGAACTTTTTGAGAAGGCGAAGCAAAATAACAATAA
    TCGGAAAACATCAAACGGTGACGATTCACTGTTCTTTTCTAACTTTAGCCTTCTTGGG
    ACGCCCGTCCTGAAGGACATAAACTTTAAGATTGAACGGGGTCAACTTCTCGCGGTC
    GCAGGGAGTACTGGAGCGGGGAAAACGAGCCTGCTGATGGTGATAATGGGGGAGTT
    GGAGCCCTCAGAAGGCAAGATCAAGCATAGTGGTAGAATTAGCTTCTGCAGTCAAT
    TTAGTTGGATTATGCCGGGCACGATCAAAGAAAATATAATCTTTGGGGTATCCTACG
    ATGAATACAGGTACCGATCAGTGATAAAAGCGTGCCAGCTTGAAGAAGACATTTCA
    AAGTTTGCTGAGAAGGATAATATCGTACTTGGAGAAGGAGGTATCACCCTGTCTGG
    GGGTCAACGAGCGAGGATCTCCCTGGCACGCGCCGTCTACAAGGACGCGGACCTCT
    ATCTGTTGGATTCACCGTTCGGATATTTGGACGTGCTTACGGAGAAAGAAATATTTG
    AGAGCTGTGTTTGCAAGCTCATGGCAAATAAAACCAGAATATTGGTTACAAGCAAG
    ATGGAGCATCTTAAGAAAGCAGATAAAATCCTGATATTGCACGAGGGCTCTTCATAC
    TTCTACGGGACGTTTTCTGAGTTGCAGAACCTCCAGCCGGATTTCAGCTCTAAGCTG
    ATGGGCTGTGATTCCTTTGATCAGTTTAGTGCGGAAAGACGAAACAGTATACTCACC
    GAAACACTGCACAGGTTCTCTCTGGAGGGCGACGCCCCGGTTTCCTGGACAGAGAC
    GAAGAAGCAGTCCTTCAAACAGACAGGCGAGTTTGGGGAGAAAAGGAAAAATAGC
    ATACTCAACCCGATTAACAGCATTCGCAAGTTCAGTATAGTACAAAAGACCCCGTTG
    CAGATGAACGGTATAGAGGAAGATTCTGATGAGCCACTGGAAAGACGGCTTTCTCT
    CGTTCCGGACAGTGAACAGGGAGAGGCAATACTGCCTCGGATCAGCGTTATCTCTAC
    AGGACCTACTTTGCAAGCTCGGCGCCGACAGTCAGTCTTGAATCTTATGACTCATAG
    TGTTAATCAAGGCCAGAATATCCATCGCAAGACCACCGCAAGTACAAGGAAAGTGA
    GCTTGGCACCTCAAGCAAACCTTACTGAACTTGATATCTACTCACGGCGACTTTCAC
    AGGAGACCGGACTTGAAATTAGTGAAGAAATTAACGAGGAGGACCTCAAGGAGTGC
    TTCTTCGATGACATGGAATCAATCCCCGCAGTCACAACCTGGAACACTTATCTGAGG
    TATATAACAGTTCACAAGAGCCTCATTTTTGTACTTATTTGGTGTTTGGTAATTTTCC
    TGGCGGAGGTTGCTGCTTCTTTGGTCGTCCTTTGGCTCCTCGGGAATACACCGCTCCA
    AGACAAAGGCAACTCTACCCATAGTAGGAACAATTCATATGCAGTGATTATAACCA
    GTACATCATCTTATTACGTTTTCTATATTTATGTCGGGGTAGCTGACACGCTGTTGGC
    GATGGGCTTCTTTAGGGGCCTCCCCTTGGTACACACCCTTATCACGGTGAGTAAAAT
    CCTGCATCACAAAATGCTTCATTCTGTACTCCAAGCGCCGATGAGTACGCTTAATAC
    GCTGAAAGCAGGAGGGATACTGAATCGGTTCAGCAAGGACATCGCCATTCTGGATG
    ACCTGCTTCCATTGACAATATTTGATTTCATTCAGCTCCTTCTCATAGTTATTGGAGC
    CATAGCGGTGGTGGCTGTGCTTCAGCCTTATATATTCGTTGCCACAGTTCCCGTTATA
    GTGGCATTTATAATGCTCAGGGCCTACTTTCTCCAGACTTCCCAGCAGTTGAAGCAA
    CTCGAATCAGAAGGAAGGTCACCTATTTTCACACATCTTGTGACTTCCTTGAAGGGC
    TTGTGGACGCTGCGGGCCTTCGGAAGACAACCATATTTTGAAACTCTCTTCCACAAA
    GCTTTGAATCTTCATACTGCGAACTGGTTCCTGTATTTGAGTACTTTGCGCTGGTTCC
    AGATGAGGATAGAAATGATATTCGTTATCTTCTTTATCGCGGTTACGTTCATAAGTA
    TCCTCACTACGGGGGAGGGTGAGGGTAGAGTGGGCATAATACTGACCCTCGCCATG
    AACATTATGTCCACCCTGCAGTGGGCGGTAAACAGCAGCATAGATGTGGATTCTTTG
    ATGCGCAGTGTGAGCAGGGTTTTTAAGTTTATCGATATGCCGACGGAAGGAAAGCC
    CACTAAAAGCACGAAACCCTATAAAAATGGACAGCTTAGCAAAGTAATGATAATCG
    AGAATAGCCATGTGAAAAAGGATGACATATGGCCTTCCGGAGGCCAAATGACTGTT
    AAAGATCTGACCGCTAAATATACCGAGGGCGGCAACGCAATACTCGAAAACATAAG
    CTTTTCCATAAGCCCCGGCCAACGCGTGGGTCTTCTGGGGAGGACTGGCTCCGGAAA
    ATCAACGTTGCTTAGCGCGTTTTTGCGGCTCCTTAACACTGAAGGTGAGATCCAAAT
    AGATGGCGTTAGTTGGGACTCTATAACACTGCAACAATGGCGGAAAGCTTTCGGCGT
    CATACCTCAGAAGGTGTTCATCTTTAGCGGAACGTTCAGGAAGAACTTGGATCCCTA
    CGAACAATGGAGTGATCAAGAAATATGGAAAGTGGCAGATGAGGTAGGCTTGCGCA
    GTGTCATTGAACAATTCCCAGGGAAACTCGACTTTGTACTGGTGGACGGCGGTTGCG
    TCTTGTCACACGGGCACAAACAGTTGATGTGTTTGGCCCGCAGTGTTTTGTCTAAGG
    CGAAGATTCTGTTGCTCGACGAACCGAGTGCTCATCTTGATCCCGTCACCTACCAAA
    TCATCAGAAGGACGTTGAAGCAAGCTTTCGCCGACTGCACTGTAATCCTTTGTGAGC
    ATAGGATCGAAGCAATGCTCGAGTGCCAACAGTTCTTGGTTATAGAGGAGAATAAG
    GTTCGGCAATACGACTCAATACAGAAACTGCTTAATGAGCGGTCACTCTTTCGACAA
    GCTATCTCTCCTAGTGACAGGGTAAAGCTTTTTCCTCATCGGAATTCCAGCAAGTGT
    AAGAGTAAACCACAGATCGCCGCCCTTAAAGAGGAGACCGAAGAAGAGGTGCAGG
    ATACGAGACTTTAG
    • EQUIVALENTS
  • Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims (30)

1. A pharmaceutical composition for treating cystic fibrosis, comprising a codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence of SEQ ID NO: 1.
2-4. (canceled)
5. The pharmaceutical composition of claim 1, wherein the codon optimized CFTR mRNA encoding the CFTR protein is encapsulated within a nanoparticle.
6. The pharmaceutical composition of claim 5, wherein the nanoparticle is a liposome.
7. The pharmaceutical composition of claim 6, wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
8. The pharmaceutical composition of claim 6, wherein the liposome comprises no more than three distinct lipid components.
9-10. (canceled)
11. A method of large scale production of codon optimized mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, comprising in vitro synthesizing codon optimized CFTR mRNA using a SP6 RNA polymerase,
wherein at least 80% of the synthesized codon optimized CFTR mRNA molecules are full-length and wherein at least 100 mg of codon optimized mRNA is synthesized at a single batch, and
wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence of SEQ ID NO: 1.
12. (canceled)
13. The method of claim 11, wherein the in vitro synthesis of codon optimized CFTR mRNA results in a secondary polynucleotide species that constitutes less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the total mRNA synthesized.
14. The method of claim 11, wherein at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the synthesized codon optimized CFTR mRNA molecules are full-length.
15. (canceled)
16. The method of claim 11, wherein at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of codon optimized CFTR mRNA is synthesized at a single batch.
17.-20. (canceled)
21. The method of claim 11, wherein the method further comprises a step of capping and/or tailing of the synthesized codon optimized CFTR mRNA.
22. (canceled)
23. A method of treating cystic fibrosis, comprising administering to a subject in need of treatment a composition comprising a codon optimized mRNA encoding an Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, wherein the codon optimized CFTR mRNA comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1.
24. The method of claim 23, wherein the codon optimized CFTR mRNA comprises SEQ ID NO: 1.
25.-26. (canceled)
27. The method of claim 23, wherein the codon optimized CFTR mRNA is encapsulated within a nanoparticle.
28. The method of claim 27, wherein the nanoparticle is a liposome.
29. The method of claim 28, wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
30-32. (canceled)
33. The method of claim 23, wherein the codon optimized CFTR mRNA is administered to the subject via pulmonary delivery.
34. The method of claim 33, wherein the pulmonary delivery is nebulization.
35. A pharmaceutical composition for treating cystic fibrosis, comprising an mRNA encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein and wherein the mRNA encoding the CFTR protein comprises a polynucleotide sequence comprising any one of SEQ ID NO: 21-40.
36-40. (canceled)
41. The pharmaceutical composition of claim 35, wherein the mRNA is encapsulated in a nanoparticle, and wherein the nanoparticle is a liposome.
42. The pharmaceutical composition of claim 41, wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
43-44. (canceled)
US17/570,352 2017-02-27 2022-01-06 Novel Codon-Optimized CFTR MRNA Pending US20220193247A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/570,352 US20220193247A1 (en) 2017-02-27 2022-01-06 Novel Codon-Optimized CFTR MRNA

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762464215P 2017-02-27 2017-02-27
US15/907,131 US11253605B2 (en) 2017-02-27 2018-02-27 Codon-optimized CFTR MRNA
US17/570,352 US20220193247A1 (en) 2017-02-27 2022-01-06 Novel Codon-Optimized CFTR MRNA

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US15/907,131 Continuation US11253605B2 (en) 2017-02-27 2018-02-27 Codon-optimized CFTR MRNA

Publications (1)

Publication Number Publication Date
US20220193247A1 true US20220193247A1 (en) 2022-06-23

Family

ID=61622756

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/907,131 Active 2040-12-26 US11253605B2 (en) 2017-02-27 2018-02-27 Codon-optimized CFTR MRNA
US17/570,352 Pending US20220193247A1 (en) 2017-02-27 2022-01-06 Novel Codon-Optimized CFTR MRNA

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US15/907,131 Active 2040-12-26 US11253605B2 (en) 2017-02-27 2018-02-27 Codon-optimized CFTR MRNA

Country Status (9)

Country Link
US (2) US11253605B2 (en)
EP (1) EP3585417B1 (en)
AU (1) AU2018224326B2 (en)
CA (1) CA3054062A1 (en)
EA (1) EA201991747A1 (en)
IL (1) IL268854A (en)
MA (1) MA47603A (en)
MX (1) MX2019010155A (en)
WO (1) WO2018157154A2 (en)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3998064A1 (en) 2011-06-08 2022-05-18 Translate Bio, Inc. Cleavable lipids
MA45053A (en) 2016-05-18 2019-03-27 Modernatx Inc POLYNUCLEOTIDES CODING FOR A CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR FOR THE TREATMENT OF CYSTIC FIBROSIS
EP4143326A1 (en) * 2020-04-27 2023-03-08 University of Iowa Research Foundation Compositions and methods for the treatment of cystic fibrosis
AU2021265768B2 (en) * 2020-04-27 2023-03-30 4D Molecular Therapeutics Inc. Adeno-associated variants, formulations and methods for pulmonary delivery
WO2021222801A2 (en) * 2020-05-01 2021-11-04 Arcturus Therapeutics, Inc. Nucleic acids and methods of treatment for cystic fibrosis
US20230181619A1 (en) * 2020-05-07 2023-06-15 Translate Bio, Inc. Improved compositions for cftr mrna therapy
AU2021268028A1 (en) * 2020-05-07 2023-01-19 Translate Bio, Inc. Generation of optimized nucleotide sequences
CA3198726A1 (en) * 2020-10-15 2022-04-21 Translate Bio, Inc. Large scale synthesis of messenger rna
EP4240326A1 (en) * 2020-11-09 2023-09-13 Translate Bio, Inc. Improved compositions for delivery of codon-optimized mrna
WO2023023487A1 (en) * 2021-08-16 2023-02-23 Translate Bio, Inc. Screening codon-optimized nucleotide sequences

Family Cites Families (435)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2647121A (en) 1951-02-02 1953-07-28 Ruth P Jacoby Diamine-bis-acetamides
US2819718A (en) 1953-07-16 1958-01-14 Isidore H Goldman Drainage tube
US2717909A (en) 1953-09-24 1955-09-13 Monsanto Chemicals Hydroxyethyl-keryl-alkylene-ammonium compounds
US2844629A (en) 1956-04-25 1958-07-22 American Home Prod Fatty acid amides and derivatives thereof
US3096560A (en) 1958-11-21 1963-07-09 William J Liebig Process for synthetic vascular implants
FR1378382A (en) 1962-12-01 1964-11-13 Sandoz Sa Amides of amino-propionic acid, usable in particular for the treatment of textile fibers
GB1072118A (en) 1962-12-01 1967-06-14 Sandoz Ag Amides of aminopropionic acid
JPS5141663B1 (en) 1966-03-12 1976-11-11
NL143127B (en) 1969-02-04 1974-09-16 Rhone Poulenc Sa REINFORCEMENT DEVICE FOR A DEFECTIVE HEART VALVE.
US3614955A (en) 1970-02-09 1971-10-26 Medtronic Inc Standby defibrillator and method of operation
US3614954A (en) 1970-02-09 1971-10-26 Medtronic Inc Electronic standby defibrillator
JPS5024216Y1 (en) 1970-12-29 1975-07-21
US3945052A (en) 1972-05-01 1976-03-23 Meadox Medicals, Inc. Synthetic vascular graft and method for manufacturing the same
US3805301A (en) 1972-07-28 1974-04-23 Meadox Medicals Inc Tubular grafts having indicia thereon
JPS49127908A (en) 1973-04-20 1974-12-07
JPS5624664B2 (en) 1973-06-28 1981-06-08
US4013507A (en) 1973-09-18 1977-03-22 California Institute Of Technology Ionene polymers for selectively inhibiting the vitro growth of malignant cells
JPS5123537A (en) 1974-04-26 1976-02-25 Adeka Argus Chemical Co Ltd KASOZAISOSEIBUTSU
GB1527592A (en) 1974-08-05 1978-10-04 Ici Ltd Wound dressing
US3995623A (en) 1974-12-23 1976-12-07 American Hospital Supply Corporation Multipurpose flow-directed catheter
JPS5813576B2 (en) 1974-12-27 1983-03-14 アデカ ア−ガスカガク カブシキガイシヤ Stabilized synthetic polymer composition
DE2520814A1 (en) 1975-05-09 1976-11-18 Bayer Ag Light stabilisation of polyurethanes - using polymeric tert. amines from aliphatic diamines and (meth)acrylic esters or amides
US4281669A (en) 1975-05-09 1981-08-04 Macgregor David C Pacemaker electrode with porous system
JPS5210847A (en) 1975-07-16 1977-01-27 Nippon Steel Corp Pinch roll
US4096860A (en) 1975-10-08 1978-06-27 Mclaughlin William F Dual flow encatheter
CA1069652A (en) 1976-01-09 1980-01-15 Alain F. Carpentier Supported bioprosthetic heart valve with compliant orifice ring
US4134402A (en) 1976-02-11 1979-01-16 Mahurkar Sakharam D Double lumen hemodialysis catheter
US4072146A (en) 1976-09-08 1978-02-07 Howes Randolph M Venous catheter device
US4335723A (en) 1976-11-26 1982-06-22 The Kendall Company Catheter having inflatable retention means
US4099528A (en) 1977-02-17 1978-07-11 Sorenson Research Co., Inc. Double lumen cannula
US4140126A (en) 1977-02-18 1979-02-20 Choudhury M Hasan Method for performing aneurysm repair
US4265745A (en) 1977-05-25 1981-05-05 Teijin Limited Permselective membrane
US4182833A (en) 1977-12-07 1980-01-08 Celanese Polymer Specialties Company Cationic epoxide-amine reaction products
US4180068A (en) 1978-04-13 1979-12-25 Motion Control, Incorporated Bi-directional flow catheter with retractable trocar/valve structure
EP0005035B1 (en) 1978-04-19 1981-09-23 Imperial Chemical Industries Plc A method of preparing a tubular product by electrostatic spinning
US4284459A (en) 1978-07-03 1981-08-18 The Kendall Company Method for making a molded catheter
US4227533A (en) 1978-11-03 1980-10-14 Bristol-Myers Company Flushable urinary catheter
US4375817A (en) 1979-07-19 1983-03-08 Medtronic, Inc. Implantable cardioverter
DE3010841A1 (en) 1980-03-21 1981-10-08 Ulrich Dr.med. 6936 Haag Uthmann CATHEDER
US4308085A (en) 1980-07-28 1981-12-29 Jenoptik Jena Gmbh Process for the preparation of high molecular thermoplastic epoxide-amine-polyadducts
US4339369A (en) 1981-04-23 1982-07-13 Celanese Corporation Cationic epoxide-amine reaction products
US4406656A (en) 1981-06-01 1983-09-27 Brack Gillium Hattler Venous catheter having collapsible multi-lumens
US4475972A (en) 1981-10-01 1984-10-09 Ontario Research Foundation Implantable material
US4401472A (en) 1982-02-26 1983-08-30 Martin Marietta Corporation Hydraulic cement mixes and processes for improving hydraulic cement mixes
US4568329A (en) 1982-03-08 1986-02-04 Mahurkar Sakharam D Double lumen catheter
US4546499A (en) 1982-12-13 1985-10-15 Possis Medical, Inc. Method of supplying blood to blood receiving vessels
US4530113A (en) 1983-05-20 1985-07-23 Intervascular, Inc. Vascular grafts with cross-weave patterns
US4550447A (en) 1983-08-03 1985-11-05 Shiley Incorporated Vascular graft prosthesis
US4647416A (en) 1983-08-03 1987-03-03 Shiley Incorporated Method of preparing a vascular graft prosthesis
US5104399A (en) 1986-12-10 1992-04-14 Endovascular Technologies, Inc. Artificial graft and implantation method
US4710169A (en) 1983-12-16 1987-12-01 Christopher T Graham Urinary catheter with collapsible urethral tube
US4571241A (en) 1983-12-16 1986-02-18 Christopher T Graham Urinary catheter with collapsible urethral tube
US4737518A (en) 1984-04-03 1988-04-12 Takeda Chemical Industries, Ltd. Lipid derivatives, their production and use
US4562596A (en) 1984-04-25 1986-01-07 Elliot Kornberg Aortic graft, device and method for performing an intraluminal abdominal aortic aneurysm repair
US4782836A (en) 1984-05-24 1988-11-08 Intermedics, Inc. Rate adaptive cardiac pacemaker responsive to patient activity and temperature
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4662382A (en) 1985-01-16 1987-05-05 Intermedics, Inc. Pacemaker lead with enhanced sensitivity
US4762915A (en) 1985-01-18 1988-08-09 Liposome Technology, Inc. Protein-liposome conjugates
US4860751A (en) 1985-02-04 1989-08-29 Cordis Corporation Activity sensor for pacemaker control
US5223263A (en) 1988-07-07 1993-06-29 Vical, Inc. Liponucleotide-containing liposomes
CA1320724C (en) 1985-07-19 1993-07-27 Koichi Kanehira Terpene amino alcohols and medicinal uses thereof
US4701162A (en) 1985-09-24 1987-10-20 The Kendall Company Foley catheter assembly
US4737323A (en) 1986-02-13 1988-04-12 Liposome Technology, Inc. Liposome extrusion method
DE3616824A1 (en) 1986-05-17 1987-11-19 Schering Ag USE OF CURABLE RESIN MIXTURES FOR SURFACE COATINGS AND PRINTING INKS AND METHOD FOR THE PRODUCTION THEREOF
EP0255899B1 (en) 1986-07-31 1992-07-15 Werner Prof. Dr.-Ing. Irnich Rate adaptive pacemaker
US4960409A (en) 1986-09-11 1990-10-02 Catalano Marc L Method of using bilumen peripheral venous catheter with adapter
JPH0829776B2 (en) 1986-10-29 1996-03-27 東燃化学株式会社 Synthetic resin container and mold for manufacturing the same
US4720517A (en) 1986-11-24 1988-01-19 Ciba-Geigy Corporation Compositions stabilized with N-hydroxyiminodiacetic and dipropionic acids and esters thereof
US4920016A (en) 1986-12-24 1990-04-24 Linear Technology, Inc. Liposomes with enhanced circulation time
DE3728917A1 (en) 1987-08-29 1989-03-09 Roth Hermann J Novel lipids containing an asymmetrically substituted disulphide bridge, processes for their preparation, and their use as medicaments
US4946683A (en) 1987-11-18 1990-08-07 Vestar, Inc. Multiple step entrapment/loading procedure for preparing lipophilic drug-containing liposomes
US5047540A (en) 1987-12-17 1991-09-10 Shionogi & Co., Ltd. Lipid derivatives
US5138067A (en) 1987-12-17 1992-08-11 Shionogi & Co. Ltd. Lipid derivatives
US4892540A (en) 1988-04-21 1990-01-09 Sorin Biomedica S.P.A. Two-leaflet prosthetic heart valve
US5176661A (en) 1988-09-06 1993-01-05 Advanced Cardiovascular Systems, Inc. Composite vascular catheter
US5024671A (en) 1988-09-19 1991-06-18 Baxter International Inc. Microporous vascular graft
US5200395A (en) 1988-10-18 1993-04-06 Ajinomoto Company, Inc. Pharmaceutical composition of BUF-5 for treating anemia
CA2001401A1 (en) 1988-10-25 1990-04-25 Claude Piantadosi Quaternary amine containing ether or ester lipid derivatives and therapeutic compositions
US6214804B1 (en) 1989-03-21 2001-04-10 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
ATE277193T1 (en) 1989-03-21 2004-10-15 Vical Inc EXPRESSION OF EXOGENEOUS POLYNUCLEOTIDE SEQUENCES IN VERTEBRATES
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
FR2645866B1 (en) 1989-04-17 1991-07-05 Centre Nat Rech Scient NEW LIPOPOLYAMINES, THEIR PREPARATION AND THEIR USE
US5194654A (en) 1989-11-22 1993-03-16 Vical, Inc. Lipid derivatives of phosphonoacids for liposomal incorporation and method of use
US5279833A (en) 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
US5101824A (en) 1990-04-16 1992-04-07 Siemens-Pacesetter, Inc. Rate-responsive pacemaker with circuitry for processing multiple sensor inputs
US5264618A (en) 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US5405379A (en) 1990-07-26 1995-04-11 Lane; Rodney J. Self expanding vascular endoprosthesis for aneurysms
US5693338A (en) 1994-09-29 1997-12-02 Emisphere Technologies, Inc. Diketopiperazine-based delivery systems
JPH0765267B2 (en) 1990-08-22 1995-07-12 花王株式会社 Softening agent
DE69118083T2 (en) 1990-10-09 1996-08-22 Cook Inc Percutaneous stent assembly
EP0491082B1 (en) 1990-12-19 1995-04-12 Peter Dr. Ing. Osypka Pacemaker lead with an inner duct and with an electrode head
US5116360A (en) 1990-12-27 1992-05-26 Corvita Corporation Mesh composite graft
US5405363A (en) 1991-03-15 1995-04-11 Angelon Corporation Implantable cardioverter defibrillator having a smaller displacement volume
US5330768A (en) 1991-07-05 1994-07-19 Massachusetts Institute Of Technology Controlled drug delivery using polymer/pluronic blends
US5545449A (en) 1991-10-02 1996-08-13 Weyerhaeuser Company Polyether-reinforced fiber-based materials
US5151105A (en) 1991-10-07 1992-09-29 Kwan Gett Clifford Collapsible vessel sleeve implant
US5284491A (en) 1992-02-27 1994-02-08 Medtronic, Inc. Cardiac pacemaker with hysteresis behavior
US5352461A (en) 1992-03-11 1994-10-04 Pharmaceutical Discovery Corporation Self assembling diketopiperazine drug delivery system
SE9200951D0 (en) 1992-03-27 1992-03-27 Kabi Pharmacia Ab PHARMACEUTICAL COMPOSITION CONTAINING A DEFINED LIPID SYSTEM
DE69325096T2 (en) 1992-04-06 1999-09-30 Biosite Diagnostics Inc MORPHINE DERIVATIVES AND THEIR CONJUGATES AND LABELS WITH PROTEINS AND POLYPEPTIDES
US6670178B1 (en) 1992-07-10 2003-12-30 Transkaryotic Therapies, Inc. In Vivo production and delivery of insulinotropin for gene therapy
EP0657534A4 (en) 1992-08-04 1997-06-04 Green Cross Corp Antiallergic agent.
US5334761A (en) 1992-08-28 1994-08-02 Life Technologies, Inc. Cationic lipids
US5461223A (en) 1992-10-09 1995-10-24 Eastman Kodak Company Bar code detecting circuitry
US5300022A (en) 1992-11-12 1994-04-05 Martin Klapper Urinary catheter and bladder irrigation system
US5496362A (en) 1992-11-24 1996-03-05 Cardiac Pacemakers, Inc. Implantable conformal coil patch electrode with multiple conductive elements for cardioversion and defibrillation
US5552155A (en) 1992-12-04 1996-09-03 The Liposome Company, Inc. Fusogenic lipsomes and methods for making and using same
US5716395A (en) 1992-12-11 1998-02-10 W.L. Gore & Associates, Inc. Prosthetic vascular graft
US6020317A (en) 1993-02-19 2000-02-01 Nippon Shinyaku Co. Ltd. Glycerol derivative, device and pharmaceutical composition
US5395619A (en) 1993-03-03 1995-03-07 Liposome Technology, Inc. Lipid-polymer conjugates and liposomes
US5697953A (en) 1993-03-13 1997-12-16 Angeion Corporation Implantable cardioverter defibrillator having a smaller displacement volume
US5624976A (en) 1994-03-25 1997-04-29 Dentsply Gmbh Dental filling composition and method
US5314430A (en) 1993-06-24 1994-05-24 Medtronic, Inc. Atrial defibrillator employing transvenous and subcutaneous electrodes and method of use
DE4325848A1 (en) 1993-07-31 1995-02-02 Basf Ag Process for the preparation of N- (2-hydroxyethyl) piperazine
DE69428057T2 (en) 1993-10-06 2002-04-18 Kansai Electric Power Co Process for the separation of carbon dioxide from combustion gases
US5609624A (en) 1993-10-08 1997-03-11 Impra, Inc. Reinforced vascular graft and method of making same
SE9303481L (en) 1993-10-22 1995-04-23 Berol Nobel Ab hygiene composition
AU1091095A (en) 1993-11-08 1995-05-29 Harrison M. Lazarus Intraluminal vascular graft and method
CA2176714A1 (en) 1993-11-24 1995-06-01 Timothy D. Heath Amphiphilic derivatives of piperazine
US5595756A (en) 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
US5464924A (en) 1994-01-07 1995-11-07 The Dow Chemical Company Flexible poly(amino ethers) for barrier packaging
US5844107A (en) 1994-03-23 1998-12-01 Case Western Reserve University Compacted nucleic acids and their delivery to cells
ATE234605T1 (en) 1994-04-12 2003-04-15 Liposome Co Inc FUSOGENIC LIPOSOMES AND METHOD FOR THE PRODUCTION AND USE THEREOF
US5858747A (en) 1994-07-20 1999-01-12 Cytotherapeutics, Inc. Control of cell growth in a bioartificial organ with extracellular matrix coated microcarriers
US5820873A (en) 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5641665A (en) 1994-11-28 1997-06-24 Vical Incorporated Plasmids suitable for IL-2 expression
US6071890A (en) 1994-12-09 2000-06-06 Genzyme Corporation Organ-specific targeting of cationic amphiphile/DNA complexes for gene therapy
US5965434A (en) 1994-12-29 1999-10-12 Wolff; Jon A. Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds
US6485726B1 (en) 1995-01-17 2002-11-26 The Brigham And Women's Hospital, Inc. Receptor specific transepithelial transport of therapeutics
US5830430A (en) 1995-02-21 1998-11-03 Imarx Pharmaceutical Corp. Cationic lipids and the use thereof
EP0822835A1 (en) 1995-04-17 1998-02-11 Imarx Pharmaceutical Corp. Hybrid magnetic resonance contrast agents
US5772694A (en) 1995-05-16 1998-06-30 Medical Carbon Research Institute L.L.C. Prosthetic heart valve with improved blood flow
US5783383A (en) 1995-05-23 1998-07-21 The Board Of Trustees Of The Leland Stanford Junior University Method of detecting cytomegalovirus (CMV)
US5981501A (en) 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US7422902B1 (en) 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
EP1489184A1 (en) 1995-06-07 2004-12-22 Inex Pharmaceutical Corp. Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5705385A (en) 1995-06-07 1998-01-06 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5609629A (en) 1995-06-07 1997-03-11 Med Institute, Inc. Coated implantable medical device
US5607385A (en) 1995-08-17 1997-03-04 Medtronic, Inc. Device and algorithm for a combined cardiomyostimulator and a cardiac pacer-carioverter-defibrillator
US5744335A (en) 1995-09-19 1998-04-28 Mirus Corporation Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein
FR2740978B1 (en) 1995-11-10 1998-01-02 Ela Medical Sa IMPLANTABLE DEFIBRILLATOR / CARDIOVERVER ACTIVE MEDICAL DEVICE
US5874105A (en) 1996-01-31 1999-02-23 Collaborative Laboratories, Inc. Lipid vesicles formed with alkylammonium fatty acid salts
EP0910576B1 (en) 1996-04-11 2004-08-11 University Of British Columbia Fusogenic liposomes
US5935936A (en) 1996-06-03 1999-08-10 Genzyme Corporation Compositions comprising cationic amphiphiles and co-lipids for intracellular delivery of therapeutic molecules
US5913848A (en) 1996-06-06 1999-06-22 Luther Medical Products, Inc. Hard tip over-the-needle catheter and method of manufacturing the same
US5677124A (en) 1996-07-03 1997-10-14 Ambion, Inc. Ribonuclease resistant viral RNA standards
US5736573A (en) 1996-07-31 1998-04-07 Galat; Alexander Lipid and water soluble derivatives of drugs
US7288266B2 (en) 1996-08-19 2007-10-30 United States Of America As Represented By The Secretary, Department Of Health And Human Services Liposome complexes for increased systemic delivery
CA2264140A1 (en) 1996-08-26 1998-03-05 Transgene S.A. Cationic lipid-nucleic acid complexes
KR100507660B1 (en) 1996-09-13 2005-08-10 리폭센 테크놀로지즈 리미티드 Liposomes
TW520297B (en) 1996-10-11 2003-02-11 Sequus Pharm Inc Fusogenic liposome composition and method
CA2270396C (en) 1996-11-04 2008-03-11 Qiagen Gmbh Cationic reagents for transfection
US6887665B2 (en) 1996-11-14 2005-05-03 Affymetrix, Inc. Methods of array synthesis
US5985930A (en) 1996-11-21 1999-11-16 Pasinetti; Giulio M. Treatment of neurodegenerative conditions with nimesulide
US6204297B1 (en) 1996-11-26 2001-03-20 Rhodia Inc. Nonionic gemini surfactants
JPH10197978A (en) 1997-01-09 1998-07-31 Mitsubishi Paper Mills Ltd Silver halide photographic sensitive material
EP0853123A1 (en) 1997-01-10 1998-07-15 Roche Diagnostics GmbH Purification of DNA by 'cross-flow-filtration'
FR2760193B1 (en) 1997-02-28 1999-05-28 Transgene Sa LIPIDS AND COMPLEXES OF CATIONIC LIPIDS AND ACTIVE SUBSTANCES, IN PARTICULAR FOR THE TRANSFECTION OF CELLS
US5837283A (en) 1997-03-12 1998-11-17 The Regents Of The University Of California Cationic lipid compositions targeting angiogenic endothelial cells
US5945326A (en) 1997-03-20 1999-08-31 New England Biolabs, Inc. Method for cloning and producing the Spel restriction endonuclease
US20030104044A1 (en) 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
JP4656675B2 (en) 1997-05-14 2011-03-23 ユニバーシティー オブ ブリティッシュ コロンビア High rate encapsulation of charged therapeutic agents in lipid vesicles
US6835395B1 (en) 1997-05-14 2004-12-28 The University Of British Columbia Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles
JPH115786A (en) 1997-06-13 1999-01-12 Pola Chem Ind Inc Novel aminohydroxypropylpiperazine derivative
US6067471A (en) 1998-08-07 2000-05-23 Cardiac Pacemakers, Inc. Atrial and ventricular implantable cardioverter-defibrillator and lead system
JPH1180142A (en) 1997-09-05 1999-03-26 Pola Chem Ind Inc Production of diphenylalkyl compound
WO1999014346A2 (en) 1997-09-19 1999-03-25 Sequitur, Inc. SENSE mRNA THERAPY
US6165763A (en) 1997-10-30 2000-12-26 Smithkline Beecham Corporation Ornithine carbamoyltransferase
US6096075A (en) 1998-01-22 2000-08-01 Medical Carbon Research Institute, Llc Prosthetic heart valve
US6617171B2 (en) 1998-02-27 2003-09-09 The General Hospital Corporation Methods for diagnosing and treating autoimmune disease
US6271209B1 (en) 1998-04-03 2001-08-07 Valentis, Inc. Cationic lipid formulation delivering nucleic acid to peritoneal tumors
US6176877B1 (en) 1998-04-20 2001-01-23 St. Jude Medical, Inc. Two piece prosthetic heart valve
DE19822602A1 (en) 1998-05-20 1999-11-25 Goldschmidt Ag Th Process for the preparation of polyamino acid esters by esterification of acidic polyamino acids or transesterification of polyamino acid esters
NO313244B1 (en) 1998-07-08 2002-09-02 Crew Dev Corp Process for the isolation and production of magnesite or magnesium chloride
US6055454A (en) 1998-07-27 2000-04-25 Cardiac Pacemakers, Inc. Cardiac pacemaker with automatic response optimization of a physiologic sensor based on a second sensor
KR20010072816A (en) 1998-08-20 2001-07-31 쿡 인코포레이티드 Coated implantable medical device
US6210892B1 (en) 1998-10-07 2001-04-03 Isis Pharmaceuticals, Inc. Alteration of cellular behavior by antisense modulation of mRNA processing
US6656498B1 (en) 1998-11-25 2003-12-02 Vanderbilt University Cationic liposomes for gene transfer
US6248725B1 (en) 1999-02-23 2001-06-19 Amgen, Inc. Combinations and methods for promoting in vivo liver cell proliferation and enhancing in vivo liver-directed gene transduction
US6379698B1 (en) 1999-04-06 2002-04-30 Isis Pharmaceuticals, Inc. Fusogenic lipids and vesicles
EP1173600A2 (en) 1999-04-20 2002-01-23 The University Of British Columbia Cationic peg-lipids and methods of use
US6342244B1 (en) 1999-04-23 2002-01-29 Alza Corporation Releasable linkage and compositions containing same
US6169923B1 (en) 1999-04-23 2001-01-02 Pacesetter, Inc. Implantable cardioverter-defibrillator with automatic arrhythmia detection criteria adjustment
CZ20014123A3 (en) 1999-05-19 2002-06-12 Lexigen Pharmaceuticals Corp. Expression and export of alpha interferons as Fc fusion proteins
US6696424B1 (en) 1999-05-28 2004-02-24 Vical Incorporated Cytofectin dimers and methods of use thereof
US6346382B1 (en) 1999-06-01 2002-02-12 Vanderbilt University Human carbamyl phosphate synthetase I polymorphism and diagnostic methods related thereto
EP1202714A1 (en) 1999-07-16 2002-05-08 Purdue Research Foundation Vinyl ether lipids with cleavable hydrophilic headgroups
HUP0202295A3 (en) 1999-07-23 2005-01-28 Genentech Inc Method for rnase- and organic solvent-free plasmid dna purification using tangential flow filtration
US6358278B1 (en) 1999-09-24 2002-03-19 St. Jude Medical, Inc. Heart valve prosthesis with rotatable cuff
US6371983B1 (en) 1999-10-04 2002-04-16 Ernest Lane Bioprosthetic heart valve
US7060291B1 (en) 1999-11-24 2006-06-13 Transave, Inc. Modular targeted liposomal delivery system
IL150484A0 (en) 1999-12-30 2002-12-01 Novartis Ag Novel colloid synthetic vectors for gene therapy
US20010036454A1 (en) 2000-02-17 2001-11-01 Chester Li Genetic modification of the lung as a portal for gene delivery
US6370434B1 (en) 2000-02-28 2002-04-09 Cardiac Pacemakers, Inc. Cardiac lead and method for lead implantation
US6565960B2 (en) 2000-06-01 2003-05-20 Shriners Hospital Of Children Polymer composite compositions
EP1297169B1 (en) 2000-06-26 2012-08-08 Ethris Gmbh Method for transfecting cells using a magnetic field
IL138474A0 (en) 2000-09-14 2001-10-31 Epox Ltd Highly branched water-soluble polyamine oligomers, process for their preparation and applications thereof
USRE43612E1 (en) 2000-10-10 2012-08-28 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
US7427394B2 (en) 2000-10-10 2008-09-23 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
US6998115B2 (en) 2000-10-10 2006-02-14 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
WO2002034236A2 (en) 2000-10-25 2002-05-02 The University Of British Columbia Lipid formulations for target delivery
GB0028361D0 (en) 2000-11-21 2001-01-03 Glaxo Group Ltd Method of separating extra chromosomal dna from other cellular components
US20020094528A1 (en) 2000-11-29 2002-07-18 Salafsky Joshua S. Method and apparatus using a surface-selective nonlinear optical technique for detection of probe-target interations
JP2002167368A (en) 2000-12-01 2002-06-11 Nitto Denko Corp Alkyl group-substituted dendrimer and method for preparing the same
US20050004058A1 (en) 2000-12-07 2005-01-06 Patrick Benoit Sequences upstream of the carp gene, vectors containing them and uses thereof
DE10109897A1 (en) 2001-02-21 2002-11-07 Novosom Ag Optional cationic liposomes and their use
US20020192721A1 (en) 2001-03-28 2002-12-19 Engeneos, Inc. Modular molecular clasps and uses thereof
TW588032B (en) 2001-04-23 2004-05-21 Shinetsu Chemical Co New tertiary amine compound having ester structure and method for producing the same
US6585410B1 (en) 2001-05-03 2003-07-01 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Radiant temperature nulling radiometer
ATE490267T1 (en) 2001-06-05 2010-12-15 Curevac Gmbh STABILIZED MRNA WITH INCREASED G/C CONTENT CODING A VIRAL ANTIGEN
EP2386637B1 (en) 2001-09-28 2018-05-16 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Microrna molecules
AU2002340490A1 (en) 2001-11-09 2003-05-19 Bayer Healthcare Ag Isotopically coded affinity markers 3
DE10162480A1 (en) 2001-12-19 2003-08-07 Ingmar Hoerr The application of mRNA for use as a therapeutic agent against tumor diseases
DE10207178A1 (en) 2002-02-19 2003-09-04 Novosom Ag Components for the production of amphoteric liposomes
DE10214983A1 (en) 2002-04-04 2004-04-08 TransMIT Gesellschaft für Technologietransfer mbH Nebulisable liposomes and their use for pulmonary application of active substances
US20030215395A1 (en) 2002-05-14 2003-11-20 Lei Yu Controllably degradable polymeric biomolecule or drug carrier and method of synthesizing said carrier
US7601367B2 (en) 2002-05-28 2009-10-13 Mirus Bio Llc Compositions and processes using siRNA, amphipathic compounds and polycations
EP2338478B1 (en) 2002-06-28 2014-07-23 Protiva Biotherapeutics Inc. Method for producing liposomes
DE10229872A1 (en) 2002-07-03 2004-01-29 Curevac Gmbh Immune stimulation through chemically modified RNA
US20040028804A1 (en) 2002-08-07 2004-02-12 Anderson Daniel G. Production of polymeric microarrays
WO2004018654A2 (en) 2002-08-22 2004-03-04 Celltran Limited Cell culture surface
EP1565512B1 (en) 2002-11-04 2018-07-04 Momentive Performance Materials GmbH Linear polyamino and/or polyammonium polysiloxane copolymers i
AU2003281978A1 (en) 2002-11-22 2004-06-18 Boehringer Ingelheim International Gmbh 2,5-diketopiperazines for the treatment of obesity
US7169892B2 (en) 2003-01-10 2007-01-30 Astellas Pharma Inc. Lipid-peptide-polymer conjugates for long blood circulation and tumor specific drug delivery systems
US8054291B2 (en) 2003-01-20 2011-11-08 Asahi Kasei Emd Corporation Pointing device
JP2006520611A (en) 2003-03-05 2006-09-14 セネスコ テクノロジーズ，インコーポレイティド Use of antisense oligonucleotides or siRNA to suppress the expression of eIF-5A1
US20040224912A1 (en) 2003-05-07 2004-11-11 Isis Pharmaceuticals Inc. Modulation of PAI-1 mRNA-binding protein expression
US7619017B2 (en) 2003-05-19 2009-11-17 Wacker Chemical Corporation Polymer emulsions resistant to biodeterioration
US7507859B2 (en) 2003-06-16 2009-03-24 Fifth Base Llc Functional synthetic molecules and macromolecules for gene delivery
EP1675943A4 (en) 2003-09-15 2007-12-05 Massachusetts Inst Technology Nanoliter-scale synthesis of arrayed biomaterials and screening thereof
NZ581166A (en) 2003-09-15 2011-06-30 Protiva Biotherapeutics Inc Polyethyleneglycol-modified lipid compounds and uses thereof
US20050069590A1 (en) 2003-09-30 2005-03-31 Buehler Gail K. Stable suspensions for medicinal dosages
DK1685251T3 (en) 2003-10-10 2014-03-24 Powderject Vaccines Inc Nucleic acid
WO2005037226A2 (en) 2003-10-17 2005-04-28 Georgia Tech Research Corporation Genetically engineered enteroendocrine cells for treating glucose-related metabolic disorders
CA2543237A1 (en) 2003-11-10 2005-05-19 Nippon Kayaku Kabushiki Kaisha Diimonium salt compound and use thereof
US7022214B2 (en) 2004-01-21 2006-04-04 Bio-Rad Laboratories, Inc. Carrier ampholytes of high pH range
US7556684B2 (en) 2004-02-26 2009-07-07 Construction Research & Technology Gmbh Amine containing strength improvement admixture
US20060228404A1 (en) 2004-03-04 2006-10-12 Anderson Daniel G Compositions and methods for treatment of hypertrophic tissues
WO2005121348A1 (en) 2004-06-07 2005-12-22 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
CA2569645C (en) 2004-06-07 2014-10-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
US7670595B2 (en) 2004-06-28 2010-03-02 Merck Patent Gmbh Fc-interferon-beta fusion proteins
GB0418172D0 (en) 2004-08-13 2004-09-15 Ic Vec Ltd Vector
DE102004042546A1 (en) 2004-09-02 2006-03-09 Curevac Gmbh Combination therapy for immune stimulation
DE102004043342A1 (en) 2004-09-08 2006-03-09 Bayer Materialscience Ag Blocked polyurethane prepolymers as adhesives
JP2008518998A (en) 2004-11-05 2008-06-05 ノヴォソム アクチェンゲゼルシャフト Improvements in or relating to pharmaceutical compositions comprising oligonucleotides as active substances
GB0502482D0 (en) 2005-02-07 2005-03-16 Glaxo Group Ltd Novel compounds
WO2007086883A2 (en) 2005-02-14 2007-08-02 Sirna Therapeutics, Inc. Cationic lipids and formulated molecular compositions containing them
WO2006105043A2 (en) 2005-03-28 2006-10-05 Dendritic Nanotechnologies, Inc. Janus dendrimers and dendrons
CN101346468B (en) 2005-06-15 2016-03-30 麻省理工学院 Containing amine lipid and its purposes
US9012219B2 (en) 2005-08-23 2015-04-21 The Trustees Of The University Of Pennsylvania RNA preparations comprising purified modified RNA for reprogramming cells
EP4174179A3 (en) 2005-08-23 2023-09-27 The Trustees of the University of Pennsylvania Rna containing modified nucleosides and methods of use thereof
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
EP2395012B8 (en) 2005-11-02 2018-06-06 Arbutus Biopharma Corporation Modified siRNA molecules and uses thereof
US7238791B1 (en) 2005-12-16 2007-07-03 Roche Diagnostics Operations, Inc. 6-monoacetylmorphine derivatives useful in immunoassay
US20090221684A1 (en) 2005-12-22 2009-09-03 Trustees Of Boston University Molecules for Gene Delivery and Gene Therapy, and Methods of Use Thereof
CN100569877C (en) 2005-12-30 2009-12-16 财团法人工业技术研究院 Contain the dendritic structural compounds of branch and the application thereof of many UV crosslinking reactive group
CA2649325C (en) 2006-04-14 2019-01-08 Epicentre Technologies Corporation Kits and methods for generating 5' capped rna
US9085778B2 (en) 2006-05-03 2015-07-21 VL27, Inc. Exosome transfer of nucleic acids to cells
US20070275923A1 (en) 2006-05-25 2007-11-29 Nastech Pharmaceutical Company Inc. CATIONIC PEPTIDES FOR siRNA INTRACELLULAR DELIVERY
WO2007143659A2 (en) 2006-06-05 2007-12-13 Massachusetts Institute Of Technology Crosslinked, degradable polymers and uses thereof
US20090186805A1 (en) 2006-07-06 2009-07-23 Aaron Thomas Tabor Compositions and Methods for Genetic Modification of Cells Having Cosmetic Function to Enhance Cosmetic Appearance
ES2293834B1 (en) 2006-07-20 2009-02-16 Consejo Superior Investig. Cientificas COMPOSED WITH INHIBITING ACTIVITY OF UBC13-UEV INTERACTIONS, PHARMACEUTICAL COMPOSITIONS THAT INCLUDE IT AND ITS THERAPEUTIC APPLICATIONS.
CA2658768C (en) 2006-07-21 2016-05-17 Massachusetts Institute Of Technology End-modified poly(beta-amino esters) and uses thereof
AU2007303205A1 (en) 2006-10-03 2008-04-10 Tekmira Pharmaceuticals Corporation Lipid containing formulations
AU2007308130A1 (en) 2006-10-12 2008-04-17 Copernicus Therapeutics Inc. Codon optimized CFTR
DE102006051516A1 (en) 2006-10-31 2008-05-08 Curevac Gmbh (Base) modified RNA to increase the expression of a protein
WO2008140615A2 (en) 2006-12-21 2008-11-20 Novozymes, Inc. Modified messenger rna stabilizing sequences for expressing genes in bacterial cells
DE102007001370A1 (en) 2007-01-09 2008-07-10 Curevac Gmbh RNA-encoded antibodies
WO2008097926A2 (en) 2007-02-02 2008-08-14 Yale University Transient transfection with rna
EP2139461A2 (en) 2007-03-20 2010-01-06 Recepticon Aps Amino derivatives to prevent nephrotoxicity and cancer
JP5186126B2 (en) 2007-03-29 2013-04-17 公益財団法人地球環境産業技術研究機構 Novel triazine derivatives, their production and their use as gas separation membranes
NZ580973A (en) 2007-04-18 2011-10-28 Cornerstone Pharmaceuticals Inc Pharmaceutical formulations containing lipoic acid derivatives
WO2008137470A1 (en) 2007-05-01 2008-11-13 Pgr-Solutions Multi-chain lipophilic polyamines
US20090163705A1 (en) 2007-05-21 2009-06-25 Alnylam Pharmaceuticals, Inc. Cationic lipids
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
WO2009036280A1 (en) 2007-09-12 2009-03-19 Copernicus Therapeutics, Inc. Long-term in vivo transgene expression
JP5697450B2 (en) 2007-10-02 2015-04-08 マリーナ バイオテック，インコーポレイテッド Lipopeptide for delivery of nucleic acids
WO2009046739A1 (en) 2007-10-09 2009-04-16 Curevac Gmbh Composition for treating prostate cancer (pca)
EP2500427B1 (en) 2007-11-22 2014-07-30 Japan Science and Technology Agency Translation regulation system in cell or artifical cell model by using low-molecular-weight RNA
CA2708153C (en) 2007-12-04 2017-09-26 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugates as delivery agents for oligonucleotides
EP2238251B1 (en) 2007-12-27 2015-02-11 Protiva Biotherapeutics Inc. Silencing of polo-like kinase expression using interfering rna
US20110038941A1 (en) 2007-12-27 2011-02-17 The Ohio State University Research Foundation Lipid Nanoparticle Compositions and Methods of Making and Using the Same
CA2711236A1 (en) 2008-01-02 2009-07-16 Alnylam Pharmaceuticals, Inc. Screening method for selected amino lipid-containing compositions
WO2009126933A2 (en) 2008-04-11 2009-10-15 Alnylam Pharmaceuticals, Inc. Site-specific delivery of nucleic acids by combining targeting ligands with endosomolytic components
PL2279254T3 (en) 2008-04-15 2017-11-30 Protiva Biotherapeutics Inc. Novel lipid formulations for nucleic acid delivery
US20090263407A1 (en) 2008-04-16 2009-10-22 Abbott Laboratories Cationic Lipids and Uses Thereof
WO2009127230A1 (en) 2008-04-16 2009-10-22 Curevac Gmbh MODIFIED (m)RNA FOR SUPPRESSING OR AVOIDING AN IMMUNOSTIMULATORY RESPONSE AND IMMUNOSUPPRESSIVE COMPOSITION
EP2297322A1 (en) 2008-06-04 2011-03-23 The Board of Regents of The University of Texas System Modulation of gene expression through endogenous small rna targeting of gene promoters
JP5024216B2 (en) 2008-07-23 2012-09-12 トヨタ自動車株式会社 Ignition timing control device and ignition timing control method for internal combustion engine
US20100035249A1 (en) 2008-08-05 2010-02-11 Kabushiki Kaisha Dnaform Rna sequencing and analysis using solid support
JP5492207B2 (en) 2008-08-27 2014-05-14 ライフ テクノロジーズ コーポレーション Biological sample processing apparatus and processing method
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
US9139554B2 (en) 2008-10-09 2015-09-22 Tekmira Pharmaceuticals Corporation Amino lipids and methods for the delivery of nucleic acids
CN104382853A (en) 2008-10-16 2015-03-04 玛瑞纳生物技术有限公司 Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
US9080211B2 (en) 2008-10-24 2015-07-14 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2010062322A2 (en) 2008-10-27 2010-06-03 Massachusetts Institute Of Technology Modulation of the immune response
AU2009311667B2 (en) 2008-11-07 2016-04-14 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
EP4241767A3 (en) 2008-11-10 2023-11-01 Arbutus Biopharma Corporation Novel lipids and compositions for the delivery of therapeutics
TW201019969A (en) 2008-11-17 2010-06-01 Enzon Pharmaceuticals Inc Branched cationic lipids for nucleic acids delivery system
WO2010083615A1 (en) 2009-01-26 2010-07-29 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein c-iii expression
US20100222489A1 (en) 2009-02-27 2010-09-02 Jiang Dayue D Copolymer composition, membrane article, and methods thereof
NZ594995A (en) 2009-03-12 2013-06-28 Alnylam Pharmaceuticals Inc LIPID FORMULATED COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF HUMAN KINESIN FAMILY MEMBER 11 (Eg5) AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) GENES
CN102334237B (en) 2009-04-02 2015-05-13 西蒙公司 Telecommunications patch panel
JP5713325B2 (en) 2009-04-17 2015-05-07 アイシス イノヴェイション リミテッド Composition for delivery of genetic material
EP2421506B1 (en) 2009-04-22 2015-08-19 Emory University Nanocarrier therapy for treating invasive tumors
NZ621981A (en) 2009-05-05 2015-09-25 Tekmira Pharmaceuticals Corp Lipid compositions
HUE056773T2 (en) 2009-06-10 2022-03-28 Arbutus Biopharma Corp Improved lipid formulation
US9051567B2 (en) 2009-06-15 2015-06-09 Tekmira Pharmaceuticals Corporation Methods for increasing efficacy of lipid formulated siRNA
MX2011013421A (en) 2009-06-15 2012-03-16 Alnylam Pharmaceuticals Inc Lipid formulated dsrna targeting the pcsk9 gene.
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8236943B2 (en) 2009-07-01 2012-08-07 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein B
US8716464B2 (en) 2009-07-20 2014-05-06 Thomas W. Geisbert Compositions and methods for silencing Ebola virus gene expression
US9040701B2 (en) 2009-07-30 2015-05-26 Laboratorios Salvat, S.A. Apaf-1 inhibitor compounds
DK2459231T3 (en) 2009-07-31 2016-09-05 Ethris Gmbh RNA with a combination of unmodified and modified nucleotides for protein expression
DE102009043342A1 (en) 2009-09-29 2011-03-31 Bayer Technology Services Gmbh Substances for self-organized carriers for the controlled release of an active substance
US8326547B2 (en) 2009-10-07 2012-12-04 Nanjingjinsirui Science & Technology Biology Corp. Method of sequence optimization for improved recombinant protein expression using a particle swarm optimization algorithm
NZ600616A (en) 2009-12-01 2014-11-28 Shire Human Genetic Therapies Delivery of mrna for the augmentation of proteins and enzymes in human genetic diseases
KR102505097B1 (en) 2009-12-07 2023-03-02 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 Rna preparations comprising purified modified rna for reprogramming cells
WO2011069529A1 (en) 2009-12-09 2011-06-16 Curevac Gmbh Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
ES2749426T3 (en) 2009-12-18 2020-03-20 Univ British Columbia Nucleic Acid Administration Methods and Compositions
EP2338520A1 (en) 2009-12-21 2011-06-29 Ludwig Maximilians Universität Conjugate with targeting ligand and use of same
CN102905763B (en) 2009-12-23 2015-06-17 诺华股份有限公司 Lipids, lipid compositions, and methods of using them
CA2799091A1 (en) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use thereof
IL300109A (en) 2010-06-03 2023-03-01 Alnylam Pharmaceuticals Inc Biodegradable lipids for the delivery of active agents
CN101863544B (en) 2010-06-29 2011-09-28 湖南科技大学 Cyanuric acid-based heavy metal chelating flocculant and preparation method thereof
WO2012000104A1 (en) 2010-06-30 2012-01-05 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
EP3578205A1 (en) 2010-08-06 2019-12-11 ModernaTX, Inc. A pharmaceutical formulation comprising engineered nucleic acids and medical use thereof
WO2012019630A1 (en) 2010-08-13 2012-02-16 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein
WO2012027675A2 (en) 2010-08-26 2012-03-01 Massachusetts Institute Of Technology Poly(beta-amino alcohols), their preparation, and uses thereof
CA2813466A1 (en) 2010-10-01 2012-04-05 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids that disrupt major groove binding partner interactions
US8853377B2 (en) 2010-11-30 2014-10-07 Shire Human Genetic Therapies, Inc. mRNA for use in treatment of human genetic diseases
WO2012113413A1 (en) 2011-02-21 2012-08-30 Curevac Gmbh Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
DK2691443T3 (en) 2011-03-28 2021-05-03 Massachusetts Inst Technology CONJUGIATED LIPOMERS AND USES OF THESE
WO2012133737A1 (en) 2011-03-31 2012-10-04 公益財団法人地球環境産業技術研究機構 Crosslinkable amine compound, polymer membrane using crosslinkable amine compound, and method for producing polymer membrane
CA2831613A1 (en) 2011-03-31 2012-10-04 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
EP2705152B1 (en) 2011-05-04 2017-10-25 The Broad Institute, Inc. Multiplexed genetic reporter assays and compositions
JP2014520084A (en) 2011-05-17 2014-08-21 モデルナ セラピューティクス インコーポレイテッド Modified nucleic acids for non-human vertebrates and methods of use thereof
EP2532649B1 (en) 2011-06-07 2015-04-08 Incella GmbH Amino lipids, their synthesis and uses thereof
EP3998064A1 (en) 2011-06-08 2022-05-18 Translate Bio, Inc. Cleavable lipids
EP4043025A1 (en) 2011-06-08 2022-08-17 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mrna delivery
WO2013003475A1 (en) 2011-06-27 2013-01-03 Cellscript, Inc. Inhibition of innate immune response
EP2755693A4 (en) 2011-09-12 2015-05-20 Moderna Therapeutics Inc Engineered nucleic acids and methods of use thereof
EP2755986A4 (en) 2011-09-12 2015-05-20 Moderna Therapeutics Inc Engineered nucleic acids and methods of use thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CN110511939A (en) 2011-10-03 2019-11-29 现代泰克斯公司 Nucleosides, nucleotide and nucleic acid of modification and application thereof
CA2850792A1 (en) 2011-10-05 2013-04-11 Protiva Biotherapeutics Inc. Compositions and methods for silencing aldehyde dehydrogenase
EP4074694A1 (en) 2011-10-27 2022-10-19 Massachusetts Institute Of Technology Amino acid-, peptide- an polypeptide-lipids, isomers, compositions, an uses thereof
US20140343129A1 (en) 2011-12-14 2014-11-20 Moderna Therapeutics, Inc. Modified nucleic acids, and acute care uses thereof
US20140378538A1 (en) 2011-12-14 2014-12-25 Moderma Therapeutics, Inc. Methods of responding to a biothreat
EP2791160B1 (en) 2011-12-16 2022-03-02 ModernaTX, Inc. Modified mrna compositions
US20130165504A1 (en) 2011-12-21 2013-06-27 modeRNA Therapeutics Methods of increasing the viability or longevity of an organ or organ explant
US20140371302A1 (en) 2011-12-29 2014-12-18 Modema Therapeutics, Inc. Modified mrnas encoding cell-penetrating polypeptides
PL3421601T3 (en) 2011-12-30 2020-06-01 Cellscript, Llc Making and using in vitro-synthesized ssrna for introducing into mammalian cells to induce a biological or biochemical effect
WO2013120497A1 (en) 2012-02-15 2013-08-22 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
BR112014020824B1 (en) 2012-02-24 2022-10-04 Protiva Biotherapeutics Inc LIPID, LIPID PARTICLE AND PHARMACEUTICAL COMPOSITION
US9546128B2 (en) 2012-03-29 2017-01-17 Shire Human Genetic Therapies, Inc. Ionizable cationic lipids
US9877919B2 (en) 2012-03-29 2018-01-30 Translate Bio, Inc. Lipid-derived neutral nanoparticles
US20150050354A1 (en) 2012-04-02 2015-02-19 Moderna Therapeutics, Inc. Modified polynucleotides for the treatment of otic diseases and conditions
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
EP2833923A4 (en) 2012-04-02 2016-02-24 Moderna Therapeutics Inc Modified polynucleotides for the production of proteins
US20140275229A1 (en) 2012-04-02 2014-09-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding udp glucuronosyltransferase 1 family, polypeptide a1
CN108949772A (en) 2012-04-02 2018-12-07 现代泰克斯公司 For generating the modification polynucleotides of biological agent relevant to human diseases and protein
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US20150267192A1 (en) 2012-06-08 2015-09-24 Shire Human Genetic Therapies, Inc. Nuclease resistant polynucleotides and uses thereof
CN104519915A (en) 2012-06-08 2015-04-15 夏尔人类遗传性治疗公司 Pulmonary delivery of mRNA to non-lung target cells
US20150126589A1 (en) 2012-06-08 2015-05-07 Ethris Gmbh Pulmonary Delivery of Messenger RNA
WO2014028487A1 (en) 2012-08-13 2014-02-20 Massachusetts Institute Of Technology Amine-containing lipidoids and uses thereof
WO2014093924A1 (en) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Modified nucleic acid molecules and uses thereof
PT2922554T (en) 2012-11-26 2022-06-28 Modernatx Inc Terminally modified rna
EP2929035A1 (en) 2012-12-07 2015-10-14 Shire Human Genetic Therapies, Inc. Lipidic nanoparticles for mrna delivering
WO2014093574A1 (en) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Modified polynucleotides for altering cell phenotype
JP2016504050A (en) 2013-01-17 2016-02-12 モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Signal sensor polynucleotide for modification of cell phenotype
WO2014158795A1 (en) 2013-03-12 2014-10-02 Moderna Therapeutics, Inc. Diagnosis and treatment of fibrosis
WO2014159813A1 (en) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Long-lived polynucleotide molecules
ES2708561T3 (en) 2013-03-14 2019-04-10 Translate Bio Inc Methods for the purification of messenger RNA
ES2692363T3 (en) 2013-03-14 2018-12-03 Translate Bio, Inc. Therapeutic compositions of mRNA and its use to treat diseases and disorders
PE20151773A1 (en) 2013-03-14 2015-12-20 Shire Human Genetic Therapies CFTR mRNA COMPOSITIONS, METHODS AND RELATED USES
AU2014239250A1 (en) 2013-03-14 2015-08-27 Shire Human Genetic Therapies, Inc. Quantitative assessment for cap efficiency of messenger RNA
EP2971098B1 (en) 2013-03-14 2018-11-21 Translate Bio, Inc. Quantitative assessment for cap efficiency of messenger rna
EP2971010B1 (en) 2013-03-14 2020-06-10 ModernaTX, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
JP6567494B2 (en) 2013-03-14 2019-08-28 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド Ribonucleic acids having 4'-thio-modified nucleotides and related methods
MX2015011947A (en) 2013-03-14 2015-12-01 Shire Human Genetic Therapies Methods and compositions for delivering mrna coded antibodies.
WO2014144039A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Characterization of mrna molecules
WO2014144767A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Ion exchange purification of mrna
US20160017313A1 (en) 2013-03-15 2016-01-21 Moderna Therapeutics, Inc. Analysis of mrna heterogeneity and stability
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
EP3578663A1 (en) 2013-03-15 2019-12-11 ModernaTX, Inc. Manufacturing methods for production of rna transcripts
ES2795249T3 (en) 2013-03-15 2020-11-23 Translate Bio Inc Synergistic enhancement of nucleic acid delivery through mixed formulations
WO2014152030A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Removal of dna fragments in mrna production process
EP3578652B1 (en) 2013-03-15 2023-07-12 ModernaTX, Inc. Ribonucleic acid purification
US9315472B2 (en) 2013-05-01 2016-04-19 Massachusetts Institute Of Technology 1,3,5-triazinane-2,4,6-trione derivatives and uses thereof
WO2014210356A1 (en) 2013-06-26 2014-12-31 Massachusetts Institute Of Technology Multi-tailed lipids and uses thereof
AU2014287009B2 (en) 2013-07-11 2020-10-29 Modernatx, Inc. Compositions comprising synthetic polynucleotides encoding CRISPR related proteins and synthetic sgRNAs and methods of use
CN105555757A (en) 2013-07-23 2016-05-04 普洛体维生物治疗公司 Compositions and methods for delivering messenger RNA
JP2016530294A (en) 2013-09-03 2016-09-29 モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． Chimeric polynucleotide
EP3041938A1 (en) 2013-09-03 2016-07-13 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
WO2015051169A2 (en) 2013-10-02 2015-04-09 Moderna Therapeutics, Inc. Polynucleotide molecules and uses thereof
WO2015051173A2 (en) 2013-10-02 2015-04-09 Moderna Therapeutics, Inc Polynucleotide molecules and uses thereof
CA2927393A1 (en) 2013-10-18 2015-04-23 Moderna Therapeutics, Inc. Compositions and methods for tolerizing cellular systems
EP3060257B1 (en) * 2013-10-22 2021-02-24 Translate Bio, Inc. Lipid formulations for delivery of messenger rna
EP4276176A3 (en) 2013-10-22 2024-01-10 Translate Bio, Inc. Mrna therapy for argininosuccinate synthetase deficiency
EA201690588A1 (en) 2013-10-22 2016-09-30 Шир Хьюман Дженетик Терапис, Инк. DELIVERY OF MRNA IN THE CNS AND ITS APPLICATION
MX2016005239A (en) 2013-10-22 2016-08-12 Shire Human Genetic Therapies Mrna therapy for phenylketonuria.
EP3076994A4 (en) 2013-12-06 2017-06-07 Modernatx, Inc. Targeted adaptive vaccines
US20150167017A1 (en) 2013-12-13 2015-06-18 Moderna Therapeutics, Inc. Alternative nucleic acid molecules and uses thereof
CN105874072A (en) 2013-12-30 2016-08-17 库瑞瓦格股份公司 Artificial nucleic acid molecules
US20170002060A1 (en) 2014-01-08 2017-01-05 Moderna Therapeutics, Inc. Polynucleotides for the in vivo production of antibodies
EP3590529A1 (en) 2014-03-12 2020-01-08 CureVac AG Combination of vaccination and ox40 agonists
EP3981437A1 (en) 2014-04-23 2022-04-13 ModernaTX, Inc. Nucleic acid vaccines
KR20220158867A (en) 2014-04-25 2022-12-01 샤이어 휴먼 지네틱 테라피즈 인크. Methods for purification of messenger rna
AU2015328012A1 (en) 2014-10-02 2017-05-11 Arbutus Biopharma Corporation Compositions and methods for silencing Hepatitis B virus gene expression
WO2016071857A1 (en) 2014-11-07 2016-05-12 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus expression
EP4324473A2 (en) 2014-11-10 2024-02-21 ModernaTX, Inc. Multiparametric nucleic acid optimization
EP3041948B1 (en) 2014-11-10 2019-01-09 Modernatx, Inc. Alternative nucleic acid molecules containing reduced uracil content and uses thereof
US20180000953A1 (en) 2015-01-21 2018-01-04 Moderna Therapeutics, Inc. Lipid nanoparticle compositions
EP3247398A4 (en) 2015-01-23 2018-09-26 Moderna Therapeutics, Inc. Lipid nanoparticle compositions
WO2016154127A2 (en) 2015-03-20 2016-09-29 Protiva Biotherapeutics, Inc. Compositions and methods for treating hypertriglyceridemia
WO2016164762A1 (en) 2015-04-08 2016-10-13 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor egf-a and intracellular domain mutants and methods of using the same
WO2016183366A2 (en) 2015-05-12 2016-11-17 Protiva Biotherapeutics, Inc. Compositions and methods for silencing expression of hepatitis d virus rna
US20180245074A1 (en) 2015-06-04 2018-08-30 Protiva Biotherapeutics, Inc. Treating hepatitis b virus infection using crispr
US10626393B2 (en) 2015-06-04 2020-04-21 Arbutus Biopharma Corporation Delivering CRISPR therapeutics with lipid nanoparticles
EP3307305A4 (en) 2015-06-10 2019-05-22 Modernatx, Inc. Targeted adaptive vaccines
EP3329003A2 (en) 2015-07-29 2018-06-06 Arbutus Biopharma Corporation Compositions and methods for silencing hepatitis b virus gene expression
US11434486B2 (en) 2015-09-17 2022-09-06 Modernatx, Inc. Polynucleotides containing a morpholino linker
CA2998370A1 (en) 2015-09-17 2017-03-23 Moderna Therapeutics, Inc. Polynucleotides containing a stabilizing tail region
WO2017049074A1 (en) 2015-09-18 2017-03-23 Moderna Therapeutics, Inc. Polynucleotide formulations for use in the treatment of renal diseases
CA3020343A1 (en) 2016-04-08 2017-10-12 Translate Bio, Inc. Multimeric coding nucleic acid and uses thereof
JP2019533707A (en) * 2016-11-10 2019-11-21 トランスレイト バイオ， インコーポレイテッド Improved process for preparing MRNA-supported lipid nanoparticles
AU2017357748B2 (en) * 2016-11-10 2023-11-09 Translate Bio, Inc. Improved ice-based lipid nanoparticle formulation for delivery of mRNA
US11173190B2 (en) 2017-05-16 2021-11-16 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mRNA encoding CFTR

Also Published As

Publication number Publication date
AU2018224326A1 (en) 2019-09-19
MX2019010155A (en) 2020-12-10
EP3585417B1 (en) 2023-02-22
WO2018157154A3 (en) 2018-10-04
AU2018224326B2 (en) 2024-01-04
MA47603A (en) 2020-01-01
WO2018157154A2 (en) 2018-08-30
CA3054062A1 (en) 2018-08-30
IL268854A (en) 2019-10-31
EP3585417A2 (en) 2020-01-01
US11253605B2 (en) 2022-02-22
US20180256741A1 (en) 2018-09-13
EA201991747A1 (en) 2020-06-04

Similar Documents

Publication Publication Date Title
US20220193247A1 (en) Novel Codon-Optimized CFTR MRNA
US20230050301A1 (en) Mrna therapy for phenylketonuria
US20240123084A1 (en) Liver specific delivery of messenger rna
US11712463B2 (en) MRNA therapy for pompe disease
US20230181619A1 (en) Improved compositions for cftr mrna therapy
KR20210091120A (en) Improved Process for Making mRNA-Loaded Lipid Nanoparticles
AU2020348376A1 (en) mRNA encoding engineered CFTR
US20220160633A1 (en) Compositions for delivery of codon-optimized mrna
US20220287966A1 (en) Stable Liquid Lipid Nanoparticle Formulations
CN116782878A (en) Improved compositions for delivery of codon optimized mRNA

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: TRANSLATE BIO MA, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DIAS, ANUSHA;ABYSALH, JONATHAN;DEROSA, FRANK;AND OTHERS;SIGNING DATES FROM 20180309 TO 20180315;REEL/FRAME:060368/0118

Owner name: TRANSLATE BIO, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:TRANSLATE BIO MA, INC.;REEL/FRAME:060368/0099

Effective date: 20180309

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED