Compositions and methods for treating conditions associated with bsep deficiency

Abstract

This application provides polynucleotides comprising a coding sequence for a functionally active bile salt export pump (BSEP) protein or a functionally active fragment thereof. The application further provides compositions comprising said polynucleotides and their use in methods of preventing or treating conditions associated with BSEP deficiency in a subject, such as progressive familial intrahepatic cholestasis type 2 (PFIC2).

Description

COMPOSITIONS AND METHODS FOR TREATING CONDITIONS ASSOCIATED WITH BSEP DEFICIENCYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Application No. 63 / 515,692 filed 26 July 2023, the entire disclosure of which is hereby incorporated by reference herein in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING XML

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. Said XML file, created 01 July 2024, is named 104542-00046_SL.xml, and is 69,731 bytes in size.TECHNICAL FIELD OF THE INVENTION

[0003] This disclosure relates to nucleic acid molecules encoding the bile salt export pump (BSEP) protein and compositions comprising the same for use in the treatment of conditions associated with BSEP deficiency.BACKGROUND OF THE INVENTION

[0004] Progressive familial intrahepatic cholestasis type 2 (PFIC2), also known as bile salt export pump deficiency (BSEP deficiency), is a rare genetic disorder that affects the liver’s ability to transport bile out of liver cells. This condition results in an elevation of bile salts in the peripheral blood, liver damage and impaired bile flow.

[0005] The disorder is primarily caused by mutations in the ABCB11 gene, which provides instructions for making the bile salt export pump (BSEP) protein. This protein is responsible for transporting bile salts from liver cells into the bile canaliculi for eventual elimination from the body.

[0006] PFIC2 typically manifests in early infancy or childhood. Common symptoms include jaundice, severe itching (pruritis), pale stools, hepatosplenomegaly (enlargement of the liver and spleen), and failure to thrive. Children with PFIC2 may also experience recurrent episodes of cholestasis (obstruction of bile flow), which can lead to liver damage and cirrhosis over time.

[0007] The management of PFIC2 aims to alleviate symptoms, prevent complications, and slow the progression of liver disease. Treatment options may include medications to reduce itching (such as antihistamines or bile acid sequestrants), supplements to replace fat-soluble vitamins, surgical procedures (such as partial external biliary diversion or liver transplantation),and specialized diets. Liver transplantation is often required in severe cases where the liver function is significantly impaired. Currently, there is no therapy that addresses the underlying cause of the disease, z.e., deficient BSEP activity.

[0008] The present disclosure addresses this need by providing novel nucleic acid molecules that can be translated to provide functional BSEP, which can ameliorate, prevent or treat conditions associated with BSEP deficiency such as PFIC2.SUMMARY OF THE INVENTION

[0009] This invention provides compositions comprising novel nucleic acid molecules that can be used to express functionally active proteins, or fragments thereof. The invention further provides methods of using these compositions comprising novel nucleic acid molecules for the prevention or treatment of various disorders, including progressive familial intrahepatic cholestasis type 2 (PFIC2). More specifically, embodiments of this invention provide compositions comprising translatable nucleic acid molecules to provide a functionally active bile salt export pump (BSEP) protein, or a functionally active fragment thereof, and methods of their use for the treatment of PFIC2. In some embodiments, the nucleic acid molecules of the invention can be expressible to provide a BSEP polypeptide that is functionally active for ameliorating, preventing or treating a disease or condition associated with BSEP deficiency, such as PFIC2.

[0010] In a first aspect, the application relates to a polynucleotide comprising an mRNA coding sequence for BSEP or a fragment thereof. In one embodiment, the polynucleotide comprises a mixture of natural and modified nucleotides. Thus, in some embodiments, the application relates to a polynucleotide for expressing BSEP, or a fragment thereof, wherein the polynucleotide comprises natural and modified nucleotides and is expressible to provide the BSEP or a fragment thereof having BSEP activity.

[0011] In one embodiment, the mRNA coding sequence for BSEP is a wild-type coding sequence. In an alternative embodiment, the mRNA coding sequence for BSEP is a codon- optimized sequence. In one exemplary embodiment, the mRNA coding sequence for BSEP is codon-optimized for expression in humans.

[0012] In some embodiments, the BSEP protein is encoded by the wild-type coding sequence shown in SEQ ID NO: 1. In alternative embodiments, the BSEP protein is encoded by a codon- optimized coding sequence that is less than 95% identical to the wild-type coding sequence shown in SEQ ID NO: 1. In some exemplary embodiments, the BSEP protein is encoded by a codon-optimized coding sequence that comprises or consists of a nucleic acid at least 95%identical to a nucleobase selected from SEQ ID NOs: 2-6 as shown in Example 6 herein. In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein further comprises a stop codon (UGA, UAA, or UAG) immediately downstream of the codon-optimized coding sequence. In some embodiments, the expressed BSEP protein comprises or consists of an amino acid sequence of SEQ ID NO: 7 (GenBank Accession No. NM 0037-12. -4, UniProtKB Accession No. 095342, 1321 amino acids). In some embodiments, the expressed polypeptide is a fragment of SEQ ID NO: 7 that retains functional BSEP activity.

[0013] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof further comprises a 5 ’-cap. In one embodiment, the 5’- cap comprises N7-Methyl-Gppp.

[0014] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof further comprises a 5’ untranslated region (5’ UTR) sequence. In one embodiment, the 5’ UTR sequence is selected from SEQ ID NOs: 8-9. In an exemplary embodiment, the 5’ UTR sequence comprises or consists of SEQ ID NO: 8.

[0015] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof further comprises a 3’ untranslated region (3’ UTR) sequence. In one embodiment, the 3’ UTR sequence is selected from SEQ ID NOs: 10-11. In an exemplary embodiment, the 3’ UTR sequence comprises or consists of SEQ ID NO: 10.

[0016] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof further comprises a 3' polyA tail sequence. In some embodiments, the length of the polyA tail sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a 3’ polyA tail sequence contains about 5 to 500 adenosine nucleotides (e.g., about 30 to 250 adenosine nucleotides, about 60 to 220 adenosine nucleotides, about 80 to 200 adenosine nucleotides, about 90 to about 150 adenosine nucleotides, or about 100 to about 120 adenosine nucleotides). In some embodiments, the 3’ polyA tail sequence is 60 to 220 adenosine nucleotides (SEQ ID NO: 20). In an exemplary embodiment, the 3’ polyA tail sequence is about 80 nucleotides in length. In another exemplary embodiment, the 3’ polyA tail sequence is about 100 nucleotides in length. In yet another exemplary embodiment, the 3’ polyA tail sequence is about 115 nucleotides in length.

[0017] In one embodiment, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof contains one or more modified nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5 -carboxy cytidine, 5-formylcytidine, 5 -methoxycytidine, 5 -propynyl cytidine, 2-thiocytidine, 5-hydroxyuridine, 5- methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5- methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine,4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5- formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5- fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N^hydroxypseudouridine, N1- methylpseudouridine, 2'-O-methyl-N1-methylpseudouridine, N^ethylpseudouridine, N1- hydroxym ethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8 -oxoadenosine, inosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, and 6-O-methylguanine.

[0018] In one embodiment, the polynucleotide comprises one or more pseudouridines. In some embodiments, the pseudouridine residue is selected from N'-methylpseudouridine, N1- ethylpseudouridine, N'-propylpseudouridine, N'-cyclopropylpseudouridine, N1- phenylpseudouridine, N'-aminomethylpseudouridine, N3-methylpseudouridine, N1- hydroxypseudouridine, and N'-hydroxymethylpseudouridine. In an exemplary embodiment, the polynucleotide is fully modified to comprise N'-methylpseudouridine residues in place of uridine residues.

[0019] In an alternative embodiment, the polynucleotide comprises one or more modified nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine, 5- propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6- methyluridine. In an exemplary embodiment, the polynucleotide is fully modified to comprise5-methoxyuridine residues in place of uridine residues.

[0020] In some embodiments, the polynucleotide may comprise a mixture of modified nucleotides, e.g., a mixture of 5-methoxyuridine and N'-methylpseudouridine residues in place of uridine residues.

[0021] In another aspect, the application provides novel codon-optimized mRNA sequences encoding BSEP. In some embodiments, the codon-optimized nucleic acid sequence encoding BSEP is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 2-6. In an exemplary embodiment, the codon-optimized nucleic acid sequence encoding BSEP is at least 97%, 98%, 99%, 99.5%, or at least 99.9% identical to a sequence selected from SEQ ID NOs: 2-6. In some embodiments, the application providesnucleic acid sequences encoding BSEP which are less than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to the wild-type coding sequence shown in SEQ ID NO: 1. In exemplary embodiments, the application provides a nucleic acid sequence encoding BSEP that comprises or consists of a sequence selected from SEQ ID NOs: 2-6. Further provided are fragments of the nucleic acid sequences shown in SEQ ID NOs: 2-6 which encode a polypeptide having functional BSEP activity. In some embodiments, the nucleic acid sequence may further comprise a stop codon (UGA, UAA, or UAG) at the 3’ end.

[0022] In yet another aspect, the application relates to a polynucleotide comprising or consisting of a nucleobase sequence that is less than 95% identical to the wild-type BSEP coding sequence over the full length BSEP coding sequence of SEQ ID NO: 1, and wherein the BSEP coding sequence is at least 97%, 98%, 99%, 99.5%, or at least 99.9% identical to a sequence selected from SEQ ID NOs: 2-6. In an exemplary embodiment, the application relates to a polynucleotide comprising a nucleobase sequence of SEQ ID NO: 2.

[0023] In yet another aspect, the application relates to a polynucleotide comprising or consisting of a nucleobase sequence is at least 95% identical to a sequence selected from SEQ ID NOs: 2-6. In one embodiment, the application relates to a polynucleotide that comprises or consists of a nucleobase sequence is at least 96% identical to a sequence selected from SEQ ID NOs: 2-6. In another embodiment, the application relates to a polynucleotide that comprises or consists of a nucleobase sequence is at least 97% identical to a sequence selected from SEQ ID NOs: 2-6. In yet another embodiment, the application relates to a polynucleotide that comprises or consists of a nucleobase sequence is at least 98% identical to a sequence selected from SEQ ID NOs: 2-6. In yet another embodiment, the application relates to a polynucleotide that comprises or consists of a nucleobase sequence is at least 99% identical to a sequence selected from SEQ ID NOs: 2-6. In yet another embodiment, the application relates to a polynucleotide that comprises or consists of a nucleobase sequence selected from SEQ ID NOs: 2-6.

[0024] In additional aspects, the application provides novel codon-optimized DNA sequences that can be transcribed to provide mRNA sequences encoding BSEP. Accordingly, the application additionally relates to nucleic acid sequences which are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to SEQ ID NOs: 12-16. In exemplary embodiments, the application provides a nucleic acid sequence selected from SEQ ID NOs: 12-16 that can be transcribed to provide an mRNA sequence encoding BSEP. Further provided are fragments of the nucleic acid sequences shown in SEQ ID NOs: 12-16 which can be transcribed to providean mRNA sequence encoding a polypeptide having functional BSEP activity. In some embodiments, the codon-optimized DNA sequence may further comprise a stop codon (TGA, TA A, or TAG) at the 3’ end.

[0025] In another aspect, the application relates to pharmaceutical compositions comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a transfection reagent, a nanoparticle (e.g. , a lipid nanoparticle), or a liposome.

[0026] In an exemplary embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle. In an exemplary embodiment, the lipid nanoparticle comprises a cationic lipid, an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), a non-cationic lipid (such as a neutral lipid), and a sterol. In a further exemplary embodiment, the lipid nanoparticle comprises at least one cationic lipid, a non-cationic lipid, a sterol, e.g., cholesterol, and a PEG-lipid, in a molar ratio of about 20-60% cationic lipid: 5-25% noncationic lipid: 25-55% sterol: 0.5-15% PEG-lipid. In yet another embodiment, the cationic lipid is selected from ATX-002, ATX-081, ATX-095, and ATX-126 as described in the detailed description that follows.

[0027] In further aspects, the application relates to the use of a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier in medical therapy, e.g., in the treatment of the human or animal body.

[0028] In another aspect, the application relates to a composition comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof and a pharmaceutically acceptable carrier in the treatment of a disease or disorder associated with reduced activity of the BSEP protein in a subject in need thereof. In some cases, the disease is progressive familial intrahepatic cholestasis type 2 (PFIC2).

[0029] In another aspect, the application relates to the use of a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier for preparing or manufacturing a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of BSEP in a subject need thereof. In one embodiment, the disease or disorder is PFIC2.

[0030] In yet another aspect, the application relates to a method for ameliorating, preventing,delaying onset, or treating a disease or disorder associated with reduced activity of BSEP in a subject need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier. In one embodiment, the disease or disorder is PFIC2.

[0031] In yet another aspect, the application relates to a method for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with BSEP deficiency in a subject need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier. In one embodiment, the disease or disorder is PFIC2.

[0032] In yet another aspect, the application relates to methods of treating PFIC2 in a human subject comprising administering to the human subject a therapeutically effective amount of a pharmaceutical composition of the invention, e.g., a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier. In one embodiment, the application provides a method of treating PFIC2 in a human subject comprising administering to the human subject a pharmaceutical composition comprising (1) a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and (2) a pharmaceutically acceptable carrier. In an exemplary embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle. In a further exemplary embodiment, the nanoparticle comprises a cationic lipid, an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), a non-cationic lipid (such as a neutral lipid), and a sterol. In another further exemplary embodiment, the nanoparticle comprises at least one cationic lipid, a non-cationic lipid, a sterol, e.g., cholesterol, and a PEG-lipid, in a molar ratio of about 20-60% cationic lipid: 5-25% noncationic lipid: 25-55% sterol: 0.5-15% PEG-lipid. In some embodiments, the cationic lipid is selected from ATX-002, ATX-081, ATX-095, and ATX-126. In some embodiments, the pharmaceutical composition comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding BSEP which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 2-6. In an exemplary embodiment, the pharmaceutical composition comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding BSEP which is at least 95% identical to SEQ ID NO: 2. In anotherexemplary embodiment, the pharmaceutical composition comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding BSEP which is at least 97% identical to SEQ ID NO: 2. In yet another exemplary embodiment, the pharmaceutical composition comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding BSEP which is at least 99% identical to SEQ ID NO: 2.

[0033] In yet another aspect, the application relates to methods of treating PFIC2 in a human subject comprising administering to a human subject diagnosed with at least one mutation in ABCB11 a therapeutically effective amount of a pharmaceutical composition described herein.

[0034] In some embodiments, a pharmaceutical composition of the invention is administered via intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, nasal, or inhalational administration.

[0035] In some embodiments, a pharmaceutical composition of the invention is administered once daily, weekly, every two weeks, monthly, every two months, quarterly, or yearly.

[0036] In some embodiments, a pharmaceutical composition of the invention administered at a dose of about 0.01 to about 10 mg / kg. In some embodiments, a pharmaceutical composition of the invention is administered at a dose of about 0.1, 0.3, 0.5, 1, 3, 5, or about 10 mg / kg.

[0037] In yet another aspect, the application relates to a kit for expressing a BSEP protein in vivo. In one embodiment, the kit comprises a 0.1 to 500 mg dose of one or more polynucleotides of the invention and a device for administering the dose. In one embodiment, the device is an injection needle, an intravenous needle, or an inhalation device.

[0038] These and other aspects and features of the invention are described in the following sections of the application.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 summarizes mRNA integrity for Nlm'P and 5M0U modified BSEP-coding sequences according to SEQ ID NOs: 2-5.

[0040] FIG. 2 is an anti-BSEP immunoblot showing dose-dependent BSEP protein expression in wild-type (“WT”) asx Abcbll knockout (“KO”) mouse liver homogenates.

[0041] FIG. 3 shows normalized BSEP protein expression levels observed in mouse liver homogenates from wild-type (“WT”) asx Abcbll knockout (“KO”) mouse liver.

[0042] FIG. 4 shows immunostaining for BSEP protein m Abcbll knockout (“KO”) mouse livers with delivery of the ABCB11 mRNA-containing LNP or vehicle control.

[0043] FIG. 5 shows BSEP expression from LNP-encapsulated ABCB11 mRNA administered to Abcbll KO mice at 4 mg / kg via tail vein injection 4, 12, 24, 48, 120, and 192hours post-dosing.

[0044] FIG. 6 shows LC-MS / MS results for bile acid species taurine-conjugated beta muricholic acid (T-bMCA) at 12 hours post dose.

[0045] FIG. 7 shows increased BSEP expression from LNP-encapsulated A8C 77 mRNA correlates with lower levels of total bile acid in the liver of Abcbll KO mice on bile acid supplemented diet.

[0046] FIG. 8 shows a significant decrease in cholic acid levels within the liver of Abcbll KO mice administered a LNP-encapsulated A8C 77 mRNA.DETAILED DESCRIPTION OF THE INVENTION

[0047] This disclosure provides a range of novel agents and compositions to be used for therapeutic applications. In some embodiments, the nucleic acid molecules and compositions of this invention can be used for ameliorating, preventing or treating PFIC2 and / or any additional diseases associated with reduced presence or function of the BSEP protein in a subject.

[0048] In some embodiments, this disclosure encompasses synthetic, purified, translatable polynucleotide molecules for expressing a BSEP protein, e.g., a human BSEP protein. The molecules may contain natural and modified nucleotides, and encode BSEP, or a fragment thereof having BSEP activity.I. Definitions

[0049] As used herein, the term “about” or “approximately” 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 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).

[0050] As used herein, the term “translatable” may be used interchangeably with the term “expressible” and refers to the ability of a polynucleotide, or a portion thereof, to be converted to a polypeptide by a host cell. As is understood in the art, translation is the process by which ribosomes in a cell’s cytoplasm create polypeptides. In translation, messenger RNA (mRNA) is decoded by tRNAs in a ribosome complex to produce a specific amino acid chain, or polypeptide. Furthermore, the term “translatable” when used in this specification in reference to an oligomer, means that at least a portion of the oligomer, e.g., the coding region of anoligomer sequence (also known as the coding sequence or CDS), is capable of being converted to a protein or a fragment thereof.

[0051] As used herein, the term “monomer” refers to a single unit, e.g., a single nucleoside, which may be joined with another molecule of the same or different type to form an oligomer.

[0052] Meanwhile, the term “polynucleotide” refers to an “oligomer,” i.e., a molecule comprising at least two monomers and includes oligonucleotides such as DNA oligonucleotides, RNA oligonucleotides, and mixed DNA / RNA oligonucleotides, as well as polynucleotides including DNA polynucleotides, RNA polynucleotides, and mixed DNA / RNA polynucleotides, as well as polynucleotides bearing synthetic or modified monomers such as modified nucleotides described herein. In the case of polynucleotides containing RNA monomers, the polynucleotides of the present disclosure may contain sequences in addition to a coding sequence (CDS). These additional sequences may be untranslated sequences, i.e., sequences which are not converted to protein by a host cell. These untranslated sequences can include a 5 ’-cap or a portion thereof, a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), and a tail region, e.g., a polyA tail region. In the context of the present disclosure, a “translatable oligomer”, a “translatable molecule”, “translatable polynucleotide”, or “translatable compound” refers to a polynucleotide that comprises a sequence or region, e.g., the coding region of an RNA (e.g., the coding sequence of BSEP or a codon-optimized version thereof), that is capable of being converted to a protein or a fragment thereof, e.g., the BSEP protein or a fragment thereof.

[0053] As used herein, the term “codon-optimized” means a purposefully designed variant of a synthetic or naturally occurring coding sequence which has been designed by choosing different codons without altering the encoded amino acid sequence. Codon-optimizing a polynucleotide sequence may result in increasing the protein expression levels (Gustafsson et al., 2004, Trends Biotechnol 22: 346-53). Variables such as high codon adaptation index (CAI), LowU method, mRNA secondary structures, cis-regulatory sequences, GC content and many other similar variables have been shown to somewhat correlate with protein expression levels (Villalobos et al., 2006, BMC Bioinformatics 7:285). The high CAI (codon adaptation index) method picks a most frequently used synonymous codon for an entire protein coding sequence, with the most frequently used codon for each amino acid being deduced from protein-coding genes from the human genome. The LowU method targets only U-containing codons that can be replaced with a synonymous codon with fewer U moieties. If there are a few choices for the replacement, the more frequently used codon will be selected. The remaining codons in thesequence are not changed by the LowU method. This method may be used in conjunction with the disclosed mRNAs to design coding sequences that are to be synthesized with one or more modified nucleotides such as N'-methylpseudouridine or 5-methoxyuridine.

[0054] 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" unless the context requires otherwise. 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.II. Overview

[0055] As will be appreciated by the skilled artisan equipped with the present disclosure, the polynucleotides of the present disclosure and compositions comprising the same may be used to ameliorate, prevent, or treat any disease or disorder associated with reduced activity (e.g., resulting from reduced concentration, presence, and / or function) of the BSEP protein in a subject. In some embodiments, the polynucleotides of this disclosure can be used in methods for ameliorating, preventing or treating PFIC2. The disease or disorder to be treated herein (e.g., PFIC2) may be associated with impaired bile flow, which can lead to a variety of disease manifestations including pruritis, liver damage and the development of fibrosis and / or cirrhosis over time. In some embodiments, the polynucleotides of the present disclosure and compositions comprising the same may be used to ameliorate, prevent, or treat any or all of these aforementioned symptoms.

[0056] A polynucleotide of this disclosure encoding a functional BSEP protein or a functional fragment thereof can be administered to a patient in need (e.g. , a patient with PFIC2) and elevate functionally active BSEP levels in the patient. The polynucleotide and compositions comprising the same can be used for preventing, treating, ameliorating or reversing any symptoms of PFIC2 in the patient. In an exemplary embodiment, the patient is a human. In some embodiments, a composition comprising a polynucleotide of the disclosure can be delivered to liver cells.

[0057] Embodiments of this disclosure further encompass processes for making a polynucleotide capable of expressing a BSEP protein. The processes include transcribing in vitro an ABCB11 DNA template in the presence of natural and modified nucleoside triphosphates to form a product mixture and purifying the product mixture to isolate thepolynucleotide. In some embodiments, a polynucleotide of the disclosure may be made by methods known in the art. In some embodiments, the polynucleotides of this disclosure can display a sequence of nucleobases designed to express a polypeptide or protein, in vitro, ex vivo, or in vivo.

[0058] In some embodiments, a polynucleotide of this disclosure may comprise a 5 ’-cap, a 5’ untranslated region of monomers, a coding region of monomers, a 3’ untranslated region of monomers, and a tail region of monomers.

[0059] In some embodiments, a polynucleotide of the disclosure can be from about 500 to about 8,000 monomers in length. In certain embodiments, a polynucleotide of the disclosure can be from 2,000 to 7,000 monomers in length, from 3,000 to 6,000 monomers in length, or from 4,000 to 5,000 monomers in length. In an exemplary embodiment, the polynucleotide of the disclosure is from 4,000 to 4,500 monomers in length. In a further exemplary embodiment, the polynucleotide of the disclosure is about 4,100, about 4,200, about 4,300, about 4,400, or about 4,500 monomers in length. In a further exemplary embodiment, the polynucleotide of the disclosure is or about 4,211 monomers in length.

[0060] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a mixture of natural and modified nucleotides and is expressible to provide a BSEP protein or a fragment thereof having BSEP activity. In some embodiments, the modified nucleotide is N'-methylpseudouridine. In an exemplary embodiment, the polynucleotide is fully modified to comprise N1- methylpseudouridine residues in place of uridine residues. In some embodiments, the modified nucleotide is 5 -methoxyuridine. In an exemplary embodiment, the polynucleotide is fully modified to comprise 5 -methoxyuridine residues in place of uridine residues. In some embodiments, the polynucleotide is modified to comprise a mixture of 5-methoxyuridine and N'-methylpseudouridine residues in place of uridine residues.

[0061] In some embodiments, the polynucleotides of this disclosure may be translatable molecules containing RNA monomers and / or alternative monomers such as unlocked nucleic acid (UNA) and locked nucleic acid (LNA) monomers.

[0062] In some embodiments, a translatable polynucleotide can contain from 1 to about 80 unlocked nucleic acid (UNA) monomers. In certain embodiments, a translatable polynucleotide can contain from 1 to 50 UNA monomers, or 1 to 20 UNA monomers, or 1 to 10 UNA monomers.

[0063] In some embodiments, a translatable polynucleotide can contain from 1 to about 80locked nucleic acid (LNA) monomers. In certain embodiments, a translatable polynucleotide can contain from 1 to 50 LNA monomers, or 1 to 20 LNA monomers, or 1 to 10 LNA monomers.

[0064] In some embodiments, one or more polynucleotides of the disclosure can be delivered to a cell, in vitro, ex vivo, or in vivo. Viral and non-viral transfer methods as are known in the art can be used to introduce polynucleotides of the disclosures into mammalian cells. In exemplary embodiments, polynucleotides of the disclosure may be delivered with a pharmaceutically acceptable vehicle, for example, with nanoparticles or liposomes. In a further exemplary embodiment, polynucleotides of the disclosure are delivered via nanoparticles, e.g., lipid nanoparticles (LNPs).

[0065] In additional embodiments, this disclosure provides methods for treating a disease or condition in a subject by administering to the subject a composition or formulation containing a polynucleotide composition of the disclosure.

[0066] In some aspects, a composition comprising a polynucleotide of the disclosure may be used for ameliorating, preventing or treating a disease or disorder, e.g., a disease or disorder associated with reduced activity (e.g., resulting from reduced concentration, presence, and / or function) of the BSEP protein in a subject. In some embodiments, a composition comprising a polynucleotide of this disclosure can be administered to regulate, modulate, or increase the concentration or effectiveness of the BSEP protein in a subject. In some embodiments, the BSEP protein to be expressed can be an unmodified, natural protein for which the patient is deficient (e.g., a patient with a mutated version of ABCB11 which partially or totally abolishes functional BSEP activity). In some aspects, the BSEP protein expressed by a polynucleotide of the disclosure can be identical to an unmodified, natural, functionally active BSEP protein which can be used to treat PFIC2 in a patient harboring a mutated version of the BSEP protein. In exemplary embodiments, a composition comprising a polynucleotide of this disclosure may be used for ameliorating, preventing or treating PFIC2.

[0067] In some embodiments, a polynucleotide of the disclosure may be delivered to cells or subjects and translated to increase BSEP protein levels in the cell or subject.

[0068] In an exemplary embodiment, a subject of the present disclosure is a subject with reduced activity (e.g., resulting from reduced concentration, presence, and / or function) of BSEP. In a further exemplary embodiment, the subject is a human.

[0069] In some embodiments, administering a composition comprising a polynucleotide of the disclosure can result in an increase in the level of functionally active BSEP protein in atreated subject. In some embodiments, administering a composition comprising a polynucleotide of the disclosure results in about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more increase in the level of functionally active BSEP protein relative to a baseline level in the subject prior to treatment. In an exemplary embodiment, administering a composition comprising a polynucleotide of the disclosure results in an increase BSEP levels relative to baseline BSEP levels in the subject prior to treatment. In some embodiments, the increase in liver BSEP levels can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, or more.

[0070] In some embodiments, the BSEP protein which is expressed from a polynucleotide of the disclosure is detectable in liver cells, e.g., hepatocytes, of a treated subject.

[0071] In some embodiments, administering a composition comprising a polynucleotide of the disclosure results in the expression of a natural, non-mutated BSEP (z.e., normal or wild type BSEP as opposed to abnormal or mutated BSEP) protein level at or above about 10 ng / mg, about 20 ng / mg, about 50 ng / mg, about 100 ng / mg, about 150 ng / mg, about 200 ng / mg, about 250 ng / mg, about 300 ng / mg, about 350 ng / mg, about 400 ng / mg, about 450 ng / mg, about 500 ng / mg, about 600 ng / mg, about 700 ng / mg, about 800 ng / mg, about 900 ng / mg, about 1000 ng / mg, about 1200 ng / mg or about 1500 ng / mg of the total protein in the liver of a treated subject.

[0072] In some embodiments, the expression of the natural, non-mutated, functionally active BSEP protein, or functionally active fragment thereof, is detectable after administration of a composition comprising a polynucleotide of the disclosure. In some embodiments, functionally active BSEP protein is detectable 2, 4, 6, 12, 18, 24, 30, 36, 48, 60, and / or 72 hours after administration of a composition comprising a polynucleotide of the disclosure. In some embodiments, functionally active BSEP protein is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after administration of a composition comprising a polynucleotide of the disclosure. In some embodiments, functionally active BSEP protein is detectable 1 week, 2 weeks, 3 weeks, and / or 4 weeks after administration of a composition comprising a polynucleotide of the disclosure. In some embodiments, functionally active BSEP protein is detectable in liver cells, e.g., in hepatocytes, after administration of a composition comprising a polynucleotide of the disclosure.III. Human ABCB11, Encoding Human BSEP (hBSEP)

[0073] The human ABCB11 gene encodes a 1321 amino acid transporter protein known as the bile salt export pump (BSEP), which has a molecular mass of approximately 160 kDa.BSEP plays a crucial role in the transport of bile salts from hepatocytes into the bile canaliculi, the small channels that form the bile ducts. Bile salts are essential for the digestion and absorption of dietary fats and fat-soluble vitamins. BSEP uses energy derived from ATP hydrolysis to actively pump bile salts against their concentration gradient from the cytoplasm of hepatocytes into the bile canaliculi.

[0074] The consensus human ABCB11 mRNA sequence that encodes BSEP has a sequence of 3,963 nucleobases (absent the stop codon) and is shown in SEQ ID NO: 1. When translated, the consensus human ABCB11 mRNA sequence encodes the 1321 amino acid, wild-type BSEP protein of SEQ ID NO: 7.

[0075] As is understood by the skilled artisan, the protein known as bile salt export pump (BSEP) may be referred to by alternative names in the art, including, ATP -binding cassette subfamily B member 11 (ABCB11), PFIC-2, ABC16, SPGP, and PGY4. See GeneCards ID: GC02M168922, which is incorporated herein by reference in its entirety. Accordingly, BSEP may be used interchangeably with any of these alternative names in the specification, the examples, the drawings, and the claims.IV. Polynucleotide Compositions

[0076] In some embodiments, a polynucleotide of the disclosure comprises an mRNA sequence capable of being translated into a functionally active BSEP protein or a fragment thereof which exhibits functional BSEP activity. The polynucleotides of the disclosure expressing a functionally active BSEP protein may be suitable for use in methods for ameliorating, preventing or treating disease associated with deficiency of normal BSEP activity.

[0077] In some embodiments, a polynucleotide of the disclosure may comprise a 5 ’-cap, a 5’ UTR, a BSEP coding sequence (CDS), a 3’UTR, and / or a tail region. In an exemplary embodiment, the polynucleotide may include a 5’-cap (e.g., N7-Methyl-Gppp), a 5’ UTR comprising or consisting of SEQ ID NO: 8, a BSEP CDS, a 3’ UTR comprising or consisting of SEQ ID NO: 10, and / or a tail region. In further exemplary embodiments, the BSEP CDS may comprise a codon-optimized sequence; for example, a sequence of SEQ ID NOs: 2-6, described in further detail below. In any of these and other embodiments described herein, the polynucleotide may comprise one or more modified nucleotides, e.g., N*-m ethylpseudouridine and / or 5-methoxyuridine, in place of one or more (e.g., all) uridine residues.

[0078] In some embodiments, the translation efficiency of the molecule can be increased as compared to a native mRNA of BSEP. As used throughout the description in the context of a nucleic acid, “native” may refer to a wild type and / or non-codon-optimized and / or non-modified mRNA. For example, in some embodiments, the translational expression of a molecule can be increased by 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or more relative to a native (z.e., wild type, non-codon-optimized, or non-modified) mRNA of B SEP.

[0079] In some embodiments, a suitable mRNA sequence for the present disclosure comprises an mRNA sequence encoding the BSEP protein. The sequence of the naturally occurring, functionally active human BSEP protein is shown in SEQ ID NO: 7.

[0080] In some embodiments, a suitable mRNA sequence may be an mRNA sequence that encodes a homolog, variant, or isoform of human BSEP. As used herein, a homolog or a variant of human BSEP protein may be a modified human BSEP protein containing one or more amino acid substitutions, deletions, and / or insertions as compared to a wild type or naturally occurring human BSEP protein while retaining substantial functional BSEP protein activity. As used herein, an isoform of human BSEP may be any one of a number of different structurally similar proteins that are created as the result of alternative splicing. In some embodiments, an mRNA suitable for the present disclosure encodes a protein substantially identical to human BSEP protein. In some embodiments, an mRNA suitable for the present disclosure encodes a BSEP protein having an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 7, wherein said BSEP protein exhibits substantially equivalent or increased functional activity relative to the BSEP protein having the amino acid sequence of SEQ ID NO: 7. In some embodiments, an mRNA suitable for the present disclosure encodes a functionally active fragment, a functionally active portion, or functionally active portions of a human BSEP protein.

[0081] In some embodiments, an mRNA suitable for the present disclosure encodes a fragment of the human BSEP protein, wherein the fragment of the protein still maintains BSEP activity similar to that of the wild type protein. In some embodiments, the disclosure may be used to deliver fragments of the BSEP, which comprise at least 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1310, or 1320 amino acid residues and retain one or more activities associated with the full-length polypeptide (e.g., bile transport activity in the case of BSEP). Such fragments may be obtained by recombinant techniques that are routine and well-known in the art.

[0082] In some embodiments, an mRNA suitable for the present disclosure encodes a truncated form of the BSEP protein, wherein the truncation (a) deletes a 5’ portion of BSEP, (b) deletes a 3’ portion of BSEP, (c) deletes an internal portion of BSEP, or (d) deletes internal portions of BSEP, or a combination of at least two deletions selected from (a)-(d).

[0083] Fragments of B SEP and truncations of B SEP may be tested for bile transport activity by assays known to the skilled artisan. For instance, characterization of bile transport activity of full-length BSEP or fragments / truncations of BSEP can be conducted using a Transwell culture system as described in Hayashi et al.. 2021, Stem Cell Reports 16: 309-23.

[0084] The disclosure further includes nucleic acid molecules which encode the abovedescribed polypeptide fragments and truncations. Furthermore, the disclosure encompasses the portions of the codon-optimized sequences of SEQ ID NOs: 2-6 which would encode the above-described polypeptide fragments and truncations.

[0085] In some embodiments, an mRNA suitable for the present disclosure comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 2-6. In an exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 2-6. In another exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 96% identical to a sequence selected from SEQ ID NOs: 2-6. In another exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 2-6 In another exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 98% identical to a sequence selected from SEQ ID NOs: 2-6. In another exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 2-6. In a further exemplary embodiment, an mRNA suitable for the present disclosure comprises a sequence that is at least 99.9% identical to SEQ ID SEQ ID NOs: 2-6.

[0086] In some embodiments, a polynucleotide of the present disclosure comprises a coding sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 2-6. In some embodiments, a polynucleotide comprising a coding sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 2-6 further comprises one or more sequences selected from a 5’- cap, a 5’ UTR, a 3’ UTR, and a tail region.

[0087] In some embodiments, a polynucleotide of the present disclosure comprises a nucleobase sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to SEQ ID NO: 19. In some embodiments, the polynucleotide further comprises a 5’ cap. In some embodiments, the first base of the polynucleotide, z.e., the 5’ base, is a guanine (G) which is supplied by the 5’ cap. In some embodiments, the 5’ guanine (G) is methylated at the N7 position. In some embodiments, the second base of the polynucleotide, z.e., the second base from the 5’ end, is an adenine which is a 2’-O-Methyl-A. In some embodiments, the 5’ G at position 1 of the polynucleotide is attached to the A of position 2 by a ppp linkage, constituting a 5’ cap.

[0088] In some embodiments, a polynucleotide of the present disclosure comprises a coding sequence that is less than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild type human BSEP coding sequence over the full length human BSEP coding sequence of SEQ ID NO: 1, and expresses a functionally active BSEP protein. In an exemplary embodiment, a polynucleotide of the present disclosure comprises a coding sequence that is less than 75%, 80%, 85%, 90% or 95% identical to the wild type human BSEP coding sequence over the full length human BSEP coding sequence of SEQ ID NO: 1, and expresses a functional BSEP protein. In another exemplary embodiment, a polynucleotide of the present disclosure comprises a coding sequence that is less than 95% identical to the wild type human BSEP coding sequence over the full length human BSEP coding sequence of SEQ ID NO: 1, and expresses a functional BSEP protein, wherein the coding sequence is at least 95% identical to a sequence selected from SEQ ID NOs: 2-6. Accordingly, in some embodiments, the present application provides a polynucleotide comprising or consisting of a nucleobase sequence that is less than 95% identical to the wild type human BSEP coding sequence over the full length human BSEP coding sequence of SEQ ID NO: 1, and wherein the BSEP coding sequence is at least 95%, 96%, 97%, 98%, 99%, 99.9% or more identical to a sequence selected from SEQ ID NOs: 2-6. In an exemplary embodiment, the present application provides a polynucleotide comprising of or consisting of a nucleobase sequence that is less than 95% identical to the wild type human BSEP coding sequence over the full length human BSEP coding sequence of SEQ ID NO: 1, and wherein the BSEP coding sequence is at least 95% identical to a sequence selected from SEQ ID NOs: 2-6. In some embodiments, the polynucleotide may comprise a sequence selected from SEQ ID NOs: 2-6 and a stop codon (UGA, UAA, or UAG) immediately downstream of said sequence. In a specific embodiment, the present application provides a polynucleotide comprising a nucleobase sequence of SEQ ID NO: 2. In yet another specific embodiment, the presentapplication provides a polynucleotide comprising a nucleobase sequence of SEQ ID NO: 19.

[0089] In some embodiments, the application further provides novel codon-optimized DNA sequences that can be transcribed to provide mRNA sequences encoding BSEP. Accordingly, the application additionally relates to nucleic acid sequences which are at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more identical to a sequence selected from SEQ ID NOs: 12-16. In exemplary embodiments, the application provides a nucleic acid sequence that can be transcribed to provide an mRNA sequence encoding BSEP selected from SEQ ID NOs: 12-16. Further provided are fragments of the nucleic acid sequences shown in SEQ ID NOs: 12-16 which can be transcribed to provide an mRNA sequence encoding a polypeptide having functional BSEP activity. In some embodiments, the polynucleotide may comprise a sequence selected from SEQ ID NOs: 12-16 and a stop codon (TGA, TAA, or TAG) immediately downstream of said sequence. In a specific embodiment, the present application provides a polynucleotide comprising a DNA sequence of SEQ ID NO: 12.

[0090] In some embodiments, a polynucleotide of the disclosure may comprise one or more unlocked nucleomonomers (z.e., UNA monomers) such as those described, for example, in US Patent No. 9,944,929.

[0091] In some embodiments, a polynucleotide of the disclosure may comprise one or more locked nucleic acids (z.e., LNA monomers) or other nucleotide analogues according to the descriptions set forth in, for example, US Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, 7,399,845, and 8,314,227.

[0092] In some embodiments, a polynucleotide of the disclosure encodes a fusion protein comprising a full length, fragment or portion of a BSEP protein fused to another sequence (e.g., an N or C terminal fusion). In some embodiments, the N or C terminal sequence is a signal sequence or a cellular targeting sequence.A. Modified Nucleotides

[0093] In various embodiments described herein, a polynucleotide of the disclosure may comprise a combination of natural and modified nucleic acid monomers (z.e., nucleotides). Various examples of modified nucleotides which may be incorporated into the polynucleotides of the disclosure are disclosed in WO / 2018 / 222926, which is herein incorporated by reference in its entirety.

[0094] In some embodiments, an alkyl, cycloalkyl, or phenyl substituent may be unsubstituted, or further substituted with one or more alkyl, halo, haloalkyl, amino, or nitro substituents.

[0095] In some embodiments, a polynucleotide of the disclosure comprises one or more pseudouridines. Examples of pseudouridines include N'-alkylpseudouridines, N1- cycloalkylpseudouridines, Nkhydroxypseudouridines, Nkhydroxyalkylpseudouridines, N1- phenylpseudouridines, N'-phenylalkylpseudouridines, N'-aminoalkylpseudouridines, N3- alkylpseudouridines, N6-alkylpseudouridines, N6-alkoxypseudouridines, N6- hydroxypseudouridines, N6-hydroxyalkylpseudouridines, N6-morpholinopseudouridines, N6- phenylpseudouridines, and N6-halopseudouridines. Examples of pseudouridines include N1- alkyl-N6-alkylpseudouridines, N1-alkyl-N6-alkoxypseudouridines, N'-alkyl-N6- hydroxypseudouridines, N'-alkyl-N6-hydroxyalkylpseudouridines, N'-alkyl-N6- morpholinopseudouridines, N1-alkyl-N6-phenylpseudouridines, and N'-alkyl-N6- halopseudouridines. In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted, or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents. Examples of pseudouridines further include N'-methylpseudouridine, N'-ethylpseudouridine, N'-propylpseudouridine, N'-cyclopropylpseudouridine, N'-phenylpseudouridine, N1- aminomethylpseudouridine, N3-methylpseudouridine, Nkhydroxypseudouridine, and N1- hydroxymethylpseudouridine.

[0096] In some embodiments, the pseudouridine residue is selected from N1- methylpseudouridine, N'-ethylpseudouridine, N'-propylpseudouridine, N1- cyclopropylpseudouridine, N1-phenylpseudouridine, N'-aminomethylpseudouridine, N3- methylpseudouridine, N'-hydroxypseudouridine, and N'-hydroxymethylpseudouridine. In an exemplary embodiment, a polynucleotide of the disclosure is fully modified to comprise N1- methylpseudouridine residues in place of uridine residues.

[0097] In some embodiments, a polynucleotide of the disclosure comprises one or more modified nucleotides selected from 5-hydroxyuridine, 5 -methyluridine, 5- hydroxymethyluridine, 5 -carboxyuridine, 5-carboxymethylesteruridine, 5 -formyluridine, 5- methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2- thiouridine, and 6-methyluridine. In an exemplary embodiment, a polynucleotide of the disclosure is fully modified to comprise 5 -methoxyuridine residues in place of uridine residues.

[0098] In some embodiments, a polynucleotide of the disclosure may comprise one or more modified nucleotides selected from 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.

[0099] In some embodiments, a polynucleotide of the disclosure may comprise one or more modified nucleotides selected from 3'-end stabilized nucleotides, 3'-glyceryl nucleotides, 3'- inverted abasic nucleotides, and 3'-inverted thymidine.

[0100] In some embodiments, a polynucleotide of the disclosure may comprise one or more modified nucleotides selected from unlocked nucleic acid nucleotides (UNA), locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'- methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'-O-methyl nucleotides. In one exemplary embodiment, the modified nucleotide is an unlocked nucleic acid nucleotide (UNA). A detailed summary of unlocked nucleic acids and methods for their incorporation into polynucleotides is found in WO / 2018 / 222926, which is herein incorporated by reference in its entirety. In another exemplary embodiment, the modified nucleotide is a locked nucleic acid nucleotide (LNA).

[0101] In some embodiments, a polynucleotide of the disclosure may comprise one or more modified nucleotides selected from 2 ',4 '-constrained 2'-O-methoxyethyl (cMOE) and 2'-O- Ethyl (cEt) modified DNAs.

[0102] In some embodiments, a polynucleotide of the disclosure may comprise one or more modified nucleotides selected from 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0103] Examples of base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications.B. Molecular Cap Structure

[0104] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof further comprises a 5 ’-cap.

[0105] 5' -caps and their analogues are known in the art. Some examples of 5 ’-cap structures which may be incorporated into the polynucleotides of the disclosure are given in WO / 2017 / 053297, WO / 2015 / 051169, WO / 2015 / 061491, and US Patent Nos. 8,093,367 and 8,304,529.

[0106] In some embodiments, the application provides 5 ’-capped RNAs, wherein the initiating capped oligonucleotide primers have the general formm / Gppp[N2'Ome]n[N]mwhereinm / G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, "n" can be any integer from 0 to 4 and "m" can be an integer from 1 to 9. Compositions and methods for synthesizing such 5 ’-capped RNAs are described in

[0107] In an exemplary embodiment, the 5 '-cap comprises N7-Methyl-Gppp.

[0108] In another exemplary embodiment, the 5 ’-cap has the following structure:

[0109] In another exemplary embodiment, the 5 ’-capping of mRNA involves the use of CleanCap® AG from TriLink BioTechnologies, Catalog No. N-7113, which is incorporated herein by reference.

[0110] In some embodiments, the 5’-cap may be a m7GpppGm cap. In further embodiments, the 5 ’-cap may be selected from m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), a trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7, 2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (See, e.g., Jemielity et al., 2003, RNA 9: 1108-1122). In other embodiments, the 5’-cap may be an ARCA cap (3’-OMe-m7G(5’)pppG) or an mCAP (m7G(5')ppp(5')G, N7-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine).C. 5 ’ and 3 ’ Untranslated Regions (UTRs)[OHl] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof may further comprise a 5’ untranslated region (5’ UTR) and / or a 3’ untranslated region (3’ UTR). As is understood in the art, the 5’ and / or 3’ UTR may affect an mRNA’s stability or efficiency of translation. In an exemplary embodiment, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a 5’ UTR and a 3’ UTR.

[0112] Examples of 5’ UTR and 3’ UTR sequences which may be incorporated into the polynucleotides of the disclosure may be found in US Patent No. 9,149,506 and

[0113] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof may comprise a 5’ UTR that is at least about 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 300, or 400 nucleotides. In some embodiments, a 5’ UTR contains about 15 to 75 nucleotides (e.g., about 25 to 55 nucleotides, about 30 to 50 nucleotides, or about 40 nucleotides). In an exemplary embodiment, the 5’ UTR is about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, or about 45 nucleotides in length.

[0114] In some embodiments, the 5’ UTR is derived from an mRNA molecule known in the art to be relatively stable (e.g., histone, tubulin, globin, GAPDH, actin, or citric acid cycle enzymes) to increase the stability of the polynucleotide. In some embodiments, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene. In some embodiments, the 5’ UTR comprises a sequence selected from the 5’ UTRs of human IL-6, alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human transthyretin, human haptoglobin, human alpha- 1 -anti chymotrypsin, human antithrombin, human alpha- 1 -antitrypsin, human albumin, human beta globin, human complement C3, human complement C5, SynK, AT1G58420, mouse beta globin, mouse albumin, and a tobacco etch virus, or fragments of any of the foregoing.

[0115] In some embodiments, the 5’ UTR comprises or consists of a sequence set forth in SEQ ID NO: 8. The 5’ UTR may be a fragment of a sequence set forth in SEQ ID NO: 8, such as a fragment of at least 10, 15, 20, 25, 30, or 35 contiguous nucleotides of SEQ ID NO: 8.

[0116] In an alternative embodiment, the 5’ UTR comprises or consists of a sequence set forth in SEQ ID NO: 9. In yet another exemplary embodiment, the 5’ UTR is a fragment of a sequence set forth in SEQ ID NO: 9, such as a fragment of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 125 contiguous nucleotides of SEQ ID NO: 9.

[0117] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises an internal ribosome entry site (IRES). As is understood in the art, an IRES is an RNA element that allows for translation initiation in an end-independent manner. In exemplary embodiments, the IRES is in the 5’ UTR. In other embodiments, the IRES may be outside the 5’ UTR.

[0118] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof may comprise a 3’ UTR that is at least about 25, 50, 75, 100, 125, 150, 175, 200, 300, 400, or 500 nucleotides. In some embodiments, a 3’ UTRcontains about 50 to 200 nucleotides e.g., about 60 to 150 nucleotides, about 80 to 120 nucleotides, or about 100 nucleotides). In an exemplary embodiment, the 3’ UTR is about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, or about 105 nucleotides in length.

[0119] In some embodiments, the 3’ UTR comprises a sequence selected from the 3’ UTRs of alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human haptoglobin, human antithrombin, human alpha globin, human beta globin, human complement C3, human growth factor, human hepcidin, MALAT-1, mouse beta globin, mouse albumin, and Xenopus beta globin, or fragments of any of the foregoing.

[0120] In some embodiments, the 3’ UTR comprises or consists of a sequence set forth in SEQ ID NO: 10. In some embodiments, the 3’ UTR is a fragment of a sequence set forth in SEQ ID NO: 10, such as a fragment of at least 20, 30, 40, 50, 60, 70, 80, 90, or 95 contiguous nucleotides of SEQ ID NO: 10.

[0121] In an alternative embodiment, the 3’ UTR comprises or consists of a sequence set forth in SEQ ID NO: 11. In another exemplary embodiment, the 3’ UTR is a fragment of a sequence set forth in SEQ ID NO: 11, such as a fragment of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 contiguous nucleotides of SEQ ID NO: 11.

[0122] In certain exemplary embodiments, the polynucleotide encoding BSEP comprises a 5’ UTR sequence of SEQ ID NO: 8 and a 3’ UTR sequence of SEQ ID NO: 10.D. Tail Region

[0123] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a tail region, which can serve to protect the mRNA from exonuclease degradation. In some embodiments, the tail region can be a polyA tail.

[0124] PolyA tails can be added using a variety of methods known in the art, e.g, using poly(A) polymerase to add tails to synthetic or in vitro transcribed RNA. Other methods include the use of a transcription vector to encode polyA tails or the use of a ligase (e.g., via splint ligation using a T4 RNA ligase and / or T4 DNA ligase), wherein polyA may be ligated to the 3' end of a sense RNA. In some embodiments, a combination of any of the above methods is utilized.

[0125] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a 3' polyA tail structure. In some embodiments, the length of the polyA tail can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 100, 200, or 300 nucleotides. In some embodiments, a 3’ polyA tail contains about 5 to 500 adenosine nucleotides (e.g., about 30 to 250 adenosine nucleotides, about 60 to 220 adenosine nucleotides, about 80 to 200 adenosine nucleotides, about 90 to about 150 adenosine nucleotides, or about 100 to about 120 adenosine nucleotides). In an exemplary embodiment, the 3’ polyA tail is about 80 nucleotides in length. In another exemplary embodiment, the 3’ polyA tail is about 100 nucleotides in length. In yet another exemplary embodiment, the 3’ polyA tail is about 115 nucleotides in length. In yet another exemplary embodiment, the 3’ polyA tail is about 250 nucleotides in length.

[0126] In some embodiments, the 3’ polyA tail comprises one or more UNA monomers. In some embodiments, the 3’ polyA tail contains 2, 3, 4, 5, 10, 15, 20, or more UNA monomers (SEQ ID NO: 21). In an exemplary embodiment, the 3’ polyA tail contains 2 UNA monomers. In a further exemplary embodiment, the 3’ polyA tail contains 2 UNA monomers which are found consecutively, z.e., contiguous to each other in the 3’ polyA tail.

[0127] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a 3' polyC tail structure. In some embodiments, the length of the polyC tail can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a 3’ polyC tail contains about 5 to 500 cytosine nucleotides (e.g., about 30 to 250 cytosine nucleotides, about 60 to 220 cytosine nucleotides, about 80 to about 200 cytosine nucleotides, about 90 to 150 cytosine nucleotides, or about 100 to about 120 cytosine nucleotides). In an exemplary embodiment, the 3’ polyC tail is about 80 nucleotides in length. In another exemplary embodiment, the 3’ polyC tail is about 100 nucleotides in length. In yet another exemplary embodiment, the 3’ polyC tail is about 115 nucleotides in length. In yet another exemplary embodiment, the 3’ polyC tail is about 250 nucleotides in length. The polyC tail may be added to the polyA tail or may substitute the polyA tail. The polyC tail may be added to the 5’ end of the polyA tail or the 3’ end of the polyA tail.

[0128] In some embodiments, the length of the polyA and / or polyC tail is adjusted to control the stability of a modified polynucleotide of the disclosure and, thus, the transcription of protein. For example, since the length of the polyA tail can influence the half-life of a polynucleotide, the length of the polyA tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and / or polypeptideproduction in a target cell.E. Triple Stop Codon

[0129] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof may comprise a sequence immediately downstream of the CDS that creates a triple stop codon. The triple stop codon may be incorporated to enhance the efficiency of translation. In some embodiments, the translatable oligomer may comprise the sequence AUAAGUGAA immediately downstream of a BSEP CDS described herein.F. Translation Initiation Sites

[0130] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof may comprise a translation initiation site. Such sequences are known in the art and include the Kozak sequence. See, e.g., Kozak, Marilyn, 1988, Mol. and Cell Biol. 8: 2737-2744; Kozak, Marilyn, 1991, J. Biol. Chem. 266: 19867- 19870; Kozak, Marilyn, 1990, PNAS USA 87:8301-8305; and Kozak, Marilyn, 1989, J. Cell Biol. 108: 229-241. As is understood in the art, a Kozak sequence is a short consensus sequence centered around the translational initiation site of eukaryotic mRNAs that allows for efficient initiation of translation of the mRNA. The ribosomal translation machinery recognizes the AUG initiation codon in the context of the Kozak sequence.

[0131] In some embodiments, the translation initiation site, e.g., a Kozak sequence, is inserted upstream of the coding sequence for BSEP. In some embodiments, the translation initiation site is inserted downstream of a 5’ UTR. In certain exemplary embodiments, the translation initiation site is inserted upstream of the coding sequence for BSEP and downstream of a 5’ UTR.

[0132] As is understood in the art, the length of the Kozak sequence may vary. Generally, increasing the length of the leader sequence enhances translation.

[0133] In some embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a Kozak sequence having the sequence (e.g., GCCACC). In certain exemplary embodiments, the polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof comprises a Kozak sequence having the sequence (e.g., GCCACC), wherein the Kozak sequence is immediately downstream of a 5’ UTR and immediately upstream of the coding sequence for BSEP.V. Synthesis Methods

[0134] In various aspects, this disclosure provides methods for synthesis of polynucleotides comprising an mRNA coding sequence for the BSEP protein or a fragment thereof.

[0135] Polynucleotides of this disclosure can be synthesized and isolated using methods disclosed herein, as well as any pertinent techniques known in the art.

[0136] Some methods for preparing nucleic acids are given in, for example, Merino, Chemical Synthesis of Nucleoside Analogues, (2013); Gait, Oligonucleotide synthesis: a practical approach (1984); Herdewijn, Oligonucleotide Synthesis, Methods in Molecular Biology, Vol. 288 (2005).

[0137] In some embodiments, a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof can be made by an in vitro transcription (IVT) reaction. A mix of nucleoside triphosphates (NTP) can be polymerized using T7 reagents, for example, to yield RNA from a DNA template. The DNA template can be degraded with RNase-free DNase, and the RNA column-separated.

[0138] In some embodiments, a ligase can be used to link a synthetic oligomer to the 3' end of an RNA molecule or an RNA transcript to form a polynucleotide of the disclosure. The synthetic oligomer that is ligated to the 3' end can provide the functionality of a polyA tail, and advantageously provide resistance to its removal by 3'-exoribonucleases. The ligated product can have increased specific activity and provide increased levels of protein expression.

[0139] In certain embodiments, the ligated product can be made with an RNA transcript that has native specificity. The ligated product can be a synthetic molecule that retains the structure of the RNA transcript at the 5' end to ensure compatibility with the native specificity.

[0140] In further embodiments, the ligated product be made with an exogenous RNA transcript or non-natural RNA. The ligated product can be a synthetic molecule that retains the structure of the RNA.

[0141] Without wishing to be bound by theory, the canonical mRNA degradation pathway in cells includes the steps: (i) the polyA tail is gradually cut back to a stub by 3’ exonucleases, shutting down the looping interaction required for efficient translation and leaving the cap open to attack; (ii) decapping complexes remove the 5 ’-cap; (iii) the unprotected and translationally incompetent residuum of the transcript is degraded by 5’ and 3’ exonuclease activity.

[0142] Embodiments of this disclosure involve new polynucleotide structures which can have increased translational activity over a native transcript. Among other things, the polynucleotides provided herein may prevent exonucleases from trimming back the polyA tail in the process of de-adenylation.VI. Lipid-Based Formulations

[0143] Lipid-based formulations have been increasingly recognized as one of the mostpromising delivery systems for RNA due to their biocompatibility and their ease of large-scale production. Cationic lipids have been widely studied as synthetic materials for delivery of RNA. After mixing together, nucleic acids are condensed by cationic lipids to form lipid / nucleic acid complexes known as lipoplexes. These lipid complexes are able to protect genetic material from the action of nucleases and to deliver it into cells by interacting with the negatively charged cell membrane. Lipoplexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

[0144] Conventional liposomes consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, which encloses an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. Liposome characteristics and behavior in vivo can be modified by addition of a hydrophilic polymer coating, e.g., polyethylene glycol (PEG), to the liposome surface to confer steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains.

[0145] Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. They may present as spherical vesicles and can range in size from 20 nm to a few microns. Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm. Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids.

[0146] Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin R A-shRNA). Cationic lipids, such as DOTAP, (l,2-dioleoyl-3- trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high in vitro transfection efficiency. Furthermore, neutral lipid-based nanoliposomes for RNA delivery as, e.g., neutrall,2-dioleoyl-sn-glycero-3- phosphatidylcholine (DOPC)-based nanoliposomes have been developed.

[0147] According to some embodiments, the polynucleotides described herein that encode BSEP are lipid formulated. The lipid formulation may include formats including, but not limited to, liposomes, lipoplexes, copolymers, such as PLGA, and lipid nanoparticles. In an exemplary embodiment, the lipid formulation is a lipid nanoparticle. In a further exemplary embodiment, the polynucleotides are encapsulated in a lipid nanoparticle, wherein the lipid nanoparticles are part of a pharmaceutical formulation that is free of liposomes.

[0148] In some embodiments, a lipid nanoparticle (LNP) comprises:(a) a nucleic acid e.g., a polynucleotide encoding BSEP),(b) a cationic lipid,(c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG- modified lipid),(d) optionally a non-cationic lipid (such as a neutral lipid), and(e) optionally, a sterol.

[0149] In some embodiments, the lipid nanoparticle formulation consists of (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.A. Thiocarbamate and Carbamate-Containing Lipid Formulations

[0150] Some examples of lipids and lipid formulations for delivery of a polynucleotide encoding BSEP are given in WO / 2015 / 074085 and U.S. Patent Publication Nos. US 2018 / 0169268 and US 20180170866. In certain embodiments, the lipid is a compound of the following Formula I:Formula IwhereinRi and R2 both consist of a linear alkyl consisting of 1 to 14 carbons, or an alkenyl or alkynyl consisting of 2 to 14 carbons;Li and L2 both consist of a linear alkylene or alkenyl ene consisting of 5 to 18 carbons, or forming a heterocycle with N;X is S;L3 consists of a bond or a linear alkylene consisting of 1 to 6 carbons, or forming a heterocycle with N;R3 consists of a linear or branched alkylene consisting of 1 to 6 carbons; andR4 and R5 are the same or different, each consisting of a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; or a pharmaceutically acceptable salt thereof.

[0151] The lipid formulation may contain one or more ionizable cationic lipids selected from among the following:ATX-081B. Cationic Lipids

[0152] The lipid nanoparticle (LNP) encapsulating a polynucleotide of the disclosure may include a cationic lipid suitable for forming a lipid nanoparticle. The cationic lipid may carry a net positive charge at about physiological pH.

[0153] The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2- dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1, 2-Dioleyl oxy-3 - trimethylaminopropane chloride salt), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy- N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), 1,2-Dilinoleylcarbamoyl oxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2- Dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1, 2-Dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- Dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA), 1-Linoleoyl -2-linoleyl oxy-3 - dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyl oxy-3 -trimethylaminopropane chloride salt (DLin-TMA.CI), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI), l,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N- Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP),1.2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH- cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl4- (dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (C 12-200), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28 31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutan-l-amine (MC4 Ether), or any combination of any of the foregoing. Other cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N' -dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dileoyl-sn-3- phosphoethanolamine (DOPE), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and2.2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO / BRL).

[0154] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Patent Nos. 8,158,601; and Love et al., 2010, PNAS 107(5): 1864-69. Other suitable amino lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N- ethyl- N-methylamino-, and N-propyl-N-ethylamino-). In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containingunsaturated fatty acids with carbon chain lengths in the range of C14 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0155] In certain embodiments, amino or cationic lipids of the disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, e.g., at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipids be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0156] The cationic lipid can comprise from about 20 mol% to about 70 mol% or 75 mol% or from about 45 mol% to about 65 mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the particle. In some embodiments, the lipid nanoparticles include from about 25% to about 75% on a molar basis of cationic lipid, e.g., from about 20% to about 70%, from about 35% to about 65%, from about 45% to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle). In some embodiments, the ratio of cationic lipid to nucleic acid is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.VII. Pharmaceutical Formulations

[0157] In some aspects, this application provides pharmaceutical formulations containing a polynucleotide of the disclosure capable of encoding a functionally active BSEP protein or functional fragment thereof and a pharmaceutically acceptable carrier.

[0158] A pharmaceutical formulation can be capable of local or systemic administration. In some aspects, a pharmaceutical formulation can be capable of any mode of administration. In certain aspects, the administration can be by any route, including intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, inhalation or nasal administration.

[0159] Embodiments of this disclosure include pharmaceutical formulations containing a BSEP-encoding polynucleotide formulated according to a lipid-based formulation as described herein.

[0160] Some embodiments of this disclosure include pharmaceutical formulations containing a BSEP-encoding polynucleotide in a lipid formulation, e.g., a lipid nanoparticle (LNP).

[0161] In some embodiments, a pharmaceutical formulation may comprise one or more lipids selected from cationic lipids, anionic lipids, sterols, pegylated lipids, and any combination of the foregoing. In some embodiments, the pharmaceutical formulation containing a BSEP-encoding polynucleotide comprises a cationic lipid, a phospholipid, cholesterol, and a pegylated lipid.

[0162] In certain exemplary embodiments, a pharmaceutical formulation of the disclosure is free of liposomes.

[0163] In further embodiments, a pharmaceutical formulation can include nanoparticles.

[0164] In certain exemplary embodiments, a pharmaceutical formulation of the disclosure comprises a BSEP-encoding polynucleotide of the disclosure encapsulated in lipid nanoparticles (LNPs) and is free of liposomes.

[0165] Some examples of lipids and lipid compositions for preparing formulations of a BSEP-encoding polynucleotide of this disclosure are given in WO / 2015 / 074085, which is hereby incorporated by reference in its entirety. In certain embodiments, the lipid is a cationic lipid. In some embodiment, the cationic lipid comprises a compound of Formula II:Formula II, in which Ri and R2 are the same or different, each a linear or branched alkyl, alkenyl, or alkynyl, Li and L2 are the same or different, each a linear alkyl having at least five carbon atoms, or form a heterocycle with the N, Xi is a bond, or is — CO— O— whereby L2-CO— O— R2 is formed X2 is S or O, L3 is a bond or a lower alkyl, R3 is a lower alkyl, R4 and R5 are the same or different, each a lower alkyl. What is also described herein is the compound of Formula II, in which L3 is absent, Ri and R2 each consists of at least seven carbon atoms, R3 is ethylene or n- propylene, R4 and R5 are methyl or ethyl, and Li and L2 each consists of a linear alkyl havingat least five carbon atoms. What is also described herein is the compound of Formula II, in which L3 is absent, Ri and R2 each consists of at least seven carbon atoms, R3 is ethylene or n- propylene, R4 and R5 are methyl or ethyl, and Li and L2 each consists of a linear alkyl having at least five carbon atoms. What is also described herein is the compound of Formula II, in which L3 is absent, Ri and R2 each consists of an alkenyl of at least nine carbon atoms, R3 is ethylene or n-propylene, R4 and R5 are methyl or ethyl, and Li and L2 each consists of a linear alkyl having at least five carbon atoms. What is also described herein is the compound of Formula II, in which L3 is methylene, Ri and R2 each consists of at least seven carbon atoms, R3 is ethylene or n-propylene, R4 and R5 are methyl or ethyl, and Li and L2 each consists of a linear alkyl having at least five carbon atoms. What is also described herein is the compound of Formula II, in which L3 is methylene, Ri and R2 each consists of at least nine carbon atoms, R3 is ethylene or n-propylene, R4 and R5 are each methyl, Li and L2 each consists of a linear alkyl having at least seven carbon atoms. What is also described herein is the compound of Formula II, in which L3 is methylene, Ri consists of an alkenyl having at least nine carbon atoms and R2 consists of an alkenyl having at least seven carbon atoms, R3 is n-propylene, R4 and Rs are each methyl, Li and L2 each consists of a linear alkyl having at least seven carbon atoms. What is also described herein is the compound of Formula II, in which L3 is methylene, Ri and R2 each consists of an alkenyl having at least nine carbon atoms, R3 is ethylene, R4 and Rs are each methyl, Li and L2 each consists of a linear alkyl having at least seven carbon atoms.

[0166] In exemplary embodiments, the cationic lipid comprises a compound of selected from the group consisting of ATX-001, ATX-002, ATX-003, ATX-004, ATX-005, ATX-006, ATX- 007, ATX-008, ATX-009, ATX-010, ATX-011, ATX-012, ATX-013, ATX-014, ATX-015, ATX-016, ATX-017, ATX-018, ATX-019, ATX-020, ATX-021, ATX-022, ATX-023, ATX- 024, ATX-025, ATX-026, ATX-027, ATX-028, ATX-029, ATX-030, ATX-031, ATX-032, ATX-081, ATX-095, and ATX-126, or a pharmaceutically acceptable salt thereof.

[0167] In exemplary embodiments, the cationic lipid is selected from ATX-002, ATX-081, ATX-095, or ATX-126.

[0168] In some embodiments, the cationic lipid or a pharmaceutically acceptable salt thereof, may be presented in a lipid formulation, comprising a nanoparticle or a bilayer of lipid molecules. The lipid bilayer may further comprise a neutral lipid or a polymer. The lipid formulation may comprise a liquid medium. The formulation may further encapsulate a polynucleotide comprising a BSEP coding sequence of the present disclosure. The lipid formulation may further comprise a polynucleotide of the present disclosure and a neutral lipidor a polymer. The lipid formulation may encapsulate the polynucleotide comprising a BSEP coding sequence.

[0169] In further embodiments, the cationic lipid comprises a compound of Formula III:Formula III, wherein Ri and R2 are the same or different, each a linear or branched alkyl consisting of 1 to 9 carbons, an alkenyl or alkynyl consisting of 2 to 11 carbons, or cholesteryl, Li and L2 are the same or different, each a linear alkylene or alkenyl ene consisting of 5 to 18 carbons, Xi is — CO— O-- whereby -L2-CO— O— R2 is formed, X2 is S or O, X3 is — CO— O— whereby -Li-CO— O— Ri is formed, L3 is a bond, R3 is a linear or branched alkylene consisting of 1 to 6 carbons, and R4 and R5 are the same or different, each hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; or a pharmaceutically acceptable salt thereof. In some embodiments, X2 is S. In some embodiments, R3 is selected from ethylene, n-propylene, or isobutylene. In yet another embodiment, R4 and Rs are separately methyl, ethyl, or isopropyl. In yet another embodiment, Li and L2 are the same. In yet another embodiment, Li and L2 differ. In yet another embodiment, Li or L2 consists of a linear alkylene having seven carbons. In yet another embodiment, Li or L2 consists of a linear alkylene having nine carbons. In yet another embodiment, Ri and R2 are the same. In yet another embodiment, Ri and R2 differ. In yet another embodiment, Ri and R2 each consists of an alkenyl. In yet another embodiment, Ri and R2 each consists of an alkyl. In yet another embodiment, the alkenyl consists of a single double bond. In yet another embodiment, Ri or R2 consists of nine carbons. In yet another embodiment, Ri or R2 consists of eleven carbons. In yet another embodiment, Ri or R2 consists of seven carbons. In yet another embodiment, L3 is a bond, R3 is ethylene, X2 is S, and R4 and R5 are each methyl. In yet another embodiment, L3 is a bond, R3 is n-propylene, X2 is S, R4 and R5 are each methyl.In yet another embodiment, L3 is a bond, R3 is ethylene, X2 is S, and R4 and R5 are each ethyl.

[0170] As will be appreciated by the skilled artisan, the compounds of formulas II and III form salts that are also within the scope of this disclosure. Reference to a compound of formulas II and III herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. In addition, when a compound of Formula II or III contains both a basic moiety, such as, but not limited to, a pyridine or imidazole, and an acidic moiety, such as, but not limited to, a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. The salts can be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts, although other salts are also useful. Salts of the compounds of the Formula II or III may be formed, for example, by reacting a compound of Formula II or III with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0171] Exemplary acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-napthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3 -phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates (such as those mentioned herein), tartarates, thiocyanates, toluenesulfonates (also known as tosylates) undecanoates, and the like. Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by Berge et al., 1977, J. Pharmaceutical Sciences 66(1) 1-19; P. Gould, 1986, International J. Pharmaceutics 33 201- 217; Anderson et al., 1996, The Practice of Medicinal Chemistry Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C.).

[0172] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acidssuch as arginine, lysine, and the like. Basic nitrogen-containing groups may be quartemized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e g, dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0173] All such acid and base salts are intended to be pharmaceutically acceptable salts within the scope of the disclosure and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure. Compounds of Formula II or III can exist in unsolvated and solvated forms, including hydrated forms. In general, the solvated forms, with pharmaceutically acceptable solvents such as water, ethanol, and the like, are equivalent to the unsolvated forms for the purposes of this disclosure. Compounds of Formula II or III and salts, solvates thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present disclosure.

[0174] The cationic lipid compounds described herein may be combined with a polynucleotide encoding BSEP to form microparticles, nanoparticles, liposomes, or micelles. The polynucleotide of the disclosure to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid. The cationic lipid compound and the polynucleotide may be combined with other cationic lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical formulation.

[0175] In certain embodiments, the cationic lipid compounds are relatively non- cytotoxic. The cationic lipid compounds may be biocompatible and biodegradable. The cationic lipid may have a pKa in the range of approximately 5.5 to approximately 7.5, such as between approximately 6.0 and approximately 7.0. It may be designed to have a desired pKa between approximately 3.0 and approximately 9.0, or between approximately 5.0 and approximately 8.0.

[0176] A formulation containing a cationic lipid compound may be 30-70% cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid and 1-10% polyethylene glycol (PEG). The formulation may comprise 30-40% cationic lipid compound, 40-50% cholesterol, and 10- 20% PEG. In other embodiments, the formulation is 50-75% cationic lipid compound, 20-40% cholesterol, and 5 to 10% phospholipid, and 1-10% PEG. The formulation may contain 60-70% cationic lipid compound, 25-35% cholesterol, and 5-10% PEG. The formulation may contain up to 90% cationic lipid compound and 2 to 15% helper lipid. The formulation may be a lipid particle formulation, for example containing 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2 to 25% cholesterol, 10 to 35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1 to 15% cholesterol, 2 to 35% cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipids, or even 100% cationic lipid.

[0177] In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol and DC-Chol (N,N-dimethyl-N- ethylcarboxamidocholesterol), and l,4-bis(3-N-oleylamino-propyl)piperazine. In an exemplary embodiment, the cholesterol-based lipid is cholesterol.

[0178] In some embodiments, the one or more pegylated lipids, z.e., PEG-modified lipids. In some embodiments, the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified lipid is a derivatized ceramide such as N-Octanoyl- Sphingosine-1 -[Succinyl (Methoxy Polyethylene Glycol)-2000], In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or Dimyristoyl glycerol (DMG)- PEG-2K. In an exemplary embodiment, the PEG-modified lipid is PEGylated cholesterol.

[0179] In additional embodiments, a pharmaceutical formulation can contain a BSEP- encoding polynucleotide of the disclosure (e.g., a polynucleotide comprising a sequence selected from SEQ ID NOs: 2-6 and 12-16) within a viral or bacterial vector.

[0180] A pharmaceutical formulation of this disclosure may include carriers, diluents or excipients as are known in the art. Examples of pharmaceutical formulations and methods are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985), and Remington, The Science and Practice of Pharmacy, 21st Edition (2005).

[0181] Examples of excipients for a pharmaceutical formulation include antioxidants, suspending agents, dispersing agents, preservatives, buffering agents, tonicity agents, and surfactants.VIII. Methods of Administration

[0182] An effective dose of an agent or pharmaceutical formulation of this disclosure can be an amount that is sufficient to cause translation of a BSEP-encoding polynucleotide in a cell.

[0183] A therapeutically effective dose can be an amount of an agent or formulation that is sufficient to cause a therapeutic effect. A therapeutically effective dose can be administered inone or more separate administrations, and by different routes. As will be appreciated in the art, a therapeutically effective dose or a therapeutically effective amount is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical formulations of the present disclosure. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and / or ameliorating PFIC2). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., a polynucleotide encoding BSEP or a functionally active fragment thereof) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

[0184] Methods provided herein contemplate single as well as multiple administrations of a therapeutically effective amount of the polynucleotide (e.g., a polynucleotide encoding BSEP or a functionally active fragment thereof) described herein. Pharmaceutical formulations comprising a polynucleotide encoding BSEP can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition (e.g., the severity of a subject’s disease state and the associated symptoms of PFIC2, and / or the subject’s BSEP activity levels). In some embodiments, a therapeutically effective amount of the polynucleotide (e.g., a polynucleotide encoding BSEP or a fragment thereof) of the present disclosure may be administered periodically at regular intervals (e.g., once every year, once every six months, once every four months, once every three months, once every two months, once a month), once every two weeks, weekly, daily, twice a day, three times a day, four times a day, five times a day, six times a day, or continuously. In an exemplary embodiment, a therapeutically effective amount of the polynucleotide (e.g., a polynucleotide encoding BSEP or a fragment thereof) of the present disclosure is administered weekly, once every two weeks, or monthly.

[0185] In some embodiments, the pharmaceutical formulations of the present disclosure are formulated such that they are suitable for extended-release of the polynucleotide encoding BSEP contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For instance, in some embodiments, the pharmaceutical formulations of the present disclosure are administered to a subject twice a day, daily or every other day. In some embodiments, the pharmaceutical formulations of the presentdisclosure are administered to a subject twice a week, once a week, every 10 days, every two weeks, every 28 days, every month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every nine months or once a year. Also contemplated herein are pharmaceutical formulations which are formulated for depot administration (e.g., subcutaneously, intramuscularly) to either deliver or release a polynucleotide encoding BSEP over extended periods of time. The extended-release means employed may be combined with modifications made to the polynucleotide encoding BSEP to enhance stability.

[0186] In some embodiments, administering a therapeutically effective dose of a composition comprising a polynucleotide of the disclosure can result in increased levels of functional BSEP protein in the liver of a treated subject. In some embodiments, administering a composition comprising a polynucleotide of the disclosure results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% increase in levels of functional BSEP protein in the liver relative to a baseline functional BSEP protein level in the subject prior to treatment. In certain embodiments, administering a therapeutically effective dose of a composition comprising a polynucleotide of the disclosure will result an increase in levels of functional BSEP protein relative to baseline functional BSEP levels in the liver of the subject prior to treatment. In some embodiments, the increase in functional BSEP levels in the liver relative to baseline functional BSEP levels in the liver will be at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 200%, or more.

[0187] In some embodiments, a therapeutically effective dose, when administered regularly, results in increased expression of functional BSEP levels in the liver as compared to baseline levels prior to treatment. In some embodiments, administering a therapeutically effective dose of a composition comprising a polynucleotide of the disclosure results in the expression of a functional BSEP protein level at or above about 10 ng / mg, about 20 ng / mg, about 50 ng / mg, about 100 ng / mg, about 150 ng / mg, about 200 ng / mg, about 250 ng / mg, about 300 ng / mg, about 350 ng / mg, about 400 ng / mg, about 450 ng / mg, about 500 ng / mg, about 600 ng / mg, about 700 ng / mg, about 800 ng / mg, about 900 ng / mg, about 1000 ng / mg, about 1200 ng / mg or about 1500 ng / mg of the total protein in the liver of a treated subject.

[0188] In some embodiments, administering a therapeutically effective dose of a composition comprising a polynucleotide encoding BSEP will result in reduced levels of bile acids in the peripheral blood. In some embodiments, administering a therapeutically effective dose of a composition comprising a polynucleotide encoding BSEP reduces bile acid levels inthe peripheral blood by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% as compared to bile acid levels in the peripheral blood before treatment.

[0189] A therapeutically effective dose of an active agent (e.g., a composition comprising a polynucleotide encoding BSEP) in vivo can be a dose of about 0.001 to about 500 mg / kg body weight. For instance, the therapeutically effective dose may be about 0.001-0.01 mg / kg body weight, or 0.01-0.1 mg / kg, or 0.1-1 mg / kg, or 1-10 mg / kg, or 10-100 mg / kg. In some embodiments, a composition comprising a polynucleotide encoding BSEP is provided at a dose ranging from about 0.01 to about 10 mg / kg body weight, e.g., from about 0.1 to about 5 mg / kg, from about 0.3 to about 4.5 mg / kg, or from about 0.5 to about 3 mg / kg.

[0190] A therapeutically effective dose of an active agent (e.g., a composition comprising a polynucleotide encoding BSEP) in vivo can be a dose of at least about 0.001 mg / kg body weight, or at least about 0.01 mg / kg, or at least about 0.1 mg / kg, or at least about 1 mg / kg, or at least about 2 mg / kg, or at least about 3 mg / kg, or at least about 4 mg / kg, or at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 50 mg / kg, or more. In some embodiments, a composition comprising a polynucleotide encoding BSEP is provided at a dose of about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 mg / kg. In an exemplary embodiment, a composition comprising a polynucleotide encoding BSEP is provided at a dose of about 0.3 mg / kg. In an exemplary embodiment, a composition comprising a polynucleotide encoding BSEP is provided at a dose of about 0.5 mg / kg. In yet another exemplary embodiment, a composition comprising a polynucleotide encoding BSEP is provided at a dose of about 1 mg / kg. In yet another exemplary embodiment, a composition comprising a polynucleotide encoding BSEP is provided at a dose of about 3 mg / kg.IX. Methods of Treatment

[0191] As described above, in some embodiments, the application relates to a method for ameliorating, preventing, delaying onset, or treating PFIC2 in a subject in need thereof, the method comprising administering to the subject a composition or a pharmaceutical formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and a pharmaceutically acceptable carrier. In some embodiments, the methodmay further comprise the administration of one or more additional therapeutics (z.e., a “second therapeutic agent”) before, simultaneous with, or after administration of the composition or pharmaceutical formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, and a pharmaceutically acceptable carrier. In some embodiments, a second therapeutic agent and the composition or pharmaceutical formulation may be administered simultaneously. In some embodiments, a second therapeutic agent and the composition or pharmaceutical formulation may be administered sequentially. In some embodiments, a second therapeutic agent and the composition or pharmaceutical formulation may be administered separately.

[0192] Methods for ameliorating, preventing, delaying onset, or treating PFIC2 in a subject in need thereof may comprise administering a composition or formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof in combination with an inhibitor of apical sodium-dependent bile acid transporter (ASBT, also known as ileal bile acid transporter or IBAT), i.e., an “IBAT inhibitor”. IBAT inhibitors may provide PFIC2 patients a benefit by blocking bile acid reuptake in the ileum in subjects, thereby reducing the size of the bile acid pool in subjects. The combined administration of an IBAT inhibitor with the pharmaceutical formulations comprising a polynucleotide encoding BSEP described herein may provide an additive benefit to PFIC2 patients by reducing bile acid load while also increasing bile acid efflux from hepatocytes into the bile, thereby alleviating or preventing cholestasis.

[0193] Some BSEP-deficient subjects (including, e.g., PFIC2 patients) are considered nonresponders to treatment with IBAT inhibitors due to their genetic mutations resulting in no residual or active BSEP protein. Similarly, some BSEP-deficient subjects have limited or low response to treatment with IBAT inhibitors; in other words, these subjects exhibit a muted or deficient response to a therapeutically effective dose of an IBAT inhibitor. Such subjects are referred to herein collectively as IBAT inhibitor non-responders. In some embodiments, IBAT inhibitor non-responders may exhibit a reduction of serum bile acid (sBA) of less than 30% or may achieve a total sBA concentration no less than about 70 pmol / L, no less than about 80 pmol / L, no less than about 90 pmol / L, no less than about 100 pmol / L, or no less than about 110 pmol / L after treatment with an IBAT inhibitor. (See, for example, Hupper, Maria Noelle, et al. "Surgical versus medical management of progressive familial intrahepatic cholestasis — case compilation and review of the literature." Children 10.6 (2023): 949; Nomden, Mark, et al. "Odevixibat treatment induces biliary bile acid secretion in responsive patients with bile saltexport pump deficiency." Gastroenterology 165.2 (2023): 496-498; Zhao, Xueheng, et al. "Serum bile acid profiling and mixed model analysis reveal biomarkers associated with pruritus reduction in maralixibat-treated patients with BSEP deficiency." Metabolites 12.10 (2022): 952.). Thus, increasing bile acid efflux from hepatocytes into the bile may improve the efficacy of IBAT inhibitors in blocking bile acid reuptake in the ileum of such IBAT inhibitor nonresponder subjects.

[0194] In this way, the compositions and formulations described herein comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof and their use in methods for ameliorating, preventing, delaying onset, or treating PFIC2 in a subject in need thereof may improve a subject’s response to treatment with an IBAT inhibitor. Thus, in some embodiments, administering a composition or formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof as provided herein to a subject who is an IBAT inhibitor non-responder may transform the subject to an IBAT inhibitor responder or otherwise may increase or improve the subject’s response to an IBAT inhibitor. Accordingly, provided herein are methods for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of BSEP protein in a subject in need thereof, the method comprising administering to the subject a composition or formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof, wherein the subject is an IBAT inhibitor non-responder. In some embodiments, the methods entail administering the composition or formulation comprising a polynucleotide comprising an mRNA coding sequence for the BSEP protein or a fragment thereof to a subject in combination with an IBAT inhibitor.

[0195] The combined treatment methods may comprise administering the composition or pharmaceutical formulation comprising the mRNA coding sequence for the BSEP protein or fragment thereof and any IBAT inhibitor. Examples of IBAT inhibitors are described, for example, in United States Patent Application Publication No. 20200330545A1. In some embodiments, the IBAT inhibitor for use in the combined treatment methods described herein is odevixibat (BYLAVY) or maralixibat (LIVMARLI). For instance, a therapeutically effective dose of the mRNA compositions or formulations described herein can be administered in combination with orally administered odevixibat or in combination with orally administered maralixibat. In some embodiments, the pharmaceutical formulation comprising a polynucleotide encoding BSEP can be administered at regular intervals such as daily, weekly, bi-weekly, monthly, bi-monthly, or quarterly, and the IBAT inhibitor can be administered, e.g.,daily or twice daily. In some embodiments, the pharmaceutical formulations comprising a polynucleotide encoding BSEP described herein may be administered to the subject intravenously at regular intervals and the IB AT inhibitor may be administered to the subject orally before, simultaneous with, or after administration of the pharmaceutical formulations comprising a polynucleotide encoding BSEP.

[0196] In addition to the disclosure of methods for ameliorating, preventing, delaying onset, or treating PFIC2 in a subject in need thereof, the compositions and pharmaceutical formulations of the disclosure may also find utility in treating a variety of other diseases or disorders characterized by BSEP deficiency.

[0197] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0198] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it will be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0199] Further, it will be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and disclosure(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the disclosure(s) described and depicted herein. All of the features disclosed in this specification may be combined in any combination.Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0200] It will be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects will be understood to have the same meaning unless otherwise understood from the context.

[0201] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, will be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0202] It will be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0203] The use of any and all examples, or exemplary language herein, for example, “such as” or “including” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification will be construed as indicating any non-claimed element as essential to the practice of the subject matter of the present disclosure.

[0204] It is understood that this disclosure is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be encompassed by the appended claims.EXAMPLES

[0205] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and is not intended to limit the disclosure.Example 1: Expression of BSEP Protein in Hepatocytes from ABCB11 mRNA

[0206] This example demonstrates that exogenous BSEP protein can be produced in cultured human hepatocellular carcinoma cells (HepG2 cells) following transfection oiABCBll mRNAusing a commercially available delivery agent (Lipofectamine MessengerMax).

[0207] In this example, HepG2 cells were seeded on 12-well plates at approximately 4 x 105cells / well the day before transfection. On the day of transfection, mRNA-lipid complexes were prepared following the manufacturer’s instructions. mRNA lipid-complexes were then added to respective control or treatment wells and incubated at 37°C for 48 hours. At 48 hours posttransfection, cells were collected and whole cell lysates were prepared, and protein was separated by SDS-PAGE. Protein was then transferred onto PVDF membranes, blocked for 1 hour, and contacted with anti-BSEP primary antibody at 4°C overnight. The following day, membranes were washed and then incubated in secondary antibody tagged with HRP for 1 hour, and ECL reagent was added to image protein using a ChemiDoc system. Blots were then stripped of antibodies using stripping buffer and blotting protocol was repeated using anti- GAPDH antibody. The ChemiDoc system was used to calculate the intensity of BSEP protein in samples normalized to GAPDH.

[0208] The expression in ng / pL for four synthetic ABCB11 sequences, SEQ ID NOs: 2-5 (modified to incorporate N1 -methylpseudouridine [Nlm'P] or 5-methoxyuridine [5M0U] in place of uridine) is shown in FIG. 1. In each instance, the transfected mRNA comprised SEQ ID NO: 2, 3, 4, or 5 plus 5’ cap, 5’ UTR (SEQ ID NO: 8), 3’ UTR (SEQ ID NO: 10), and a 80 bp 3’ polyA tail (SEQ ID NO: 22). All four synthetic ABCB11 mRNA sequences showed exogenous BSEP protein expression, with SEQ ID NO: 2 and SEQ ID NO: 3 showing the highest expression following transfection in HepG2 cells. In addition, amongst the ABCB11 mRNA sequences containing Nlm'P, SEQ ID NOs: 2 and 3 demonstrated a significantly longer duration (3.5 days and 3 days, respectively) than SEQ ID NOs: 4 and 5 (each 1 day).Example 2: Measurement of Full Length ABCB11 mRNA

[0209] The fraction of mRNA that is full length is referred to as mRNA integrity. IVT reactions were set up to evaluate the integrity of mRNA generated from plasmids comprising synthetic, codon-optimized versions of the BSEP coding sequence (SEQ ID NOs: 2-5). The plasmids utilized in this example were identical but for the BSEP coding sequence (SEQ ID NO: 2-5). A master mix solution containing all reagents excluding plasmid was prepared and aliquoted to mix with the plasmids. The IVT solution contained, among other components, each NTP, cap, plasmid, and T7 RNA polymerase. In each IVT run, Nl-methyl-pseudouridine [Nlm'P] or 5-methyoxyuridine [5M0U] was used in place of uridine. IVT runs were incubated at 37°C for 3 hours to allow mRNA synthesis. mRNA was purified by spin column, and integrity was assessed by fragment analyzer.

[0210] FIG. 1 summarizes fragment analyzer results which show that the codonoptimization impacted mRNA integrity differently. For both samples prepared with Nlm'P and 5M0U, SEQ ID NO: 2 exhibited the highest integrity amongst the BSEP-coding sequences evaluated.Example 3: BSEP Protein Expression in Liver of WT and Abcbll KO Mice

[0211] This example demonstrates that administering an mRNA encoding BSEP that is encapsulated in a lipid nanoparticle (LNP) can produce liver BSEP protein expression in wildtype (“WT”) and Abcbll knockout (“KO”) mice in a dose-dependent manner.

[0212] In this example, mRNA capable of encoding for human BSEP (SEQ ID NO: 19) was encapsulated in an LNP and administered to WT and Abcbll KO mice at 1 mg / kg and 3 mg / kg via tail vein injection. Approximately 48 hours after dosing, livers were harvested and assayed for BSEP levels.

[0213] Following the single dose of BSEP-encoding mRNA, dose-dependent BSEP protein expression was observed in mouse liver homogenates that were evaluated using an anti-BSEP immunoblot (FIG. 2 and FIG. 3).

[0214] Livers from Abcbll KO mice were fixed in 4% paraformaldehyde, embedded, and sectioned onto glass slides. Slides containing liver tissue were then rehydrated, blocked with 5% normal donkey serum in 0.3% PBS-Triton-X, and incubated overnight with a primary antibody against human BSEP. Slides were then washed and incubated with an appropriate secondary antibody conjugated to a fluorophore and coverslipped. Images of the stained liver sections were taken on a Zeiss Axiolmager M2 at 20X. FIG. 4 shows that human BSEP provided by the delivery of the ABCB11 mRNA-containing LNP is properly localized to the cell membrane of cells in the liver.Example 4: BSEP Protein is Expressed for At Least One Week After Dosing in WT and Abcbll KO Mice and a Decrease in Bile Acid Levels is Concurrent with Peak hBSEP Protein Expression

[0215] This example demonstrates that administering an mRNA encoding BSEP that is encapsulated in a lipid nanoparticle (LNP) can produce liver BSEP protein expression in Abcbll KO mice for at least one week, with a half-life of ~40 hours.

[0216] In this example, mRNA capable of encoding for human BSEP (SEQ ID NO: 19) was encapsulated in an LNP and administered to Abcbll KO mice at 4 mg / kg via tail vein injection. The liver was retrieved for analysis at 4, 12, 24, 48, 120, and 192 hours post-dosing.

[0217] Following the administration of the mRNA-LNP, BSEP protein expression was observed in mouse liver homogenates evaluated using an anti-BSEP immunoblot as shown inFIG. 5. LC-MS / MS was performed for taurine-conjugated beta muri cholic acid (T-bMCA), and a reduction of this bile acid species was observed at 12 hours post dose, concurrent with peak hBSEP protein expression as shown in FIG. 6.Example 5. Higher hBSEP expression in Abcbll KO mice administered LNP- encapsulated ABCB11 mRNA results in lower levels of total bile acid in the liver, and a significant decrease in the level of cholic acid is found in liver of treated mice versus control

[0218] This example demonstrates that higher levels of hBSEP protein in individual mice administered an mRNA capable of encoding for human BSEP (SEQ ID NO: 19) encapsulated in an LNP are inversely correlated with lower levels of total bile acid in the liver of Abcbll KO mice on bile acid supplemented diet (FIG. 7).

[0219] Administration of the mRNA capable of encoding for human BSEP (SEQ ID NO: 19) encapsulated in an LNP resulted in a significant decrease in cholic acid levels within the liver of treated Abcbll KO mice on bile acid supplemented diet compared to mice treated with control on the same bile acid supplemented diet (FIG. 8; p=0.0275, two-tailed t-test).

[0220] For this example, Abcbll KO mice were given a bile acid supplemented chow diet (0.1% cholic acid and 0.3% glychochenodeoxycholic acid) for five weeks and dosed with mRNA capable of encoding for human BSEP (SEQ ID NO: 19) encapsulated in an LNP or eGFP (control) at 4 mg / kg via tail vein injection. The liver of these mice was retrieved for analysis at 48 hours post-dosing. One lobe of the liver was processed and analyzed via multiple reaction monitoring (MRM), a mass spectrometry technique that enabled quantification of hBSEP derived from the mRNA-LNP administration. Another lobe of the liver was processed and analyzed by a multiplexing analytical mass spectrometry technique that enabled quantification of Cholic Acid bile acid species (inclusive of unconjugated cholic acid (CA), taurine-CA (T-CA), glycine-CA (G-CA), apocholic acid, 3-dehydro-CA, and norcholic acid) in liver homogenate tissue.Example 6. Sequences

[0221] SEQ ID NO: 1 (wild-type ABCB 11 mRNA)

[0222] AUGUCUGACUCAGUAAUUCUUCGAAGUAUAAAGAAAUUUGGAGAGGA GAAUGAUGGUUUUGAGUCAGAUAAAUCAUAUAAUAAUGAUAAGAAAUCAAGG UUACAAGAUGAGAAGAAAGGUGAUGGCGUUAGAGUUGGCUUCUUUCAAUUGU UUCGGUUUUCUUCAUCAACUGACAUUUGGCUGAUGUUUGUGGGAAGUUUGUG UGCAUUUCUCCAUGGAAUAGCCCAGCCAGGCGUGCUACUCAUUUUUGGCACAA UGACAGAUGUUUUUAUUGACUACGACGUUGAGUUACAAGAACUCCAGAUUCCAGGAAAAGCAUGUGUGAAUAACACCAUUGUAUGGACUAACAGUUCCCUCAACCAGAACAUGACAAAUGGAACACGUUGUGGGUUGCUGAACAUCGAGAGCGAAAUGAUCAAAUUUGCCAGUUACUAUGCUGGAAUUGCUGUCGCAGUACUUAUCACAGGAUAUAUUCAAAUAUGCUUUUGGGUCAUUGCCGCAGCUCGUCAGAUACAGAAAAUGAGAAAAUUUUACUUUAGGAGAAUAAUGAGAAUGGAAAUAGGGUGGUUUGACUGCAAUUCAGUGGGGGAGCUGAAUACAAGAUUCUCUGAUGAUAUUAAUAAAAUCAAUGAUGCCAUAGCUGACCAAAUGGCCCUUUUCAUUCAGCGCAUGACCUCGACCAUCUGUGGUUUCCUGUUGGGAUUUUUCAGGGGUUGGAAACUGACCUUGGUUAUUAUUUCUGUCAGCCCUCUCAUUGGGAUUGGAGCAGCCACCAUUGGUCUGAGUGUGUCCAAGUUUACGGACUAUGAGCUGAAGGCCUAUGCCAAAGCAGGGGUGGUGGCUGAUGAAGUCAUUUCAUCAAUGAGAACAGUGGCUGCUUUUGGUGGUGAGAAAAGAGAGGUUGAAAGGUAUGAGAAAAAUCUUGUGUUCGCCCAGCGUUGGGGAAUUAGAAAAGGAAUAGUGAUGGGAUUCUUUACUGGAUUCGUGUGGUGUCUCAUCUUUUUGUGUUAUGCACUGGCCUUCUGGUACGGCUCCACACUUGUCCUGGAUGAAGGAGAAUAUACACCAGGAACCCUUGUCCAGAUUUUCCUCAGUGUCAUAGUAGGAGCUUUAAAUCUUGGCAAUGCCUCUCCUUGUUUGGAAGCCUUUGCAACUGGACGUGCAGCAGCCACCAGCAUUUUUGAGACAAUAGACAGGAAACCCAUCAUUGACUGCAUGUCAGAAGAUGGUUACAAGUUGGAUCGAAUCAAGGGUGAAAUUGAAUUCCAUAAUGUGACCUUCCAUUAUCCUUCCAGACCAGAGGUGAAGAUUCUAAAUGACCUCAACAUGGUCAUUAAACCAGGGGAAAUGACAGCUCUGGUAGGACCCAGUGGAGCUGGAAAAAGUACAGCACUGCAACUCAUUCAGCGAUUCUAUGACCCCUGUGAAGGAAUGGUGACCGUGGAUGGCCAUGACAUUCGCUCUCUUAACAUUCAGUGGCUUAGAGAUCAGAUUGGGAUAGUGGAGCAAGAGCCAGUUCUGUUCUCUACCACCAUUGCAGAAAAUAUUCGCUAUGGCAGAGAAGAUGCAACAAUGGAAGACAUAGUCCAAGCUGCCAAGGAGGCCAAUGCCUACAACUUCAUCAUGGACCUGCCACAGCAAUUUGACACCCUUGUUGGAGAAGGAGGAGGCCAGAUGAGUGGUGGCCAGAAACAAAGGGUAGCUAUCGCCAGAGCCCUCAUCCGAAAUCCCAAGAUUCUGCUUUUGGACAUGGCCACCUCAGCUCUGGACAAUGAGAGUGAAGCCAUGGUGCAAGAAGUGCUGAGUAAGAUUCAGCAUGGGCACACAAUCAUUUCAGUUGCUCAUCGCUUGUCUACGGUCAGAGCUGCAGAUACCAUCAUUGGUUUUGAACAUGGCACUGCAGUGGAAAGAGGGACCCAUGAAGAAUUACUGGAAAGGAAAGGUGUUUACUUCACUCUAGUGACUUUGCAAAGCCAGGGAAAUCAAGCUCUUAAUGAAGAGGACAUAAAGGAUGCAACUGAAGAUGACAUGCUUGCGAGGACCUUUAGCAGAGGGAGCUACCAGGAUAGUUUAAGGGCUUCCAUCCGGCAACGCUCCAAGUCUCAGCUUUCUUACCUGGUGCACGAACCUCCAUUAGCUGUUGUAGAUCAUAAGUCUACCUAUGAAGAAGAUAGAAAGGACAAGGACAUUCCUGUGCAGGAAGAAGUUGAACCUGCCCCAGUUAGGAGGAUUCUGAAAUUCAGUGCUCCAGAAUGGCCCUACAUGCUGGUAGGGUCUGUGGGUGCAGCUGUGAACGGGACAGUCACACCCUUGUAUGCCUUUUUAUUCAGCCAGAUUCUUGGGACUUUUUCAAUUCCUGAUAAAGAGGAACAAAGGUCACAGAUCAAUGGUGUGUGCCUACUUUUUGUAGCAAUGGGCUGUGUAUCUCUUUUCACCCAAUUUCUACAGGGAUAUGCCUUUGCUAAAUCUGGGGAGCUCCUAACAAAAAGGCUACGUAAAUUUGGUUUCAGGGCAAUGCUGGGGCAAGAUAUUGCCUGGUUUGAUGACCUCAGAAAUAGCCCUGGAGCAUUGACAACAAGACUUGCUACAGAUGCUUCCCAAGUUCAAGGGGCUGCCGGCUCUCAGAUCGGGAUGAUAGUCAAUUCCUUCACUAACGUCACUGUGGCCAUGAUCAUUGCCUUCUCCUUUAGCUGGAAGCUGAGCCUGGUCAUCUUGUGCUUCUUCCCCUUCUUGGCUUUAUCAGGAGCCACACAGACCAGGAUGUUGACAGGAUUUGCCUCUCGAGAUAAGCAGGCCCUGGAGAUGGUGGGACAGAUUACAAAUGAAGCCCUCAGUAACAUCCGCACUGUUGCUGGAAUUGGAAAGGAGAGGCGGUUCAUUGAAGCACUUGAGACUGAGCUGGAGAAGCCCUUCAAGACAGCCAUUCAGAAAGCCAAUAUUUACGGAUUCUGCUUUGCCUUUGCCCAGUGCAUCAUGUUUAUUGCGAAUUCUGCUUCCUACAGAUAUGGAGGUUACUUAAUCUCCAAUGAGGGGCUCCAUUUCAGCUAUGUGUUCAGGGUGAUCUCUGCAGUUGUACUGAGUGCAACAGCUCUUGGAAGAGCCUUCUCUUACACCCCAAGUUAUGCAAAAGCUAAAAUAUCAGCUGCACGCUUUUUUCAACUGCUGGACCGACAACCCCCAAUCAGUGUAUACAAUACUGCAGGUGAAAAAUGGGACAACUUCCAGGGGAAGAUUGAUUUUGUUGAUUGUAAAUUUACAUAUCCUUCUCGACCUGACUCGCAAGUUCUGAAUGGUCUCUCAGUGUCGAUUAGUCCAGGGCAGACACUGGCGUUUGUUGGGAGCAGUGGAUGUGGCAAAAGCACUAGCAUUCAGCUGUUGGAACGUUUCUAUGAUCCUGAUCAAGGGAAGGUGAUGAUAGAUGGUCAUGACAGCAAAAAAGUAAAUGUCCAGUUCCUCCGCUCAAACAUUGGAAUUGUUUCCCAGGAACCAGUGUUGUUUGCCUGUAGCAUAAUGGACAAUAUCAAGUAUGGAGACAACACCAAAGAAAUUCCCAUGGAAAGAGUCAUAGCAGCUGCAAAACAGGCUCAGCUGCAUGAUUUUGUCAUGUCACUCCCAGAGAAAUAUGAAACUAACGUUGGGUCCCAGGGGUCUCAACUCUCUAGAGGGGAGAAACAACGCAUUGCUAUUGCUCGGGCCAUUGUACGAGAUCCUAAAAUCUUGCUACUAGAUGAAGCCACUUCUGCCUUAGACACAGAAAGUGAAAAGACGGUGCAGGUUGCUCUAGACAAAGCCAGAGAGGGUCGGACCUGCAUUGUCAUUGCCCAUCGCUUGUCCACCAUCCAGAACGCGGAUAUCAUUGCUGUCAUGGCACAGGGGGUGGUGAUUGAAAAGGGGACCCAUGAAGAACUGAUGGCCCAAAAAGGAGCCUACUACAAACUAGUCACCACUGGAUCCCCCAUCAGU

[0223] SEQ ID NO: 2 (Codon-Optimized mRNA - ABCB 11 mRNA LowU)

[0224] AUGAGCGACAGCGUGAUCCUGCGGAGCAUCAAGAAGUUCGGCGAGGAGAACGACGGCUUCGAGAGCGACAAGAGCUACAACAACGACAAGAAAAGCCGGCUGCAGGACGAGAAGAAGGGCGACGGCGUGCGGGUGGGCUUCUUCCAGCUGUUCCGGUUCAGCAGCAGCACCGACAUCUGGCUGAUGUUCGUGGGCAGCCUGUGCGCCUUCCUGCACGGCAUCGCCCAGCCCGGCGUGCUGCUGAUCUUCGGCACCAUGACCGACGUGUUCAUCGACUACGACGUGGAGCUGCAGGAGCUGCAGAUCCCCGGCAAGGCCUGCGUGAACAACACCAUCGUGUGGACCAACAGCAGCCUGAACCAGAACAUGACCAACGGCACCCGGUGCGGCCUGCUGAACAUCGAGAGCGAGAUGAUCAAGUUCGCCAGCUACUACGCCGGCAUCGCCGUGGCCGUGCUGAUCACCGGCUACAUCCAGAUCUGCUUCUGGGUGAUCGCCGCCGCCCGGCAGAUCCAGAAGAUGCGGAAGUUCUACUUCCGGCGGAUCAUGCGGAUGGAGAUCGGCUGGUUCGACUGCAACAGCGUGGGCGAGCUGAACACCCGGUUCAGCGACGACAUCAACAAGAUCAACGACGCCAUCGCCGACCAGAUGGCCCUGUUCAUCCAGCGGAUGACCAGCACCAUCUGCGGCUUCCUGCUGGGCUUCUUCCGGGGCUGGAAGCUGACCCUGGUGAUCAUCAGCGUGAGCCCCCUGAUCGGCAUCGGCGCCGCCACCAUCGGCCUGAGCGUGAGCAAGUUCACCGACUACGAGCUGAAGGCCUACGCCAAGGCCGGCGUGGUGGCCGACGAGGUGAUCAGCAGCAUGCGGACCGUGGCCGCCUUCGGCGGCGAGAAGCGGGAGGUGGAGCGGUACGAGAAGAACCUGGUGUUCGCCCAGCGGUGGGGCAUCCGGAAGGGCAUCGUGAUGGGCUUCUUCACCGGCUUCGUGUGGUGCCUGAUCUUCCUGUGCUACGCCCUGGCCUUCUGGUACGGCAGCACCCUGGUGCUGGACGAGGGCGAGUACACCCCCGGCACCCUGGUGCAGAUCUUCCUGAGCGUGAUCGUGGGCGCCCUGAACCUGGGCAACGCCAGCCCCUGCCUGGAGGCCUUCGCCACCGGCCGGGCCGCCGCCACCAGCAUCUUCGAGACCAUCGACCGGAAGCCCAUCAUCGACUGCAUGAGCGAGGACGGCUACAAGCUGGACCGGAUCAAGGGCGAGAUCGAGUUCCACAACGUGACCUUCCACUACCCCAGCCGGCCCGAGGUGAAGAUCCUGAACGACCUGAACAUGGUGAUCAAGCCCGGCGAGAUGACCGCCCUGGUGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCUGCAGCUGAUCCAGCGGUUCUACGACCCCUGCGAGGGCAUGGUGACCGUGGACGGCCACGACAUCCGGAGCCUGAACAUCCAGUGGCUGCGGGACCAGAUCGGCAUCGUGGAGCAGGAGCCCGUGCUGUUCAGCACCACCAUCGCCGAGAACAUCCGGUACGGCCGGGAGGACGCCACCAUGGAGGACAUCGUGCAGGCCGCCAAGGAGGCCAACGCCUACAACUUCAUCAUGGACCUGCCCCAGCAGUUCGACACCCUGGUGGGCGAGGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGCGGGUGGCCAUCGCCCGGGCCCUGAUCCGGAACCCCAAGAUCCUGCUGCUGGACAUGGCCACCAGCGCCCUGGACAACGAGAGCGAGGCCAUGGUGCAGGAGGUGCUGAGCAAGAUCCAGCACGGCCACACCAUCAUCAGCGUGGCCCACCGGCUGAGCACCGUGCGGGCCGCCGACACCAUCAUCGGCUUCGAGCACGGCACCGCCGUGGAGCGGGGCACCCACGAGGAGCUGCUGGAGCGGAAGGGCGUGUACUUCACCCUGGUGACCCUGCAGAGCCAGGGCAACCAGGCCCUGAACGAGGAGGACAUCAAGGACGCCACCGAGGACGACAUGCUGGCCCGGACCUUCAGCCGGGGCAGCUACCAGGACAGCCUGCGGGCCAGCAUCCGGCAGCGGAGCAAGAGCCAGCUGAGCUACCUGGUGCACGAGCCCCCCCUGGCCGUGGUGGACCACAAGAGCACCUACGAGGAGGACCGGAAGGACAAGGACAUCCCCGUGCAGGAGGAGGUGGAGCCCGCCCCCGUGCGGCGGAUCCUGAAGUUCAGCGCCCCCGAGUGGCCCUACAUGCUGGUGGGCAGCGUGGGCGCCGCCGUGAACGGCACCGUGACCCCCCUGUACGCCUUCCUGUUCAGCCAGAUCCUGGGCACCUUCAGCAUCCCCGACAAGGAGGAGCAGCGGAGCCAGAUCAACGGCGUGUGCCUGCUGUUCGUGGCCAUGGGCUGCGUGAGCCUGUUCACCCAGUUCCUGCAGGGCUACGCCUUCGCCAAGAGCGGCGAGCUGCUGACCAAGCGGCUGCGGAAGUUCGGCUUCCGGGCCAUGCUGGGCCAGGACAUCGCCUGGUUCGACGACCUGCGGAACAGCCCCGGCGCCCUGACCACCCGGCUGGCCACCGACGCCAGCCAGGUGCAGGGCGCCGCCGGCAGCCAGAUCGGCAUGAUCGUGAACAGCUUCACCAACGUGACCGUGGCCAUGAUCAUCGCCUUCAGCUUCAGCUGGAAGCUGAGCCUGGUGAUCCUGUGCUUCUUCCCCUUCCUGGCCCUGAGCGGCGCCACCCAGACCCGGAUGCUGACCGGCUUCGCCAGCCGGGACAAGCAGGCCCUGGAGAUGGUGGGCCAGAUCACCAACGAGGCCCUGAGCAACAUCCGGACCGUGGCCGGCAUCGGCAAGGAGCGGCGGUUCAUCGAGGCCCUGGAGACCGAGCUGGAGAAGCCCUUCAAGACCGCCAUCCAGAAGGCCAACAUCUACGGCUUCUGCUUCGCCUUCGCCCAGUGCAUCAUGUUCAUCGCCAACAGCGCCAGCUACCGGUACGGCGGCUACCUGAUCAGCAACGAGGGCCUGCACUUCAGCUACGUGUUCCGGGUGAUCAGCGCCGUGGUGCUGAGCGCCACCGCCCUGGGCCGGGCCUUCAGCUACACCCCCAGCUACGCCAAGGCCAAGAUCAGCGCCGCCCGGUUCUUCCAGCUGCUGGACCGGCAGCCCCCCAUCAGCGUGUACAACACCGCCGGCGAGAAGUGGGACAACUUCCAGGGCAAGAUCGACUUCGUGGACUGCAAGUUCACCUACCCCAGCCGGCCCGACAGCCAGGUGCUGAACGGCCUGAGCGUGAGCAUCAGCCCCGGCCAGACCCUGGCCUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACCAGCAUCCAGCUGCUGGAGCGGUUCUACGACCCCGACCAGGGCAAGGUGAUGAUCGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUGCGGAGCAACAUCGGCAUCGUGAGCCAGGAGCCCGUGCUGUUCGCCUGCAGCAUCAUGGACAACAUCAAGUACGGCGACAACACCAAGGAGAUCCCCAUGGAGCGGGUGAUCGCCGCCGCCAAGCAGGCCCAGCUGCACGACUUCGUGAUGAGCCUGCCCGAGAAGUACGAGACCAACGUGGGCAGCCAGGGCAGCCAGCUGAGCCGGGGCGAGAAGCAGCGGAUCGCCAUCGCCCGGGCCAUCGUGCGGGACCCCAAGAUCCUGCUGCUGGACGAGGCCACCAGCGCCCUGGACACCGAGAGCGAAAAGACCGUGCAGGUGGCCCUGGACAAGGCCCGGGAGGGCCGGACCUGCAUCGUGAUCGCCCACCGGCUGAGCACCAUCCAGAACGCCGACAUCAUCGCCGUGAUGGCCCAGGGCGUGGUGAUCGAGAAGGGCACCCACGAGGAGCUGAUGGCCCAGAAGGGCGCCUACUACAAGCUGGUGACCACCGGCAGCCCCAUCAGC

[0225] SEQ ID NO: 3 (Codon-Optimized mRNA - ABCB11 human ramp)

[0226] AUGAGCGAUUCGGUAAUACUACGUUCGAUAAAAAAAUUUGGUGAAGAAAAUGAUGGUUUUGAAUCGGAUAAAUCGUAUAAUAAUGAUAAAAAAUCGCGUCUACAAGAUGAAAAAAAAGGUGAUGGUGUACGUGUAGGUUUUUUUCAACUAUUCCGGUUCAGCAGCAGCACCGACAUCUGGCUGAUGUUCGUGGGCAGCCUGUGCGCCUUCCUGCACGGCAUCGCCCAGCCCGGCGUGCUGCUGAUCUUCGGCACCAUGACCGACGUGUUCAUCGACUACGACGUGGAGCUGCAGGAGCUGCAGAUCCCCGGCAAGGCCUGCGUGAACAACACCAUCGUGUGGACCAACAGCAGCCUGAACCAGAACAUGACCAACGGCACCCGGUGCGGCCUGCUGAACAUCGAGAGCGAGAUGAUCAAGUUCGCCAGCUACUACGCCGGCAUCGCCGUGGCCGUGCUGAUCACCGGCUACAUCCAGAUCUGCUUCUGGGUGAUCGCCGCCGCCCGGCAGAUCCAGAAGAUGCGGAAGUUCUACUUCCGGCGGAUCAUGCGGAUGGAGAUCGGCUGGUUCGACUGCAACAGCGUGGGCGAGCUGAACACCCGGUUCAGCGACGACAUCAACAAGAUCAACGACGCCAUCGCCGACCAGAUGGCCCUGUUCAUCCAGCGGAUGACCAGCACCAUCUGCGGCUUCCUGCUGGGCUUCUUCCGGGGCUGGAAGCUGACCCUGGUGAUCAUCAGCGUGAGCCCCCUGAUCGGCAUCGGCGCCGCCACCAUCGGCCUGAGCGUGAGCAAGUUCACCGACUACGAGCUGAAGGCCUACGCCAAGGCCGGCGUGGUGGCCGACGAGGUGAUCAGCAGCAUGCGGACCGUGGCCGCCUUCGGCGGCGAGAAGCGGGAGGUGGAGCGGUACGAGAAGAACCUGGUGUUCGCCCAGCGGUGGGGCAUCCGGAAGGGCAUCGUGAUGGGCUUCUUCACCGGCUUCGUGUGGUGCCUGAUCUUCCUGUGCUACGCCCUGGCCUUCUGGUACGGCAGCACCCUGGUGCUGGACGAGGGCGAGUACACCCCCGGCACCCUGGUGCAGAUCUUCCUGAGCGUGAUCGUGGGCGCCCUGAACCUGGGCAACGCCAGCCCCUGCCUGGAGGCCUUCGCCACCGGCCGGGCCGCCGCCACCAGCAUCUUCGAGACCAUCGACCGGAAGCCCAUCAUCGACUGCAUGAGCGAGGACGGCUACAAGCUGGACCGGAUCAAGGGCGAGAUCGAGUUCCACAACGUGACCUUCCACUACCCCAGCCGGCCCGAGGUGAAGAUCCUGAACGACCUGAACAUGGUGAUCAAGCCCGGCGAGAUGACCGCCCUGGUGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCUGCAGCUGAUCCAGCGGUUCUACGACCCCUGCGAGGGCAUGGUGACCGUGGACGGCCACGACAUCCGGAGCCUGAACAUCCAGUGGCUGCGGGACCAGAUCGGCAUCGUGGAGCAGGAGCCCGUGCUGUUCAGCACCACCAUCGCCGAGAACAUCCGGUACGGCCGGGAGGACGCCACCAUGGAGGACAUCGUGCAGGCCGCCAAGGAGGCCAACGCCUACAACUUCAUCAUGGACCUGCCCCAGCAGUUCGACACCCUGGUGGGCGAGGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGCGGGUGGCCAUCGCCCGGGCCCUGAUCCGGAACCCCAAGAUCCUGCUGCUGGACAUGGCCACCAGCGCCCUGGACAACGAGAGCGAGGCCAUGGUGCAGGAGGUGCUGAGCAAGAUCCAGCACGGCCACACCAUCAUCAGCGUGGCCCACCGGCUGAGCACCGUGCGGGCCGCCGACACCAUCAUCGGCUUCGAGCACGGCACCGCCGUGGAGCGGGGCACCCACGAGGAGCUGCUGGAGCGGAAGGGCGUGUACUUCACCCUGGUGACCCUGCAGAGCCAGGGCAACCAGGCCCUGAACGAGGAGGACAUCAAGGACGCCACCGAGGACGACAUGCUGGCCCGGACCUUCAGCCGGGGCAGCUACCAGGACAGCCUGCGGGCCAGCAUCCGGCAGCGGAGCAAGAGCCAGCUGAGCUACCUGGUGCACGAGCCCCCCCUGGCCGUGGUGGACCACAAGAGCACCUACGAGGAGGACCGGAAGGACAAGGACAUCCCCGUGCAGGAGGAGGUGGAGCCCGCCCCCGUGCGGCGGAUCCUGAAGUUCAGCGCCCCCGAGUGGCCCUACAUGCUGGUGGGCAGCGUGGGCGCCGCCGUGAACGGCACCGUGACCCCCCUGUACGCCUUCCUGUUCAGCCAGAUCCUGGGCACCUUCAGCAUCCCCGACAAGGAGGAGCAGCGGAGCCAGAUCAACGGCGUGUGCCUGCUGUUCGUGGCCAUGGGCUGCGUGAGCCUGUUCACCCAGUUCCUGCAGGGCUACGCCUUCGCCAAGAGCGGCGAGCUGCUGACCAAGCGGCUGCGGAAGUUCGGCUUCCGGGCCAUGCUGGGCCAGGACAUCGCCUGGUUCGACGACCUGCGGAACAGCCCCGGCGCCCUGACCACCCGGCUGGCCACCGACGCCAGCCAGGUGCAGGGCGCCGCCGGCAGCCAGAUCGGCAUGAUCGUGAACAGCUUCACCAACGUGACCGUGGCCAUGAUCAUCGCCUUCAGCUUCAGCUGGAAGCUGAGCCUGGUGAUCCUGUGCUUCUUCCCCUUCCUGGCCCUGAGCGGCGCCACCCAGACCCGGAUGCUGACCGGCUUCGCCAGCCGGGACAAGCAGGCCCUGGAGAUGGUGGGCCAGAUCACCAACGAGGCCCUGAGCAACAUCCGGACCGUGGCCGGCAUCGGCAAGGAGCGGCGGUUCAUCGAGGCCCUGGAGACCGAGCUGGAGAAGCCCUUCAAGACCGCCAUCCAGAAGGCCAACAUCUACGGCUUCUGCUUCGCCUUCGCCCAGUGCAUCAUGUUCAUCGCCAACAGCGCCAGCUACCGGUACGGCGGCUACCUGAUCAGCAACGAGGGCCUGCACUUCAGCUACGUGUUCCGGGUGAUCAGCGCCGUGGUGCUGAGCGCCACCGCCCUGGGCCGGGCCUUCAGCUACACCCCCAGCUACGCCAAGGCCAAGAUCAGCGCCGCCCGGUUCUUCCAGCUGCUGGACCGGCAGCCCCCCAUCAGCGUGUACAACACCGCCGGCGAGAAGUGGGACAACUUCCAGGGCAAGAUCGACUUCGUGGACUGCAAGUUCACCUACCCCAGCCGGCCCGACAGCCAGGUGCUGAACGGCCUGAGCGUGAGCAUCAGCCCCGGCCAGACCCUGGCCUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACCAGCAUCCAGCUGCUGGAGCGGUUCUACGACCCCGACCAGGGCAAGGUGAUGAUCGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUGCGGAGCAACAUCGGCAUCGUGAGCCAGGAGCCCGUGCUGUUCGCCUGCAGCAUCAUGGACAACAUCAAGUACGGCGACAACACCAAGGAGAUCCCCAUGGAGCGGGUGAUCGCCGCCGCCAAGCAGGCCCAGCUGCACGACUUCGUGAUGAGCCUGCCCGAGAAGUACGAGACCAACGUGGGCAGCCAGGGCAGCCAGCUGAGCCGGGGCGAGAAGCAGCGGAUCGCCAUCGCCCGGGCCAUCGUGCGGGACCCCAAGAUCCUGCUGCUGGACGAGGCCACCAGCGCCCUGGACACCGAGAGCGAAAAGACCGUGCAGGUGGCCCUGGACAAGGCCCGGGAGGGCCGGACCUGCAUCGUGAUCGCCCACCGGCUGAGCACCAUCCAGAACGCCGACAUCAUCGCCGUGAUGGCCCAGGGCGUGGUGAUCGAGAAGGGCACCCACGAGGAGCUGAUGGCCCAGAAGGGCGCCUACUACAAGCUGGUGACCACCGGCAGCCCCAUCAGC

[0227] SEQ ID NO: 4 (Codon-Optimized mRNA - ABCB11 human_opt2)

[0228] AUGUCUGACUCUGUGAUUCUUCGUAGCAUUAAGAAGUUCGGCGAAGAAAACGACGGCUUCGAAUCUGACAAGUCUUACAACAACGACAAGAAGUCUAGACUUCAGGACGAAAAGAAGGGCGACGGCGUGAGAGUGGGCUUCUUCCAGCUGUUCCGUUUCUCUUCUUCUACUGACAUUUGGCUGAUGUUCGUGGGCAGCCUGUGCGCUUUCCUGCACGGCAUUGCUCAGCCUGGCGUGCUUCUGAUUUUCGGCACAAUGACAGACGUGUUCAUUGACUACGACGUGGAACUUCAGGAACUGCAGAUUCCUGGCAAGGCUUGCGUGAACAACACUAUUGUGUGGACUAACAGCUCUCUGAACCAGAACAUGACAAACGGCACACGUUGCGGCCUGCUGAACAUUGAAAGCGAAAUGAUUAAGUUCGCUAGCUACUACGCUGGCAUUGCUGUGGCUGUGCUUAUUACAGGCUACAUUCAGAUUUGCUUCUGGGUGAUUGCUGCUGCUCGUCAGAUUCAGAAGAUGAGAAAGUUCUACUUCAGAAGAAUUAUGAGAAUGGAAAUUGGCUGGUUCGACUGCAACUCUGUGGGCGAACUGAACACAAGAUUCUCUGACGACAUUAACAAGAUUAACGACGCUAUUGCUGACCAGAUGGCUCUUUUCAUUCAGCGUAUGACUUCUACUAUUUGCGGCUUCCUGCUGGGCUUCUUCAGAGGCUGGAAGCUGACUCUGGUGAUUAUUUCUGUGAGCCCUCUGAUUGGCAUUGGCGCUGCUACUAUUGGCCUGAGCGUGUCUAAGUUCACAGACUACGAACUGAAGGCUUACGCUAAGGCUGGCGUGGUGGCUGACGAAGUGAUUUCUUCUAUGAGAACAGUGGCUGCUUUCGGCGGCGAAAAGAGAGAAGUGGAAAGAUACGAAAAGAACCUUGUGUUCGCUCAGCGUUGGGGCAUUAGAAAGGGCAUUGUGAUGGGCUUCUUCACUGGCUUCGUGUGGUGCCUGAUUUUCCUGUGCUACGCUCUGGCUUUCUGGUACGGCUCUACACUUGUGCUGGACGAAGGCGAAUACACACCUGGCACUCUUGUGCAGAUUUUCCUGAGCGUGAUUGUGGGCGCUCUUAACCUUGGCAACGCUUCUCCUUGCCUGGAAGCUUUCGCUACUGGCCGUGCUGCUGCUACUAGCAUUUUCGAAACAAUUGACAGAAAGCCUAUUAUUGACUGCAUGUCUGAGGACGGCUACAAGCUGGACCGUAUUAAGGGCGAAAUUGAAUUCCACAACGUGACUUUCCACUACCCUUCUAGACCUGAAGUGAAGAUUCUUAACGACCUGAACAUGGUGAUUAAGCCUGGCGAAAUGACAGCUCUGGUGGGCCCUAGCGGCGCUGGCAAGAGCACAGCUCUGCAGCUGAUUCAGCGUUUCUACGACCCUUGCGAAGGCAUGGUGACUGUGGACGGCCACGACAUUCGUUCUCUUAACAUUCAGUGGCUUAGAGACCAGAUUGGCAUUGUGGAACAGGAACCUGUGCUGUUCUCUACUACUAUUGCUGAAAACAUUCGUUACGGCAGAGAGGACGCUACAAUGGAGGACAUUGUGCAGGCUGCUAAGGAAGCUAACGCUUACAACUUCAUUAUGGACCUGCCUCAGCAGUUCGACACUCUUGUGGGCGAAGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGAGAGUGGCUAUUGCUAGAGCUCUGAUUCGUAACCCUAAGAUUCUGCUUCUGGACAUGGCUACUUCUGCUCUGGACAACGAAAGCGAAGCUAUGGUGCAGGAAGUGCUGAGCAAGAUUCAGCACGGCCACACAAUUAUUUCUGUGGCUCACCGUCUGUCUACAGUGAGAGCUGCUGACACUAUUAUUGGCUUCGAACACGGCACUGCUGUGGAAAGAGGCACUCACGAAGAACUUCUGGAAAGAAAGGGCGUGUACUUCACUCUUGUGACUCUGCAGAGCCAGGGCAACCAGGCUCUUAACGAAGAGGACAUUAAGGACGCUACUGAGGACGACAUGCUUGCUAGAACUUUCAGCAGAGGCAGCUACCAGGACAGCCUUAGAGCUUCUAUUCGUCAGCGUUCUAAGUCUCAGCUUUCUUACCUGGUGCACGAACCUCCUCUUGCUGUGGUGGACCACAAGUCUACUUACGAAGAGGACAGAAAGGACAAGGACAUUCCUGUGCAGGAAGAAGUGGAACCUGCUCCUGUGAGAAGAAUUCUGAAGUUCAGCGCUCCUGAAUGGCCUUACAUGCUGGUGGGCUCUGUGGGCGCUGCUGUGAACGGCACAGUGACACCUCUGUACGCUUUCCUUUUCAGCCAGAUUCUUGGCACUUUCUCUAUUCCUGACAAGGAAGAACAGAGAUCUCAGAUUAACGGCGUGUGCCUUCUUUUCGUGGCUAUGGGCUGCGUGUCUCUUUUCACUCAGUUCCUUCAGGGCUACGCUUUCGCUAAGUCUGGCGAACUGCUUACAAAGAGACUUCGUAAGUUCGGCUUCAGAGCUAUGCUGGGCCAGGACAUUGCUUGGUUCGACGACCUGAGAAACAGCCCUGGCGCUCUGACAACAAGACUUGCUACAGACGCUUCUCAGGUGCAGGGCGCUGCUGGCUCUCAGAUUGGCAUGAUUGUGAACUCUUUCACUAACGUGACUGUGGCUAUGAUUAUUGCUUUCUCUUUCAGCUGGAAGCUGAGCCUGGUGAUUCUGUGCUUCUUCCCUUUCCUGGCUCUUUCUGGCGCUACACAGACUAGAAUGCUGACAGGCUUCGCUUCUCGUGACAAGCAGGCUCUGGAAAUGGUGGGCCAGAUUACAAACGAAGCUCUGAGCAACAUUCGUACUGUGGCUGGCAUUGGCAAGGAAAGACGUUUCAUUGAAGCUCUUGAAACUGAACUGGAAAAGCCUUUCAAGACAGCUAUUCAGAAGGCUAACAUUUACGGCUUCUGCUUCGCUUUCGCUCAGUGCAUUAUGUUCAUUGCUAACUCUGCUUCUUACAGAUACGGCGGCUACCUUAUUUCUAACGAAGGCCUGCACUUCAGCUACGUGUUCAGAGUGAUUUCUGCUGUGGUGCUGAGCGCUACAGCUCUUGGCAGAGCUUUCUCUUACACUCCUAGCUACGCUAAGGCUAAGAUUUCUGCUGCUCGUUUCUUCCAGCUGCUGGACCGUCAGCCUCCUAUUAGCGUGUACAACACUGCUGGCGAAAAGUGGGACAACUUCCAGGGCAAGAUUGACUUCGUGGACUGCAAGUUCACAUACCCUUCUCGUCCUGACUCUCAGGUGCUGAACGGCCUGUCUGUGUCUAUUAGCCCUGGCCAGACACUGGCUUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACUAGCAUUCAGCUGCUGGAACGUUUCUACGACCCUGACCAGGGCAAGGUGAUGAUUGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUGCGUUCUAACAUUGGCAUUGUGUCUCAGGAACCUGUGCUGUUCGCUUGCAGCAUUAUGGACAACAUUAAGUACGGCGACAACACUAAGGAAAUUCCUAUGGAAAGAGUGAUUGCUGCUGCUAAGCAGGCUCAGCUGCACGACUUCGUGAUGUCUCUGCCUGAAAAGUACGAAACUAACGUGGGCUCUCAGGGCUCUCAGCUGUCUAGAGGCGAAAAGCAGCGUAUUGCUAUUGCUCGUGCUAUUGUGCGUGACCCUAAGAUUCUGCUUCUUGACGAAGCUACUUCUGCUCUUGACACAGAAAGCGAAAAGACAGUGCAGGUGGCUCUUGACAAGGCUAGAGAAGGCCGUACUUGCAUUGUGAUUGCUCACCGUCUGUCUACUAUUCAGAACGCUGACAUUAUUGCUGUGAUGGCUCAGGGCGUGGUGAUUGAAAAGGGCACUCACGAAGAACUGAUGGCUCAGAAGGGCGCUUACUACAAGCUUGUGACUACUGGCUCUCCUAUUAGC

[0229] SEQ ID NO: 5 (Codon-Optimized mRNA - ABCB 11 mRNA_LowU2)

[0230] AUGAGCGACAGCGUGAUCCUCAGGAGCAUCAAGAAGUUCGGCGAAGAAAACGACGGCUUCGAAAGCGACAAGAGCUACAACAACGACAAGAAGAGCAGGCUCCAGGACGAAAAGAAGGGCGACGGCGUGAGGGUGGGCUUCUUCCAGCUCUUCAGGUUCAGCAGCAGCACAGACAUCUGGCUCAUGUUCGUGGGCAGCCUCUGCGCCUUCCUCCACGGCAUCGCCCAGCCAGGCGUGCUCCUCAUCUUCGGCACAAUGACAGACGUGUUCAUCGACUACGACGUGGAACUCCAGGAACUCCAGAUCCCAGGCAAGGCCUGCGUGAACAACACAAUCGUGUGGACAAACAGCAGCCUCAACCAGAACAUGACAAACGGCACAAGGUGCGGCCUCCUCAACAUCGAAAGCGAAAUGAUCAAGUUCGCCAGCUACUACGCCGGCAUCGCCGUGGCCGUGCUCAUCACAGGCUACAUCCAGAUCUGCUUCUGGGUGAUCGCCGCCGCCAGGCAGAUCCAGAAGAUGAGGAAGUUCUACUUCAGGAGGAUCAUGAGGAUGGAAAUCGGCUGGUUCGACUGCAACAGCGUGGGCGAACUCAACACAAGGUUCAGCGACGACAUCAACAAGAUCAACGACGCCAUCGCCGACCAGAUGGCCCUCUUCAUCCAGAGGAUGACAAGCACAAUCUGCGGCUUCCUCCUCGGCUUCUUCAGGGGCUGGAAGCUCACACUCGUGAUCAUCAGCGUGAGCCCACUCAUCGGCAUCGGCGCCGCCACAAUCGGCCUCAGCGUGAGCAAGUUCACAGACUACGAACUCAAGGCCUACGCCAAGGCCGGCGUGGUGGCCGACGAAGUGAUCAGCAGCAUGAGGACAGUGGCCGCCUUCGGCGGCGAAAAGAGGGAAGUGGAAAGGUACGAAAAGAACCUCGUGUUCGCCCAGAGGUGGGGCAUCAGGAAGGGCAUCGUGAUGGGCUUCUUCACAGGCUUCGUGUGGUGCCUCAUCUUCCUCUGCUACGCCCUCGCCUUCUGGUACGGCAGCACACUCGUGCUCGACGAAGGCGAAUACACACCAGGCACACUCGUGCAGAUCUUCCUCAGCGUGAUCGUGGGCGCCCUCAACCUCGGCAACGCCAGCCCAUGCCUCGAAGCCUUCGCCACAGGCAGGGCCGCCGCCACAAGCAUCUUCGAAACAAUCGACAGGAAGCCAAUCAUCGACUGCAUGAGCGAGGACGGCUACAAGCUCGACAGGAUCAAGGGCGAAAUCGAAUUCCACAACGUGACAUUCCACUACCCAAGCAGGCCAGAAGUGAAGAUCCUCAACGACCUCAACAUGGUGAUCAAGCCAGGCGAAAUGACAGCCCUCGUGGGCCCAAGCGGCGCCGGCAAGAGCACAGCCCUCCAGCUCAUCCAGAGGUUCUACGACCCAUGCGAAGGCAUGGUGACAGUGGACGGCCACGACAUCAGGAGCCUCAACAUCCAGUGGCUCAGGGACCAGAUCGGCAUCGUGGAACAGGAACCAGUGCUCUUCAGCACAACAAUCGCCGAAAACAUCAGGUACGGCAGGGAGGACGCCACAAUGGAGGACAUCGUGCAGGCCGCCAAGGAAGCCAACGCCUACAACUUCAUCAUGGACCUCCCACAGCAGUUCGACACACUCGUGGGCGAAGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGAGGGUGGCCAUCGCCAGGGCCCUCAUCAGGAACCCAAAGAUCCUCCUCCUCGACAUGGCCACAAGCGCCCUCGACAACGAAAGCGAAGCCAUGGUGCAGGAAGUGCUCAGCAAGAUCCAGCACGGCCACACAAUCAUCAGCGUGGCCCACAGGCUCAGCACAGUGAGGGCCGCCGACACAAUCAUCGGCUUCGAACACGGCACAGCCGUGGAAAGGGGCACACACGAAGAACUCCUCGAAAGGAAGGGCGUGUACUUCACACUCGUGACACUCCAGAGCCAGGGCAACCAGGCCCUCAACGAAGAGGACAUCAAGGACGCCACAGAGGACGACAUGCUCGCCAGGACAUUCAGCAGGGGCAGCUACCAGGACAGCCUCAGGGCCAGCAUCAGGCAGAGGAGCAAGAGCCAGCUCAGCUACCUCGUGCACGAACCACCACUCGCCGUGGUGGACCACAAGAGCACAUACGAAGAGGACAGGAAGGACAAGGACAUCCCAGUGCAGGAAGAAGUGGAACCAGCCCCAGUGAGGAGGAUCCUCAAGUUCAGCGCCCCAGAAUGGCCAUACAUGCUCGUGGGCAGCGUGGGCGCCGCCGUGAACGGCACAGUGACACCACUCUACGCCUUCCUCUUCAGCCAGAUCCUCGGCACAUUCAGCAUCCCAGACAAGGAAGAACAGAGGAGCCAGAUCAACGGCGUGUGCCUCCUCUUCGUGGCCAUGGGCUGCGUGAGCCUCUUCACACAGUUCCUCCAGGGCUACGCCUUCGCCAAGAGCGGCGAACUCCUCACAAAGAGGCUCAGGAAGUUCGGCUUCAGGGCCAUGCUCGGCCAGGACAUCGCCUGGUUCGACGACCUCAGGAACAGCCCAGGCGCCCUCACAACAAGGCUCGCCACAGACGCCAGCCAGGUGCAGGGCGCCGCCGGCAGCCAGAUCGGCAUGAUCGUGAACAGCUUCACAAACGUGACAGUGGCCAUGAUCAUCGCCUUCAGCUUCAGCUGGAAGCUCAGCCUCGUGAUCCUCUGCUUCUUCCCAUUCCUCGCCCUCAGCGGCGCCACACAGACAAGGAUGCUCACAGGCUUCGCCAGCAGGGACAAGCAGGCCCUCGAAAUGGUGGGCCAGAUCACAAACGAAGCCCUCAGCAACAUCAGGACAGUGGCCGGCAUCGGCAAGGAAAGGAGGUUCAUCGAAGCCCUCGAAACAGAACUCGAAAAGCCAUUCAAGACAGCCAUCCAGAAGGCCAACAUCUACGGCUUCUGCUUCGCCUUCGCCCAGUGCAUCAUGUUCAUCGCCAACAGCGCCAGCUACAGGUACGGCGGCUACCUCAUCAGCAACGAAGGCCUCCACUUCAGCUACGUGUUCAGGGUGAUCAGCGCCGUGGUGCUCAGCGCCACAGCCCUCGGCAGGGCCUUCAGCUACACACCAAGCUACGCCAAGGCCAAGAUCAGCGCCGCCAGGUUCUUCCAGCUCCUCGACAGGCAGCCACCAAUCAGCGUGUACAACACAGCCGGCGAAAAGUGGGACAACUUCCAGGGCAAGAUCGACUUCGUGGACUGCAAGUUCACAUACCCAAGCAGGCCAGACAGCCAGGUGCUCAACGGCCUCAGCGUGAGCAUCAGCCCAGGCCAGACACUCGCCUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACAAGCAUCCAGCUCCUCGAAAGGUUCUACGACCCAGACCAGGGCAAGGUGAUGAUCGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUCAGGAGCAACAUCGGCAUCGUGAGCCAGGAACCAGUGCUCUUCGCCUGCAGCAUCAUGGACAACAUCAAGUACGGCGACAACACAAAGGAAAUCCCAAUGGAAAGGGUGAUCGCCGCCGCCAAGCAGGCCCAGCUCCACGACUUCGUGAUGAGCCUCCCAGAAAAGUACGAAACAAACGUGGGCAGCCAGGGCAGCCAGCUCAGCAGGGGCGAAAAGCAGAGGAUCGCCAUCGCCAGGGCCAUCGUGAGGGACCCAAAGAUCCUCCUCCUCGACGAAGCCACAAGCGCCCUCGACACAGAAAGCGAAAAGACAGUGCAGGUGGCCCUCGACAAGGCCAGGGAAGGCAGGACAUGCAUCGUGAUCGCCCACAGGCUCAGCACAAUCCAGAACGCCGACAUCAUCGCCGUGAUGGCCCAGGGCGUGGUGAUCGAAAAGGGCACACACGAAGAACUCAUGGCCCAGAAGGGCGCCUACUACAAGCUCGUGACAACAGGCAGCCCAAUCAGC

[0231] SEQ ID NO: 6 (Codon-Optimized mRNA - ABCB 11 mRNA LowU*)

[0232] AUGAGCGACAGCGUGAUCCUGCGGAGCAUCAAGAAGUUCGGCGAGGAGAACGACGGCUUCGAGAGCGACAAGAGCUACAACAACGACAAGAAGAGCCGGCUGCAGGACGAGAAGAAGGGCGACGGCGUGCGGGUGGGCUUCUUCCAGCUGUUCCGGUUCAGCAGCAGCACCGACAUCUGGCUGAUGUUCGUGGGCAGCCUGUGCGCCUUCCUGCACGGCAUCGCCCAGCCCGGCGUGCUGCUGAUCUUCGGCACCAUGACCGACGUGUUCAUCGACUACGACGUGGAGCUGCAGGAGCUGCAGAUCCCCGGCAAGGCCUGCGUGAACAACACCAUCGUGUGGACCAACAGCAGCCUGAACCAGAACAUGACCAACGGCACCCGGUGCGGCCUGCUGAACAUCGAGAGCGAGAUGAUCAAGUUCGCCAGCUACUACGCCGGCAUCGCCGUGGCCGUGCUGAUCACCGGCUACAUCCAGAUCUGCUUCUGGGUGAUCGCCGCCGCCCGGCAGAUCCAGAAGAUGCGGAAGUUCUACUUCCGGCGGAUCAUGCGGAUGGAGAUCGGCUGGUUCGACUGCAACAGCGUGGGCGAGCUGAACACCCGGUUCAGCGACGACAUCAACAAGAUCAACGACGCCAUCGCCGACCAGAUGGCCCUGUUCAUCCAGCGGAUGACCAGCACCAUCUGCGGCUUCCUGCUGGGCUUCUUCCGGGGCUGGAAGCUGACCCUGGUGAUCAUCAGCGUGAGCCCCCUGAUCGGCAUCGGCGCCGCCACCAUCGGCCUGAGCGUGAGCAAGUUCACCGACUACGAGCUGAAGGCCUACGCCAAGGCCGGCGUGGUGGCCGACGAGGUGAUCAGCAGCAUGCGGACCGUGGCCGCCUUCGGCGGCGAGAAGCGGGAGGUGGAGCGGUACGAGAAGAACCUGGUGUUCGCCCAGCGGUGGGGCAUCCGGAAGGGCAUCGUGAUGGGCUUCUUCACCGGCUUCGUGUGGUGCCUGAUCUUCCUGUGCUACGCCCUGGCCUUCUGGUACGGCAGCACCCUGGUGCUGGACGAGGGCGAGUACACCCCCGGCACCCUGGUGCAGAUCUUCCUGAGCGUGAUCGUGGGCGCCCUGAACCUGGGCAACGCCAGCCCCUGCCUGGAGGCCUUCGCCACCGGCCGGGCCGCCGCCACCAGCAUCUUCGAGACCAUCGACCGGAAGCCCAUCAUCGACUGCAUGAGCGAGGACGGCUACAAGCUGGACCGGAUCAAGGGCGAGAUCGAGUUCCACAACGUGACCUUCCACUACCCCAGCCGGCCCGAGGUGAAGAUCCUGAACGACCUGAACAUGGUGAUCAAGCCCGGCGAGAUGACCGCCCUGGUGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCUGCAGCUGAUCCAGCGGUUCUACGACCCCUGCGAGGGCAUGGUGACCGUGGACGGCCACGACAUCCGGAGCCUGAACAUCCAGUGGCUGCGGGACCAGAUCGGCAUCGUGGAGCAGGAGCCCGUGCUGUUCAGCACCACCAUCGCCGAGAACAUCCGGUACGGCCGGGAGGACGCCACCAUGGAGGACAUCGUGCAGGCCGCCAAGGAGGCCAACGCCUACAACUUCAUCAUGGACCUGCCCCAGCAGUUCGACACCCUGGUGGGCGAGGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGCGGGUGGCCAUCGCCCGGGCCCUGAUCCGGAACCCCAAGAUCCUGCUGCUGGACAUGGCCACCAGCGCCCUGGACAACGAGAGCGAGGCCAUGGUGCAGGAGGUGCUGAGCAAGAUCCAGCACGGCCACACCAUCAUCAGCGUGGCCCACCGGCUGAGCACCGUGCGGGCCGCCGACACCAUCAUCGGCUUCGAGCACGGCACCGCCGUGGAGCGGGGCACCCACGAGGAGCUGCUGGAGCGGAAGGGCGUGUACUUCACCCUGGUGACCCUGCAGAGCCAGGGCAACCAGGCCCUGAACGAGGAGGACAUCAAGGACGCCACCGAGGACGACAUGCUGGCCCGGACCUUCAGCCGGGGCAGCUACCAGGACAGCCUGCGGGCCAGCAUCCGGCAGCGGAGCAAGAGCCAGCUGAGCUACCUGGUGCACGAGCCCCCCCUGGCCGUGGUGGACCACAAGAGCACCUACGAGGAGGACCGGAAGGACAAGGACAUCCCCGUGCAGGAGGAGGUGGAGCCCGCCCCCGUGCGGCGGAUCCUGAAGUUCAGCGCCCCCGAGUGGCCCUACAUGCUGGUGGGCAGCGUGGGCGCCGCCGUGAACGGCACCGUGACCCCCCUGUACGCCUUCCUGUUCAGCCAGAUCCUGGGCACCUUCAGCAUCCCCGACAAGGAGGAGCAGCGGAGCCAGAUCAACGGCGUGUGCCUGCUGUUCGUGGCCAUGGGCUGCGUGAGCCUGUUCACCCAGUUCCUGCAGGGCUACGCCUUCGCCAAGAGCGGCGAGCUGCUGACCAAGCGGCUGCGGAAGUUCGGCUUCCGGGCCAUGCUGGGCCAGGACAUCGCCUGGUUCGACGACCUGCGGAACAGCCCCGGCGCCCUGACCACCCGGCUGGCCACCGACGCCAGCCAGGUGCAGGGCGCCGCCGGCAGCCAGAUCGGCAUGAUCGUGAACAGCUUCACCAACGUGACCGUGGCCAUGAUCAUCGCCUUCAGCUUCAGCUGGAAGCUGAGCCUGGUGAUCCUGUGCUUCUUCCCCUUCCUGGCCCUGAGCGGCGCCACCCAGACCCGGAUGCUGACCGGCUUCGCCAGCCGGGACAAGCAGGCCCUGGAGAUGGUGGGCCAGAUCACCAACGAGGCCCUGAGCAACAUCCGGACCGUGGCCGGCAUCGGCAAGGAGCGGCGGUUCAUCGAGGCCCUGGAGACCGAGCUGGAGAAGCCCUUCAAGACCGCCAUCCAGAAGGCCAACAUCUACGGCUUCUGCUUCGCCUUCGCCCAGUGCAUCAUGUUCAUCGCCAACAGCGCCAGCUACCGGUACGGCGGCUACCUGAUCAGCAACGAGGGCCUGCACUUCAGCUACGUGUUCCGGGUGAUCAGCGCCGUGGUGCUGAGCGCCACCGCCCUGGGCCGGGCCUUCAGCUACACCCCCAGCUACGCCAAGGCCAAGAUCAGCGCCGCCCGGUUCUUCCAGCUGCUGGACCGGCAGCCCCCCAUCAGCGUGUACAACACCGCCGGCGAGAAGUGGGACAACUUCCAGGGCAAGAUCGACUUCGUGGACUGCAAGUUCACCUACCCCAGCCGGCCCGACAGCCAGGUGCUGAACGGCCUGAGCGUGAGCAUCAGCCCCGGCCAGACCCUGGCCUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACCAGCAUCCAGCUGCUGGAGCGGUUCUACGACCCCGACCAGGGCAAGGUGAUGAUCGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUGCGGAGCAACAUCGGCAUCGUGAGCCAGGAGCCCGUGCUGUUCGCCUGCAGCAUCAUGGACAACAUCAAGUACGGCGACAACACCAAGGAGAUCCCCAUGGAGCGGGUGAUCGCCGCCGCCAAGCAGGCCCAGCUGCACGACUUCGUGAUGAGCCUGCCCGAGAAGUACGAGACCAACGUGGGCAGCCAGGGCAGCCAGCUGAGCCGGGGCGAGAAGCAGCGGAUCGCCAUCGCCCGGGCCAUCGUGCGGGACCCCAAGAUCCUGCUGCUGGACGAGGCCACCAGCGCCCUGGACACCGAGAGCGAAAAGACCGUGCAGGUGGCCCUGGACAAGGCCCGGGAGGGCCGGACCUGCAUCGUGAUCGCCCACCGGCUGAGCACCAUCCAGAACGCCGACAUCAUCGCCGUGAUGGCCCAGGGCGUGGUGAUCGAGAAGGGCACCCACGAGGAGCUGAUGGCCCAGAAGGGCGCCUACUACAAGCUGGUGACCACCGGCAGCCCCAUCAGC

[0233] SEQ ID NO: 7 (BSEP protein)

[0234] MSDSVILRSIKKFGEENDGFESDKSYNNDKKSRLQDEKKGDGVRVGFFQLFRFSSSTDIWLMFVGSLCAFLHGIAQPGVLLIFGTMTDVFIDYDVELQELQIPGKACVNNTIVWTNSSLNQNMTNGTRCGLLNIESEMIKFASYYAGIAVAVLITGYIQICFWVIAAARQIQKMRKFYFRRIMRMEIGWFDCNSVGELNTRFSDDINKINDAIADQMALFIQRMTSTICGFLLGFFRGWKLTLVIISVSPLIGIGAATIGLSVSKFTDYELKAYAKAGVVADEVISSMRTVAAFGGEKREVERYEKNLVFAQRWGIRKGIVMGFFTGFVWCLIFLCYALAFWYGSTLVLDEGEYTPGTLVQIFLSVIVGALNLGNASPCLEAFATGRAAATSIFETIDRKPIIDCMSEDGYKLDRIKGEIEFHNVTFHYPSRPEVKILNDLNMVIKPGEMTALVGPSGAGKSTALQLIQRFYDPCEGMVTVDGHDIRSLNIQWLRDQIGIVEQEPVLFSTTIAENIRYGREDATMEDIVQAAKEANAYNFIMDLPQQFDTLVGEGGGQMSGGQKQRVAIARALIRNPKILLLDMATSALDNESEAMVQEVLSKIQHGHTIISVAHRLSTVRAADTIIGFE HGTAVERGTHEELLERKGVYFTLVTLQSQGNQALNEEDIKDATEDDMLARTFSRGS YQDSLRASIRQRSKSQLSYLVHEPPLAVVDHKSTYEEDRKDKDIPVQEEVEPAPVRRI LKFSAPEWPYMLVGSVGAAVNGTVTPLYAFLFSQILGTFSIPDKEEQRSQINGVCLLF VAMGCVSLFTQFLQGYAFAKSGELLTKRLRKFGFRAMLGQDIAWFDDLRNSPGALT TRLATDASQVQGAAGSQIGMIVNSFTNVTVAMIIAFSFSWKLSLVILCFFPFLALSGAT QTRMLTGFASRDKQALEMVGQITNEALSNIRTVAGIGKERRFIEALETELEKPFKTAI QKANIYGFCFAFAQCIMFIANSASYRYGGYLISNEGLHFSYVFRVISAVVLSATALGR AFSYTPSYAKAKISAARFFQLLDRQPPISVYNTAGEKWDNFQGKIDFVDCKFTYPSRP DSQVLNGLSVSISPGQTLAFVGSSGCGKSTSIQLLERFYDPDQGKVMIDGHDSKKVN VQFLRSNIGIVSQEPVLFACSIMDNIKYGDNTKEIPMERVIAAAKQAQLHDFVMSLPE KYETNVGSQGSQLSRGEKQRIAIARAIVRDPKILLLDEATSALDTESEKTVQVALDKA REGRTCIVIAHRLSTIQNADIIAVMAQGVVIEKGTHEELMAQKGAYYKLVTTGSPIS

[0235] SEQ ID NO: 8 (5’ UTR)

[0236] GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA

[0237] SEQ ID NO: 9 (5’ UTR - Tobacco Etch Virus)

[0238] UCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUU CUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGC

[0239] SEQ ID NO : 10 (3 ’ UTR)

[0240] UUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUC UCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG

[0241] SEQ ID NO: 11 (3 ’ UTR - Xenopus beta-globin)

[0242] GGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGC UACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUU CGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACA

[0243] SEQ ID NO : 12 (DN A of SEQ ID NO : 2)

[0244] ATGAGCGACAGCGTGATCCTGCGGAGCATCAAGAAGTTCGGCGAGGAG AACGACGGCTTCGAGAGCGACAAGAGCTACAACAACGACAAGAAAAGCCGGCT GCAGGACGAGAAGAAGGGCGACGGCGTGCGGGTGGGCTTCTTCCAGCTGTTCCG GTTCAGCAGCAGCACCGACATCTGGCTGATGTTCGTGGGCAGCCTGTGCGCCTTC CTGCACGGCATCGCCCAGCCCGGCGTGCTGCTGATCTTCGGCACCATGACCGACG TGTTCATCGACTACGACGTGGAGCTGCAGGAGCTGCAGATCCCCGGCAAGGCCTGCGTGAACAACACCATCGTGTGGACCAACAGCAGCCTGAACCAGAACATGACCAACGGCACCCGGTGCGGCCTGCTGAACATCGAGAGCGAGATGATCAAGTTCGCCAGCTACTACGCCGGCATCGCCGTGGCCGTGCTGATCACCGGCTACATCCAGATCTGCTTCTGGGTGATCGCCGCCGCCCGGCAGATCCAGAAGATGCGGAAGTTCTACTTCCGGCGGATCATGCGGATGGAGATCGGCTGGTTCGACTGCAACAGCGTGGGCGAGCTGAACACCCGGTTCAGCGACGACATCAACAAGATCAACGACGCCATCGCCGACCAGATGGCCCTGTTCATCCAGCGGATGACCAGCACCATCTGCGGCTTCCTGCTGGGCTTCTTCCGGGGCTGGAAGCTGACCCTGGTGATCATCAGCGTGAGCCCCCTGATCGGCATCGGCGCCGCCACCATCGGCCTGAGCGTGAGCAAGTTCACCGACTACGAGCTGAAGGCCTACGCCAAGGCCGGCGTGGTGGCCGACGAGGTGATCAGCAGCATGCGGACCGTGGCCGCCTTCGGCGGCGAGAAGCGGGAGGTGGAGCGGTACGAGAAGAACCTGGTGTTCGCCCAGCGGTGGGGCATCCGGAAGGGCATCGTGATGGGCTTCTTCACCGGCTTCGTGTGGTGCCTGATCTTCCTGTGCTACGCCCTGGCCTTCTGGTACGGCAGCACCCTGGTGCTGGACGAGGGCGAGTACACCCCCGGCACCCTGGTGCAGATCTTCCTGAGCGTGATCGTGGGCGCCCTGAACCTGGGCAACGCCAGCCCCTGCCTGGAGGCCTTCGCCACCGGCCGGGCCGCCGCCACCAGCATCTTCGAGACCATCGACCGGAAGCCCATCATCGACTGCATGAGCGAGGACGGCTACAAGCTGGACCGGATCAAGGGCGAGATCGAGTTCCACAACGTGACCTTCCACTACCCCAGCCGGCCCGAGGTGAAGATCCTGAACGACCTGAACATGGTGATCAAGCCCGGCGAGATGACCGCCCTGGTGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCTGCAGCTGATCCAGCGGTTCTACGACCCCTGCGAGGGCATGGTGACCGTGGACGGCCACGACATCCGGAGCCTGAACATCCAGTGGCTGCGGGACCAGATCGGCATCGTGGAGCAGGAGCCCGTGCTGTTCAGCACCACCATCGCCGAGAACATCCGGTACGGCCGGGAGGACGCCACCATGGAGGACATCGTGCAGGCCGCCAAGGAGGCCAACGCCTACAACTTCATCATGGACCTGCCCCAGCAGTTCGACACCCTGGTGGGCGAGGGCGGCGGCCAGATGAGCGGCGGCCAGAAGCAGCGGGTGGCCATCGCCCGGGCCCTGATCCGGAACCCCAAGATCCTGCTGCTGGACATGGCCACCAGCGCCCTGGACAACGAGAGCGAGGCCATGGTGCAGGAGGTGCTGAGCAAGATCCAGCACGGCCACACCATCATCAGCGTGGCCCACCGGCTGAGCACCGTGCGGGCCGCCGACACCATCATCGGCTTCGAGCACGGCACCGCCGTGGAGCGGGGCACCCACGAGGAGCTGCTGGAGCGGAAGGGCGTGTACTTCACCCTGGTGACCCTGCAGAGCCAGGGCAACCAGGCCCTGAACGAGGAGGACATCAAGGACGCCACCGAGGACGACATGCTGGCCCGGACCTTCAGCCGGGGCAGCTACCAGGACAGCCTGCGGGCCAGCATCCGGCAGCGGAGCAAGAGCCAGCTGAGCTACCTGGTGCACGAGCCCCCCCTGGCCGTGGTGGACCACAAGAGCACCTACGAGGAGGACCGGAAGGACAAGGACATCCCCGTGCAGGAGGAGGTGGAGCCCGCCCCCGTGCGGCGGATCCTGAAGTTCAGCGCCCCCGAGTGGCCCTACATGCTGGTGGGCAGCGTGGGCGCCGCCGTGAACGGCACCGTGACCCCCCTGTACGCCTTCCTGTTCAGCCAGATCCTGGGCACCTTCAGCATCCCCGACAAGGAGGAGCAGCGGAGCCAGATCAACGGCGTGTGCCTGCTGTTCGTGGCCATGGGCTGCGTGAGCCTGTTCACCCAGTTCCTGCAGGGCTACGCCTTCGCCAAGAGCGGCGAGCTGCTGACCAAGCGGCTGCGGAAGTTCGGCTTCCGGGCCATGCTGGGCCAGGACATCGCCTGGTTCGACGACCTGCGGAACAGCCCCGGCGCCCTGACCACCCGGCTGGCCACCGACGCCAGCCAGGTGCAGGGCGCCGCCGGCAGCCAGATCGGCATGATCGTGAACAGCTTCACCAACGTGACCGTGGCCATGATCATCGCCTTCAGCTTCAGCTGGAAGCTGAGCCTGGTGATCCTGTGCTTCTTCCCCTTCCTGGCCCTGAGCGGCGCCACCCAGACCCGGATGCTGACCGGCTTCGCCAGCCGGGACAAGCAGGCCCTGGAGATGGTGGGCCAGATCACCAACGAGGCCCTGAGCAACATCCGGACCGTGGCCGGCATCGGCAAGGAGCGGCGGTTCATCGAGGCCCTGGAGACCGAGCTGGAGAAGCCCTTCAAGACCGCCATCCAGAAGGCCAACATCTACGGCTTCTGCTTCGCCTTCGCCCAGTGCATCATGTTCATCGCCAACAGCGCCAGCTACCGGTACGGCGGCTACCTGATCAGCAACGAGGGCCTGCACTTCAGCTACGTGTTCCGGGTGATCAGCGCCGTGGTGCTGAGCGCCACCGCCCTGGGCCGGGCCTTCAGCTACACCCCCAGCTACGCCAAGGCCAAGATCAGCGCCGCCCGGTTCTTCCAGCTGCTGGACCGGCAGCCCCCCATCAGCGTGTACAACACCGCCGGCGAGAAGTGGGACAACTTCCAGGGCAAGATCGACTTCGTGGACTGCAAGTTCACCTACCCCAGCCGGCCCGACAGCCAGGTGCTGAACGGCCTGAGCGTGAGCATCAGCCCCGGCCAGACCCTGGCCTTCGTGGGCAGCAGCGGCTGCGGCAAGAGCACCAGCATCCAGCTGCTGGAGCGGTTCTACGACCCCGACCAGGGCAAGGTGATGATCGACGGCCACGACAGCAAGAAGGTGAACGTGCAGTTCCTGCGGAGCAACATCGGCATCGTGAGCCAGGAGCCCGTGCTGTTCGCCTGCAGCATCATGGACAACATCAAGTACGGCGACAACACCAAGGAGATCCCCATGGAGCGGGTGATCGCCGCCGCCAAGCAGGCCCAGCTGCACGACTTCGTGATGAGCCTGCCCGAGAAGTACGAGACCAACGTGGGCAGCCAGGGCAGCCAGCTGAGCCGGGGCGAGAAGCAGCGGATCGCCATCGCCCGGGCCATCGTGCGGGACCCCAAGATCCTGCTGCTGGACGAGGCCACCAGCGCCCTGGACACCGAGAGCGAAAAGACCGTGCAGGTGGCCCTGGACAAGGCCCGGGAGGGCCGGACCTGCATCGTGATCGCCCACCGGCTGAGCACCATCCAGAACGCCGACATCATCGCCGTGATGGCCCAGGGCGTGGTGATCGAGAAGGGCACCCACGAGGAGCTGATGGCCCAGAAGGGCGCCTACTACAAGCTGGTGACCACCGGCAGCCCCATCAGC

[0245] SEQ ID NO: 13 (DNA of SEQ ID NO: 3)

[0246] ATGAGCGATTCGGTAATACTACGTTCGATAAAAAAATTTGGTGAAGAAAATGATGGTTTTGAATCGGATAAATCGTATAATAATGATAAAAAATCGCGTCTACAAGATGAAAAAAAAGGTGATGGTGTACGTGTAGGTTTTTTTCAACTATTCCGGTTCAGCAGCAGCACCGACATCTGGCTGATGTTCGTGGGCAGCCTGTGCGCCTTCCTGCACGGCATCGCCCAGCCCGGCGTGCTGCTGATCTTCGGCACCATGACCGACGTGTTCATCGACTACGACGTGGAGCTGCAGGAGCTGCAGATCCCCGGCAAGGCCTGCGTGAACAACACCATCGTGTGGACCAACAGCAGCCTGAACCAGAACATGACCAACGGCACCCGGTGCGGCCTGCTGAACATCGAGAGCGAGATGATCAAGTTCGCCAGCTACTACGCCGGCATCGCCGTGGCCGTGCTGATCACCGGCTACATCCAGATCTGCTTCTGGGTGATCGCCGCCGCCCGGCAGATCCAGAAGATGCGGAAGTTCTACTTCCGGCGGATCATGCGGATGGAGATCGGCTGGTTCGACTGCAACAGCGTGGGCGAGCTGAACACCCGGTTCAGCGACGACATCAACAAGATCAACGACGCCATCGCCGACCAGATGGCCCTGTTCATCCAGCGGATGACCAGCACCATCTGCGGCTTCCTGCTGGGCTTCTTCCGGGGCTGGAAGCTGACCCTGGTGATCATCAGCGTGAGCCCCCTGATCGGCATCGGCGCCGCCACCATCGGCCTGAGCGTGAGCAAGTTCACCGACTACGAGCTGAAGGCCTACGCCAAGGCCGGCGTGGTGGCCGACGAGGTGATCAGCAGCATGCGGACCGTGGCCGCCTTCGGCGGCGAGAAGCGGGAGGTGGAGCGGTACGAGAAGAACCTGGTGTTCGCCCAGCGGTGGGGCATCCGGAAGGGCATCGTGATGGGCTTCTTCACCGGCTTCGTGTGGTGCCTGATCTTCCTGTGCTACGCCCTGGCCTTCTGGTACGGCAGCACCCTGGTGCTGGACGAGGGCGAGTACACCCCCGGCACCCTGGTGCAGATCTTCCTGAGCGTGATCGTGGGCGCCCTGAACCTGGGCAACGCCAGCCCCTGCCTGGAGGCCTTCGCCACCGGCCGGGCCGCCGCCACCAGCATCTTCGAGACCATCGACCGGAAGCCCATCATCGACTGCATGAGCGAGGACGGCTACAAGCTGGACCGGATCAAGGGCGAGATCGAGTTCCACAACGTGACCTTCCACTACCCCAGCCGGCCCGAGGTGAAGATCCTGAACGACCTGAACATGGTGATCAAGCCCGGCGAGATGACCGCCCTGGTGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCTGCAGCTGATCCAGCGGTTCTACGACCCCTGCGAGGGCATGGTGACCGTGGACGGCCACGACATCCGGAGCCTGAACATCCAGTGGCTGCGGGACCAGATCGGCATCGTGGAGCAGGAGCCCGTGCTGTTCAGCACCACCATCGCCGAGAACATCCGGTACGGCCGGGAGGACGCCACCATGGAGGACATCGTGCAGGCCGCCAAGGAGGCCAACGCCTACAACTTCATCATGGACCTGCCCCAGCAGTTCGACACCCTGGTGGGCGAGGGCGGCGGCCAGATGAGCGGCGGCCAGAAGCAGCGGGTGGCCATCGCCCGGGCCCTGATCCGGAACCCCAAGATCCTGCTGCTGGACATGGCCACCAGCGCCCTGGACAACGAGAGCGAGGCCATGGTGCAGGAGGTGCTGAGCAAGATCCAGCACGGCCACACCATCATCAGCGTGGCCCACCGGCTGAGCACCGTGCGGGCCGCCGACACCATCATCGGCTTCGAGCACGGCACCGCCGTGGAGCGGGGCACCCACGAGGAGCTGCTGGAGCGGAAGGGCGTGTACTTCACCCTGGTGACCCTGCAGAGCCAGGGCAACCAGGCCCTGAACGAGGAGGACATCAAGGACGCCACCGAGGACGACATGCTGGCCCGGACCTTCAGCCGGGGCAGCTACCAGGACAGCCTGCGGGCCAGCATCCGGCAGCGGAGCAAGAGCCAGCTGAGCTACCTGGTGCACGAGCCCCCCCTGGCCGTGGTGGACCACAAGAGCACCTACGAGGAGGACCGGAAGGACAAGGACATCCCCGTGCAGGAGGAGGTGGAGCCCGCCCCCGTGCGGCGGATCCTGAAGTTCAGCGCCCCCGAGTGGCCCTACATGCTGGTGGGCAGCGTGGGCGCCGCCGTGAACGGCACCGTGACCCCCCTGTACGCCTTCCTGTTCAGCCAGATCCTGGGCACCTTCAGCATCCCCGACAAGGAGGAGCAGCGGAGCCAGATCAACGGCGTGTGCCTGCTGTTCGTGGCCATGGGCTGCGTGAGCCTGTTCACCCAGTTCCTGCAGGGCTACGCCTTCGCCAAGAGCGGCGAGCTGCTGACCAAGCGGCTGCGGAAGTTCGGCTTCCGGGCCATGCTGGGCCAGGACATCGCCTGGTTCGACGACCTGCGGAACAGCCCCGGCGCCCTGACCACCCGGCTGGCCACCGACGCCAGCCAGGTGCAGGGCGCCGCCGGCAGCCAGATCGGCATGATCGTGAACAGCTTCACCAACGTGACCGTGGCCATGATCATCGCCTTCAGCTTCAGCTGGAAGCTGAGCCTGGTGATCCTGTGCTTCTTCCCCTTCCTGGCCCTGAGCGGCGCCACCCAGACCCGGATGCTGACCGGCTTCGCCAGCCGGGACAAGCAGGCCCTGGAGATGGTGGGCCAGATCACCAACGAGGCCCTGAGCAACATCCGGACCGTGGCCGGCATCGGCAAGGAGCGGCGGTTCATCGAGGCCCTGGAGACCGAGCTGGAGAAGCCCTTCAAGACCGCCATCCAGAAGGCCAACATCTACGGCTTCTGCTTCGCCTTCGCCCAGTGCATCATGTTCATCGCCAACAGCGCCAGCTACCGGTACGGCGGCTACCTGATCAGCAACGAGGGCCTGCACTTCAGCTACGTGTTCCGGGTGATCAGCGCCGTGGTGCTGAGCGCCACCGCCCTGGGCCGGGCCTTCAGCTACACCCCCAGCTACGCCAAGGCCAAGATCAGCGCCGCCCGGTTCTTCCAGCTGCTGGACCGGCAGCCCCCCATCAGCGTGTACAACACCGCCGGCGAGAAGTGGGACAACTTCCAGGGCAAGATCGACTTCGTGGACTGCAAGTTCACCTACCCCAGCCGGCCCGACAGCCAGGTGCTGAACGGCCTGAGCGTGAGCATCAGCCCCGGCCAGACCCTGGCCTTCGTGGGCAGCAGCGGCTGCGGCAAGAGCACCAGCATCCAGCTGCTGGAGCGGTTCTACGACCCCGACCAGGGCAAGGTGATGATCGACGGCCACGACAGCAAGAAGGTGAACGTGCAGTTCCTGCGGAGCAACATCGGCATCGTGAGCCAGGAGCCCGTGCTGTTCGCCTGCAGCATCATGGACAACATCAAGTACGGCGACAACACCAAGGAGATCCCCATGGAGCGGGTGATCGCCGCCGCCAAGCAGGCCCAGCTGCACGACTTCGTGATGAGCCTGCCCGAGAAGTACGAGACCAACGTGGGCAGCCAGGGCAGCCAGCTGAGCCGGGGCGAGAAGCAGCGGATCGCCATCGCCCGGGCCATCGTGCGGGACCCCAAGATCCTGCTGCTGGACGAGGCCACCAGCGCCCTGGACACCGAGAGCGAAAAGACCGTGCAGGTGGCCCTGGACAAGGCCCGGGAGGGCCGGACCTGCATCGTGATCGCCCACCGGCTGAGCACCATCCAGAACGCCGACATCATCGCCGTGATGGCCCAGGGCGTGGTGATCGAGAAGGGCACCCACGAGGAGCTGATGGCCCAGAAGGGCGCCTACTACAAGCTGGTGACCACCGGCAGCCCCATCAGC

[0247] SEQ ID NO : 14 (DN A of SEQ ID NO : 4)

[0248] ATGTCTGACTCTGTGATTCTTCGTAGCATTAAGAAGTTCGGCGAAGAAAACGACGGCTTCGAATCTGACAAGTCTTACAACAACGACAAGAAGTCTAGACTTCAGGACGAAAAGAAGGGCGACGGCGTGAGAGTGGGCTTCTTCCAGCTGTTCCGTTTCTCTTCTTCTACTGACATTTGGCTGATGTTCGTGGGCAGCCTGTGCGCTTTCCTGCACGGCATTGCTCAGCCTGGCGTGCTTCTGATTTTCGGCACAATGACAGACGTGTTCATTGACTACGACGTGGAACTTCAGGAACTGCAGATTCCTGGCAAGGCTTGCGTGAACAACACTATTGTGTGGACTAACAGCTCTCTGAACCAGAACATGACAAACGGCACACGTTGCGGCCTGCTGAACATTGAAAGCGAAATGATTAAGTTCGCTAGCTACTACGCTGGCATTGCTGTGGCTGTGCTTATTACAGGCTACATTCAGATTTGCTTCTGGGTGATTGCTGCTGCTCGTCAGATTCAGAAGATGAGAAAGTTCTACTTCAGAAGAATTATGAGAATGGAAATTGGCTGGTTCGACTGCAACTCTGTGGGCGAACTGAACACAAGATTCTCTGACGACATTAACAAGATTAACGACGCTATTGCTGACCAGATGGCTCTTTTCATTCAGCGTATGACTTCTACTATTTGCGGCTTCCTGCTGGGCTTCTTCAGAGGCTGGAAGCTGACTCTGGTGATTATTTCTGTGAGCCCTCTGATTGGCATTGGCGCTGCTACTATTGGCCTGAGCGTGTCTAAGTTCACAGACTACGAACTGAAGGCTTACGCTAAGGCTGGCGTGGTGGCTGACGAAGTGATTTCTTCTATGAGAACAGTGGCTGCTTTCGGCGGCGAAAAGAGAGAAGTGGAAAGATACGAAAAGAACCTTGTGTTCGCTCAGCGTTGGGGCATTAGAAAGGGCATTGTGATGGGCTTCTTCACTGGCTTCGTGTGGTGCCTGATTTTCCTGTGCTACGCTCTGGCTTTCTGGTACGGCTCTACACTTGTGCTGGACGAAGGCGAATACACACCTGGCACTCTTGTGCAGATTTTCCTGAGCGTGATTGTGGGCGCTCTTAACCTTGGCAACGCTTCTCCTTGCCTGGAAGCTTTCGCTACTGGCCGTGCTGCTGCTACTAGCATTTTCGAAACAATTGACAGAAAGCCTATTATTGACTGCATGTCTGAGGACGGCTACAAGCTGGACCGTATTAAGGGCGAAATTGAATTCCACAACGTGACTTTCCACTACCCTTCTAGACCTGAAGTGAAGATTCTTAACGACCTGAACATGGTGATTAAGCCTGGCGAAATGACAGCTCTGGTGGGCCCTAGCGGCGCTGGCAAGAGCACAGCTCTGCAGCTGATTCAGCGTTTCTACGACCCTTGCGAAGGCATGGTGACTGTGGACGGCCACGACATTCGTTCTCTTAACATTCAGTGGCTTAGAGACCAGATTGGCATTGTGGAACAGGAACCTGTGCTGTTCTCTACTACTATTGCTGAAAACATTCGTTACGGCAGAGAGGACGCTACAATGGAGGACATTGTGCAGGCTGCTAAGGAAGCTAACGCTTACAACTTCATTATGGACCTGCCTCAGCAGTTCGACACTCTTGTGGGCGAAGGCGGCGGCCAGATGAGCGGCGGCCAGAAGCAGAGAGTGGCTATTGCTAGAGCTCTGATTCGTAACCCTAAGATTCTGCTTCTGGACATGGCTACTTCTGCTCTGGACAACGAAAGCGAAGCTATGGTGCAGGAAGTGCTGAGCAAGATTCAGCACGGCCACACAATTATTTCTGTGGCTCACCGTCTGTCTACAGTGAGAGCTGCTGACACTATTATTGGCTTCGAACACGGCACTGCTGTGGAAAGAGGCACTCACGAAGAACTTCTGGAAAGAAAGGGCGTGTACTTCACTCTTGTGACTCTGCAGAGCCAGGGCAACCAGGCTCTTAACGAAGAGGACATTAAGGACGCTACTGAGGACGACATGCTTGCTAGAACTTTCAGCAGAGGCAGCTACCAGGACAGCCTTAGAGCTTCTATTCGTCAGCGTTCTAAGTCTCAGCTTTCTTACCTGGTGCACGAACCTCCTCTTGCTGTGGTGGACCACAAGTCTACTTACGAAGAGGACAGAAAGGACAAGGACATTCCTGTGCAGGAAGAAGTGGAACCTGCTCCTGTGAGAAGAATTCTGAAGTTCAGCGCTCCTGAATGGCCTTACATGCTGGTGGGCTCTGTGGGCGCTGCTGTGAACGGCACAGTGACACCTCTGTACGCTTTCCTTTTCAGCCAGATTCTTGGCACTTTCTCTATTCCTGACAAGGAAGAACAGAGATCTCAGATTAACGGCGTGTGCCTTCTTTTCGTGGCTATGGGCTGCGTGTCTCTTTTCACTCAGTTCCTTCAGGGCTACGCTTTCGCTAAGTCTGGCGAACTGCTTACAAAGAGACTTCGTAAGTTCGGCTTCAGAGCTATGCTGGGCCAGGACATTGCTTGGTTCGACGACCTGAGAAACAGCCCTGGCGCTCTGACAACAAGACTTGCTACAGACGCTTCTCAGGTGCAGGGCGCTGCTGGCTCTCAGATTGGCATGATTGTGAACTCTTTCACTAACGTGACTGTGGCTATGATTATTGCTTTCTCTTTCAGCTGGAAGCTGAGCCTGGTGATTCTGTGCTTCTTCCCTTTCCTGGCTCTTTCTGGCGCTACACAGACTAGAATGCTGACAGGCTTCGCTTCTCGTGACAAGCAGGCTCTGGAAATGGTGGGCCAGATTACAAACGAAGCTCTGAGCAACATTCGTACTGTGGCTGGCATTGGCAAGGAAAGACGTTTCATTGAAGCTCTTGAAACTGAACTGGAAAAGCCTTTCAAGACAGCTATTCAGAAGGCTAACATTTACGGCTTCTGCTTCGCTTTCGCTCAGTGCATTATGTTCATTGCTAACTCTGCTTCTTACAGATACGGCGGCTACCTTATTTCTAACGAAGGCCTGCACTTCAGCTACGTGTTCAGAGTGATTTCTGCTGTGGTGCTGAGCGCTACAGCTCTTGGCAGAGCTTTCTCTTACACTCCTAGCTACGCTAAGGCTAAGATTTCTGCTGCTCGTTTCTTCCAGCTGCTGGACCGTCAGCCTCCTATTAGCGTGTACAACACTGCTGGCGAAAAGTGGGACAACTTCCAGGGCAAGATTGACTTCGTGGACTGCAAGTTCACATACCCTTCTCGTCCTGACTCTCAGGTGCTGAACGGCCTGTCTGTGTCTATTAGCCCTGGCCAGACACTGGCTTTCGTGGGCAGCAGCGGCTGCGGCAAGAGCACTAGCATTCAGCTGCTGGAACGTTTCTACGACCCTGACCAGGGCAAGGTGATGATTGACGGCCACGACAGCAAGAAGGTGAACGTGCAGTTCCTGCGTTCTAACATTGGCATTGTGTCTCAGGAACCTGTGCTGTTCGCTTGCAGCATTATGGACAACATTAAGTACGGCGACAACACTAAGGAAATTCCTATGGAAAGAGTGATTGCTGCTGCTAAGCAGGCTCAGCTGCACGACTTCGTGATGTCTCTGCCTGAAAAGTACGAAACTAACGTGGGCTCTCAGGGCTCTCAGCTGTCTAGAGGCGAAAAGCAGCGTATTGCTATTGCTCGTGCTATTGTGCGTGACCCTAAGATTCTGCTTCTTGACGAAGCTACTTCTGCTCTTGACACAGAAAGCGAAAAGACAGTGCAGGTGGCTCTTGACAAGGCTAGAGAAGGCCGTACTTGCATTGTGATTGCTCACCGTCTGTCTACTATTCAGAACGCTGACATTATTGCTGTGATGGCTCAGGGCGTGGTGATTGAAAAGGGCACTCACGAAGAACTGATGGCTCAGAAGGGCGCTTACTACAAGCTTGTGACTACTGGCTCTCCTATTAGC

[0249] SEQ ID NO: 15 (DNA of SEQ ID NO: 5)

[0250] ATGAGCGACAGCGTGATCCTCAGGAGCATCAAGAAGTTCGGCGAAGAAAACGACGGCTTCGAAAGCGACAAGAGCTACAACAACGACAAGAAGAGCAGGCTCCAGGACGAAAAGAAGGGCGACGGCGTGAGGGTGGGCTTCTTCCAGCTCTTCAGGTTCAGCAGCAGCACAGACATCTGGCTCATGTTCGTGGGCAGCCTCTGCGCCTTCCTCCACGGCATCGCCCAGCCAGGCGTGCTCCTCATCTTCGGCACAATGACAGACGTGTTCATCGACTACGACGTGGAACTCCAGGAACTCCAGATCCCAGGCAAGGCCTGCGTGAACAACACAATCGTGTGGACAAACAGCAGCCTCAACCAGAACATGACAAACGGCACAAGGTGCGGCCTCCTCAACATCGAAAGCGAAATGATCAAGTTCGCCAGCTACTACGCCGGCATCGCCGTGGCCGTGCTCATCACAGGCTACATCCAGATCTGCTTCTGGGTGATCGCCGCCGCCAGGCAGATCCAGAAGATGAGGAAGTTCTACTTCAGGAGGATCATGAGGATGGAAATCGGCTGGTTCGACTGCAACAGCGTGGGCGAACTCAACACAAGGTTCAGCGACGACATCAACAAGATCAACGACGCCATCGCCGACCAGATGGCCCTCTTCATCCAGAGGATGACAAGCACAATCTGCGGCTTCCTCCTCGGCTTCTTCAGGGGCTGGAAGCTCACACTCGTGATCATCAGCGTGAGCCCACTCATCGGCATCGGCGCCGCCACAATCGGCCTCAGCGTGAGCAAGTTCACAGACTACGAACTCAAGGCCTACGCCAAGGCCGGCGTGGTGGCCGACGAAGTGATCAGCAGCATGAGGACAGTGGCCGCCTTCGGCGGCGAAAAGAGGGAAGTGGAAAGGTACGAAAAGAACCTCGTGTTCGCCCAGAGGTGGGGCATCAGGAAGGGCATCGTGATGGGCTTCTTCACAGGCTTCGTGTGGTGCCTCATCTTCCTCTGCTACGCCCTCGCCTTCTGGTACGGCAGCACACTCGTGCTCGACGAAGGCGAATACACACCAGGCACACTCGTGCAGATCTTCCTCAGCGTGATCGTGGGCGCCCTCAACCTCGGCAACGCCAGCCCATGCCTCGAAGCCTTCGCCACAGGCAGGGCCGCCGCCACAAGCATCTTCGAAACAATCGACAGGAAGCCAATCATCGACTGCATGAGCGAGGACGGCTACAAGCTCGACAGGATCAAGGGCGAAATCGAATTCCACAACGTGACATTCCACTACCCAAGCAGGCCAGAAGTGAAGATCCTCAACGACCTCAACATGGTGATCAAGCCAGGCGAAATGACAGCCCTCGTGGGCCCAAGCGGCGCCGGCAAGAGCACAGCCCTCCAGCTCATCCAGAGGTTCTACGACCCATGCGAAGGCATGGTGACAGTGGACGGCCACGACATCAGGAGCCTCAACATCCAGTGGCTCAGGGACCAGATCGGCATCGTGGAACAGGAACCAGTGCTCTTCAGCACAACAATCGCCGAAAACATCAGGTACGGCAGGGAGGACGCCACAATGGAGGACATCGTGCAGGCCGCCAAGGAAGCCAACGCCTACAACTTCATCATGGACCTCCCACAGCAGTTCGACACACTCGTGGGCGAAGGCGGCGGCCAGATGAGCGGCGGCCAGAAGCAGAGGGTGGCCATCGCCAGGGCCCTCATCAGGAACCCAAAGATCCTCCTCCTCGACATGGCCACAAGCGCCCTCGACAACGAAAGCGAAGCCATGGTGCAGGAAGTGCTCAGCAAGATCCAGCACGGCCACACAATCATCAGCGTGGCCCACAGGCTCAGCACAGTGAGGGCCGCCGACACAATCATCGGCTTCGAACACGGCACAGCCGTGGAAAGGGGCACACACGAAGAACTCCTCGAAAGGAAGGGCGTGTACTTCACACTCGTGACACTCCAGAGCCAGGGCAACCAGGCCCTCAACGAAGAGGACATCAAGGACGCCACAGAGGACGACATGCTCGCCAGGACATTCAGCAGGGGCAGCTACCAGGACAGCCTCAGGGCCAGCATCAGGCAGAGGAGCAAGAGCCAGCTCAGCTACCTCGTGCACGAACCACCACTCGCCGTGGTGGACCACAAGAGCACATACGAAGAGGACAGGAAGGACAAGGACATCCCAGTGCAGGAAGAAGTGGAACCAGCCCCAGTGAGGAGGATCCTCAAGTTCAGCGCCCCAGAATGGCCATACATGCTCGTGGGCAGCGTGGGCGCCGCCGTGAACGGCACAGTGACACCACTCTACGCCTTCCTCTTCAGCCAGATCCTCGGCACATTCAGCATCCCAGACAAGGAAGAACAGAGGAGCCAGATCAACGGCGTGTGCCTCCTCTTCGTGGCCATGGGCTGCGTGAGCCTCTTCACACAGTTCCTCCAGGGCTACGCCTTCGCCAAGAGCGGCGAACTCCTCACAAAGAGGCTCAGGAAGTTCGGCTTCAGGGCCATGCTCGGCCAGGACATCGCCTGGTTCGACGACCTCAGGAACAGCCCAGGCGCCCTCACAACAAGGCTCGCCACAGACGCCAGCCAGGTGCAGGGCGCCGCCGGCAGCCAGATCGGCATGATCGTGAACAGCTTCACAAACGTGACAGTGGCCATGATCATCGCCTTCAGCTTCAGCTGGAAGCTCAGCCTCGTGATCCTCTGCTTCTTCCCATTCCTCGCCCTCAGCGGCGCCACACAGACAAGGATGCTCACAGGCTTCGCCAGCAGGGACAAGCAGGCCCTCGAAATGGTGGGCCAGATCACAAACGAAGCCCTCAGCAACATCAGGACAGTGGCCGGCATCGGCAAGGAAAGGAGGTTCATCGAAGCCCTCGAAACAGAACTCGAAAAGCCATTCAAGACAGCCATCCAGAAGGCCAACATCTACGGCTTCTGCTTCGCCTTCGCCCAGTGCATCATGTTCATCGCCAACAGCGCCAGCTACAGGTACGGCGGCTACCTCATCAGCAACGAAGGCCTCCACTTCAGCTACGTGTTCAGGGTGATCAGCGCCGTGGTGCTCAGCGCCACAGCCCTCGGCAGGGCCTTCAGCTACACACCAAGCTACGCCAAGGCCAAGATCAGCGCCGCCAGGTTCTTCCAGCTCCTCGACAGGCAGCCACCAATCAGCGTGTACAACACAGCCGGCGAAAAGTGGGACAACTTCCAGGGCAAGATCGACTTCGTGGACTGCAAGTTCACATACCCAAGCAGGCCAGACAGCCAGGTGCTCAACGGCCTCAGCGTGAGCATCAGCCCAGGCCAGACACTCGCCTTCGTGGGCAGCAGCGGCTGCGGCAAGAGCACAAGCATCCAGCTCCTCGAAAGGTTCTACGACCCAGACCAGGGCAAGGTGATGATCGACGGCCACGACAGCAAGAAGGTGAACGTGCAGTTCCTCAGGAGCAACATCGGCATCGTGAGCCAGGAACCAGTGCTCTTCGCCTGCAGCATCATGGACAACATCAAGTACGGCGACAACACAAAGGAAATCCCAATGGAAAGGGTGATCGCCGCCGCCAAGCAGGCCCAGCTCCACGACTTCGTGATGAGCCTCCCAGAAAAGTACGAAACAAACGTGGGCAGCCAGGGCAGCCAGCTCAGCAGGGGCGAAAAGCAGAGGATCGCCATCGCCAGGGCCATCGTGAGGGACCCAAAGATCCTCCTCCTCGACGAAGCCACAAGCGCCCTCGACACAGAAAGCGAAAAGACAGTGCAGGTGGCCCTCGACAAGGCCAGGGAAGGCAGGACATGCATCGTGATCGCCCACAGGCTCAGCACAATCCAGAACGCCGACATCATCGCCGTGATGGCCCAGGGCGTGGTGATCGAAAAGGGCACACACGAAGAACTCATGGCCCAGAAGGGCGCCTACTACAAGCTCGTGACAACAGGCAGCCCAATCAGC

[0251] SEQ ID NO: 16 (DNA of SEQ ID NO: 6)

[0252] ATGAGCGACAGCGTGATCCTGCGGAGCATCAAGAAGTTCGGCGAGGAGAACGACGGCTTCGAGAGCGACAAGAGCTACAACAACGACAAGAAGAGCCGGCTGCAGGACGAGAAGAAGGGCGACGGCGTGCGGGTGGGCTTCTTCCAGCTGTTCCGGTTCAGCAGCAGCACCGACATCTGGCTGATGTTCGTGGGCAGCCTGTGCGCCTTCCTGCACGGCATCGCCCAGCCCGGCGTGCTGCTGATCTTCGGCACCATGACCGACGTGTTCATCGACTACGACGTGGAGCTGCAGGAGCTGCAGATCCCCGGCAAGGCCTGCGTGAACAACACCATCGTGTGGACCAACAGCAGCCTGAACCAGAACATGACCAACGGCACCCGGTGCGGCCTGCTGAACATCGAGAGCGAGATGATCAAGTTCGCCAGCTACTACGCCGGCATCGCCGTGGCCGTGCTGATCACCGGCTACATCCAGATCTGCTTCTGGGTGATCGCCGCCGCCCGGCAGATCCAGAAGATGCGGAAGTTCTACTTCCGGCGGATCATGCGGATGGAGATCGGCTGGTTCGACTGCAACAGCGTGGGCGAGCTGAACACCCGGTTCAGCGACGACATCAACAAGATCAACGACGCCATCGCCGACCAGATGGCCCTGTTCATCCAGCGGATGACCAGCACCATCTGCGGCTTCCTGCTGGGCTTCTTCCGGGGCTGGAAGCTGACCCTGGTGATCATCAGCGTGAGCCCCCTGATCGGCATCGGCGCCGCCACCATCGGCCTGAGCGTGAGCAAGTTCACCGACTACGAGCTGAAGGCCTACGCCAAGGCCGGCGTGGTGGCCGACGAGGTGATCAGCAGCATGCGGACCGTGGCCGCCTTCGGCGGCGAGAAGCGGGAGGTGGAGCGGTACGAGAAGAACCTGGTGTTCGCCCAGCGGTGGGGCATCCGGAAGGGCATCGTGATGGGCTTCTTCACCGGCTTCGTGTGGTGCCTGATCTTCCTGTGCTACGCCCTGGCCTTCTGGTACGGCAGCACCCTGGTGCTGGACGAGGGCGAGTACACCCCCGGCACCCTGGTGCAGATCTTCCTGAGCGTGATCGTGGGCGCCCTGAACCTGGGCAACGCCAGCCCCTGCCTGGAGGCCTTCGCCACCGGCCGGGCCGCCGCCACCAGCATCTTCGAGACCATCGACCGGAAGCCCATCATCGACTGCATGAGCGAGGACGGCTACAAGCTGGACCGGATCAAGGGCGAGATCGAGTTCCACAACGTGACCTTCCACTACCCCAGCCGGCCCGAGGTGAAGATCCTGAACGACCTGAACATGGTGATCAAGCCCGGCGAGATGACCGCCCTGGTGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCTGCAGCTGATCCAGCGGTTCTACGACCCCTGCGAGGGCATGGTGACCGTGGACGGCCACGACATCCGGAGCCTGAACATCCAGTGGCTGCGGGACCAGATCGGCATCGTGGAGCAGGAGCCCGTGCTGTTCAGCACCACCATCGCCGAGAACATCCGGTACGGCCGGGAGGACGCCACCATGGAGGACATCGTGCAGGCCGCCAAGGAGGCCAACGCCTACAACTTCATCATGGACCTGCCCCAGCAGTTCGACACCCTGGTGGGCGAGGGCGGCGGCCAGATGAGCGGCGGCCAGAAGCAGCGGGTGGCCATCGCCCGGGCCCTGATCCGGAACCCCAAGATCCTGCTGCTGGACATGGCCACCAGCGCCCTGGACAACGAGAGCGAGGCCATGGTGCAGGAGGTGCTGAGCAAGATCCAGCACGGCCACACCATCATCAGCGTGGCCCACCGGCTGAGCACCGTGCGGGCCGCCGACACCATCATCGGCTTCGAGCACGGCACCGCCGTGGAGCGGGGCACCCACGAGGAGCTGCTGGAGCGGAAGGGCGTGTACTTCACCCTGGTGACCCTGCAGAGCCAGGGCAACCAGGCCCTGAACGAGGAGGACATCAAGGACGCCACCGAGGACGACATGCTGGCCCGGACCTTCAGCCGGGGCAGCTACCAGGACAGCCTGCGGGCCAGCATCCGGCAGCGGAGCAAGAGCCAGCTGAGCTACCTGGTGCACGAGCCCCCCCTGGCCGTGGTGGACCACAAGAGCACCTACGAGGAGGACCGGAAGGACAAGGACATCCCCGTGCAGGAGGAGGTGGAGCCCGCCCCCGTGCGGCGGATCCTGAAGTTCAGCGCCCCCGAGTGGCCCTACATGCTGGTGGGCAGCGTGGGCGCCGCCGTGAACGGCACCGTGACCCCCCTGTACGCCTTCCTGTTCAGCCAGATCCTGGGCACCTTCAGCATCCCCGACAAGGAGGAGCAGCGGAGCCAGATCAACGGCGTGTGCCTGCTGTTCGTGGCCATGGGCTGCGTGAGCCTGTTCACCCAGTTCCTGCAGGGCTACGCCTTCGCCAAGAGCGGCGAGCTGCTGACCAAGCGGCTGCGGAAGTTCGGCTTCCGGGCCATGCTGGGCCAGGACATCGCCTGGTTCGACGACCTGCGGAACAGCCCCGGCGCCCTGACCACCCGGCTGGCCACCGACGCCAGCCAGGTGCAGGGCGCCGCCGGCAGCCAGATCGGCATGATCGTGAACAGCTTCACCAACGTGACCGTGGCCATGATCATCGCCTTCAGCTTCAGCTGGAAGCTGAGCCTGGTGATCCTGTGCTTCTTCCCCTTCCTGGCCCTGAGCGGCGCCACCCAGACCCGGATGCTGACCGGCTTCGCCAGCCGGGACAAGCAGGCCCTGGAGATGGTGGGCCAGATCACCAACGAGGCCCTGAGCAACATCCGGACCGTGGCCGGCATCGGCAAGGAGCGGCGGTTCATCGAGGCCCTGGAGACCGAGCTGGAGAAGCCCTTCAAGACCGCCATCCAGAAGGCCAACATCTACGGCTTCTGCTTCGCCTTCGCCCAGTGCATCATGTTCATCGCCAACAGCGCCAGCTACCGGTACGGCGGCTACCTGATCAGCAACGAGGGCCTGCACTTCAGCTACGTGTTCCGGGTGATCAGCGCCGTGGTGCTGAGCGCCACCGCCCTGGGCCGGGCCTTCAGCTACACCCCCAGCTACGCCAAGGCCAAGATCAGCGCCGCCCGGTTCTTCCAGCTGCTGGACCGGCAGCCCCCCATCAGCGTGTACAACACCGCCGGCGAGAAGTGGGACAACTTCCAGGGCAAGATCGACTTCGTGGACTGCAAGTTCACCTACCCCAGCCGGCCCGACAGCCAGGTGCTGAACGGCCTGAGCGTGAGCATCAGCCCCGGCCAGACCCTGGCCTTCGTGGGCAGCAGCGGCTGCGGCAAGAGCACCAGCATCCAGCTGCTGGAGCGGTTCTACGACCCCGACCAGGGCAAGGTGATGATCGACGGCCACGACAGCAAGAAGGTGAACGTGCAGTTCCTGCGGAGCAACATCGGCATCGTGAGCCAGGAGCCCGTGCTGTTCGCCTGCAGCATCATGGACAACATCAAGTACGGCGACAACACCAAGGAGATCCCCATGGAGCGGGTGATCGCCGCCGCCAAGCAGGCCCAGCTGCACGACTTCGTGATGAGCCTGCCCGAGAAGTACGAGACCAACGTGGGCAGCCAGGGCAGCCAGCTGAGCCGGGGCGAGAAGCAGCGGATCGCCATCGCCCGGGCCATCGTGCGGGACCCCAAGATCCTGCTGCTGGACGAGGCCACCAGCGCCCTGGACACCGAGAGCGAAAAGACCGTGCAGGTGGCCCTGGACAAGGCCCGGGAGGGCCGGACCTGCATCGTGATCGCCCACCGGCTGAGCACCAT CCAGAACGCCGACATCATCGCCGTGATGGCCCAGGGCGTGGTGATCGAGAAGGG CACCCACGAGGAGCTGATGGCCCAGAAGGGCGCCTACTACAAGCTGGTGACCAC CGGCAGCCCCATCAGC

[0253] (TRIPLE STOP CODON)

[0254] AUAAGUGAA

[0255] (Kozak Sequence)

[0256] GCCACC

[0257] SEQ ID NO: 19 (Full LNP-encapsulated mRNA sequence: 5'UTR - Kozak - SEQ ID NO:2 - Stop - 3'UTR - PolyA)GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAGCGACAGCGUGAUCCUGCGGAGCAUCAAGAAGUUCGGCGAGGAGAACGACGGCUUCGAGAGCGACAAGAGCUACAACAACGACAAGAAAAGCCGGCUGCAGGACGAGAAGAAGGGCGACGGCGUGCGGGUGGGCUUCUUCCAGCUGUUCCGGUUCAGCAGCAGCACCGACAUCUGGCUGAUGUUCGUGGGCAGCCUGUGCGCCUUCCUGCACGGCAUCGCCCAGCCCGGCGUGCUGCUGAUCUUCGGCACCAUGACCGACGUGUUCAUCGACUACGACGUGGAGCUGCAGGAGCUGCAGAUCCCCGGCAAGGCCUGCGUGAACAACACCAUCGUGUGGACCAACAGCAGCCUGAACCAGAACAUGACCAACGGCACCCGGUGCGGCCUGCUGAACAUCGAGAGCGAGAUGAUCAAGUUCGCCAGCUACUACGCCGGCAUCGCCGUGGCCGUGCUGAUCACCGGCUACAUCCAGAUCUGCUUCUGGGUGAUCGCCGCCGCCCGGCAGAUCCAGAAGAUGCGGAAGUUCUACUUCCGGCGGAUCAUGCGGAUGGAGAUCGGCUGGUUCGACUGCAACAGCGUGGGCGAGCUGAACACCCGGUUCAGCGACGACAUCAACAAGAUCAACGACGCCAUCGCCGACCAGAUGGCCCUGUUCAUCCAGCGGAUGACCAGCACCAUCUGCGGCUUCCUGCUGGGCUUCUUCCGGGGCUGGAAGCUGACCCUGGUGAUCAUCAGCGUGAGCCCCCUGAUCGGCAUCGGCGCCGCCACCAUCGGCCUGAGCGUGAGCAAGUUCACCGACUACGAGCUGAAGGCCUACGCCAAGGCCGGCGUGGUGGCCGACGAGGUGAUCAGCAGCAUGCGGACCGUGGCCGCCUUCGGCGGCGAGAAGCGGGAGGUGGAGCGGUACGAGAAGAACCUGGUGUUCGCCCAGCGGUGGGGCAUCCGGAAGGGCAUCGUGAUGGGCUUCUUCACCGGCUUCGUGUGGUGCCUGAUCUUCCUGUGCUACGCCCUGGCCUUCUGGUACGGCAGCACCCUGGUGCUGGACGAGGGCGAGUACACCCCCGGCACCCUGGUGCAGAUCUUCCUGAGCGUGAUCGUGGGCGCCCUGAACCUGGGCAACGCCAGCCCCUGCCUGGAGGCCUUCGCCACCGGCCGGGCCGCCGCCACCAGCAUCUUCGAGACCAUCGACCGGAAGCCCAUCAUCGACUGCAUGAGCGAGGACGGCUACAAGCUGGACCGGAUCAAGGGCGAGAUCGAGUUCCACAACGUGACCUUCCACUACCCCAGCCGGCCCGAGGUGAAGAUCCUGAACGACCUGAACAUGGUGAUCAAGCCCGGCGAGAUGACCGCCCUGGUGGGCCCCAGCGGCGCCGGCAAGAGCACCGCCCUGCAGCUGAUCCAGCGGUUCUACGACCCCUGCGAGGGCAUGGUGACCGUGGACGGCCACGACAUCCGGAGCCUGAACAUCCAGUGGCUGCGGGACCAGAUCGGCAUCGUGGAGCAGGAGCCCGUGCUGUUCAGCACCACCAUCGCCGAGAACAUCCGGUACGGCCGGGAGGACGCCACCAUGGAGGACAUCGUGCAGGCCGCCAAGGAGGCCAACGCCUACAACUUCAUCAUGGACCUGCCCCAGCAGUUCGACACCCUGGUGGGCGAGGGCGGCGGCCAGAUGAGCGGCGGCCAGAAGCAGCGGGUGGCCAUCGCCCGGGCCCUGAUCCGGAACCCCAAGAUCCUGCUGCUGGACAUGGCCACCAGCGCCCUGGACAACGAGAGCGAGGCCAUGGUGCAGGAGGUGCUGAGCAAGAUCCAGCACGGCCACACCAUCAUCAGCGUGGCCCACCGGCUGAGCACCGUGCGGGCCGCCGACACCAUCAUCGGCUUCGAGCACGGCACCGCCGUGGAGCGGGGCACCCACGAGGAGCUGCUGGAGCGGAAGGGCGUGUACUUCACCCUGGUGACCCUGCAGAGCCAGGGCAACCAGGCCCUGAACGAGGAGGACAUCAAGGACGCCACCGAGGACGACAUGCUGGCCCGGACCUUCAGCCGGGGCAGCUACCAGGACAGCCUGCGGGCCAGCAUCCGGCAGCGGAGCAAGAGCCAGCUGAGCUACCUGGUGCACGAGCCCCCCCUGGCCGUGGUGGACCACAAGAGCACCUACGAGGAGGACCGGAAGGACAAGGACAUCCCCGUGCAGGAGGAGGUGGAGCCCGCCCCCGUGCGGCGGAUCCUGAAGUUCAGCGCCCCCGAGUGGCCCUACAUGCUGGUGGGCAGCGUGGGCGCCGCCGUGAACGGCACCGUGACCCCCCUGUACGCCUUCCUGUUCAGCCAGAUCCUGGGCACCUUCAGCAUCCCCGACAAGGAGGAGCAGCGGAGCCAGAUCAACGGCGUGUGCCUGCUGUUCGUGGCCAUGGGCUGCGUGAGCCUGUUCACCCAGUUCCUGCAGGGCUACGCCUUCGCCAAGAGCGGCGAGCUGCUGACCAAGCGGCUGCGGAAGUUCGGCUUCCGGGCCAUGCUGGGCCAGGACAUCGCCUGGUUCGACGACCUGCGGAACAGCCCCGGCGCCCUGACCACCCGGCUGGCCACCGACGCCAGCCAGGUGCAGGGCGCCGCCGGCAGCCAGAUCGGCAUGAUCGUGAACAGCUUCACCAACGUGACCGUGGCCAUGAUCAUCGCCUUCAGCUUCAGCUGGAAGCUGAGCCUGGUGAUCCUGUGCUUCUUCCCCUUCCUGGCCCUGAGCGGCGCCACCCAGACCCGGAUGCUGACCGGCUUCGCCAGCCGGGACAAGCAGGCCCUGGAGAUGGUGGGCCAGAUCACCAACGAGGCCCUGAGCAACAUCCGGACCGUGGCCGGCAUCGGCAAGGAGCGGCGGUUCAUCGAGGCCCUGGAGACCGAGCUGGAGAAGCCCUUCAAGACCGCCAUCCAGAAGGCCAACAUCUACGGCUUCUGCUUCGCCUUCGCCCAGUGCAUCA UGUUCAUCGCCAACAGCGCCAGCUACCGGUACGGCGGCUACCUGAUCAGCAAC GAGGGCCUGCACUUCAGCUACGUGUUCCGGGUGAUCAGCGCCGUGGUGCUGAGCGCCACCGCCCUGGGCCGGGCCUUCAGCUACACCCCCAGCUACGCCAAGGCCAAGAUCAGCGCCGCCCGGUUCUUCCAGCUGCUGGACCGGCAGCCCCCCAUCAGCGUGUACAACACCGCCGGCGAGAAGUGGGACAACUUCCAGGGCAAGAUCGACUUCGUGGACUGCAAGUUCACCUACCCCAGCCGGCCCGACAGCCAGGUGCUGAACGGCCUGAGCGUGAGCAUCAGCCCCGGCCAGACCCUGGCCUUCGUGGGCAGCAGCGGCUGCGGCAAGAGCACCAGCAUCCAGCUGCUGGAGCGGUUCUACGACCCCGACCAGGGCAAGGUGAUGAUCGACGGCCACGACAGCAAGAAGGUGAACGUGCAGUUCCUGCGGAGCAACAUCGGCAUCGUGAGCCAGGAGCCCGUGCUGUUCGCCUGCAGCAUCAUGGACAACAUCAAGUACGGCGACAACACCAAGGAGAUCCCCAUGGAGCGGGUGAUCGCCGCCGCCAAGCAGGCCCAGCUGCACGACUUCGUGAUGAGCCUGCCCGAGAAGUACGAGACCAACGUGGGCAGCCAGGGCAGCCAGCUGAGCCGGGGCGAGAAGCAGCGGAUCGCCAUCGCCCGGGCCAUCGUGCGGGACCCCAAGAUCCUGCUGCUGGACGAGGCCACCAGCGCCCUGGACACCGAGAGCGAAAAGACCGUGCAGGUGGCCCUGGACAAGGCCCGGGAGGGCCGGACCUGCAUCGUGAUCGCCCACCGGCUGAGCACCAUCCAGAACGCCGACAUCAUCGCCGUGAUGGCCCAGGGCGUGGUGAUCGAGAAGGGCACCCACGAGGAGCUGAUGGCCCAGAAGGGCGCCUACUACAAGCUGGUGACCACCGGCAGCCCCAUCAGCUGAGCGGCCGCUUAAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0258] All publications, patents and literature specifically mentioned herein are incorporated by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide for expressing a bile salt export pump (BSEP) protein, or a fragment thereof, wherein the polynucleotide comprises natural and modified nucleotides and is expressible to provide a BSEP protein or fragment thereof having BSEP activity.

2. The polynucleotide of claim 1, wherein the polynucleotide is codon-optimized as compared to BSEP wild-type mRNA.

3. The polynucleotide of claim 1 or 2, wherein the modified nucleotides comprise one or more of5-hydroxycytidine, 5-methylcytidine, 5 -hydroxymethyl cytidine, 5 -carboxy cytidine, 5- formylcytidine, 5 -methoxycytidine, 5 -propynyl cytidine, 2-thiocytidine;5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido- 2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5- bromouridine, 5-iodouridine, 5-fluorouridine; pseudouridine, 2'-O-methyl-pseudouridine, N^hydroxypseudouridine, N1- methylpseudouridine, 2'-O-methyl-N1-methylpseudouridine, N^ethylpseudouridine, N1- hydroxymethylpseudouridine, and arauridine;N6-methyladenosine, 2-aminoadenosine, 3 -methyl adenosine, 7-deazaad enosine, 8- oxoadenosine, inosine; thi enoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-m ethyl guanine.

4. The polynucleotide of claim 1 or 2, wherein the modified nucleotides comprise N1- methylpseudouridines.

5. The polynucleotide of claim 1 or 2, wherein the modified nucleotides comprise 5- methoxyuridines.

6. The polynucleotide of claim 1 or 2, wherein the modified nucleotides comprise pseudouridines.

7. The polynucleotide of claim 1 or 2, wherein the modified nucleotides comprise a combination of N'-methylpseudouridines and 5-methoxyuridines.

8. The polynucleotide of any of claims 1-7, wherein the polynucleotide comprises a 5’- cap, a 5’ untranslated region, a coding region, a 3’ untranslated region, and a tail region.

9. The polynucleotide of any of claims 1-8, wherein the polynucleotide is translatable in a mammalian cell to express BSEP or fragment thereof having BSEP activity.

10. The polynucleotide of any of claims 1-9, wherein the polynucleotide is translatable in a subject in vivo to express BSEP or fragment thereof having BSEP activity.

11. The polynucleotide of any of claims 2-10, wherein the polynucleotide has reduced immunogenicity as compared to a non-codon-optimized polynucleotide.

12. The polynucleotide of any of claims 2-11, wherein the polynucleotide comprises a nucleobase sequence at least 95% identical to a nucleobase sequence selected from SEQ ID NOs: 2-6.

13. The polynucleotide of claim 8, wherein the 5 ’-cap comprises N7-Methyl-Gppp(2’-O- Methyl-A).

14. The polynucleotide of claim 8, wherein the 5 ’ untranslated region comprises or consists of the nucleobase sequence of SEQ ID NO: 8.

15. The polynucleotide of claim 8, wherein the 3’ untranslated region comprises or consists of the nucleobase sequence of SEQ ID NO: 10.

16. The polynucleotide of claim 8, wherein the tail region is a polyA tail region.

17. The polynucleotide of claim 16, wherein the polyA tail region is 60 to 220 adenosine nucleotides (SEQ ID NO: 20).

18. The polynucleotide of claim 17, wherein the polyA tail region is about 80 nucleotides in length.

19. A polynucleotide comprising a nucleobase sequence that is at least 95% identical to a nucleobase sequence selected from SEQ ID NOs: 2-6.

20. The polynucleotide of claim 19, wherein the polynucleotide comprises a nucleobase sequence that is at least 97% identical to a nucleobase sequence selected from SEQ ID NOs: 2-6.

21. The polynucleotide of claim 19, wherein the polynucleotide comprises a nucleobase sequence that is at least 99% identical to a nucleobase sequence selected from SEQ ID NOs: 2-6.

22. The polynucleotide of claim 19, wherein the polynucleotide comprises a nucleobase sequence that is at least 99.9% identical to a nucleobase sequence selected from SEQ ID NOs: 2-6.

23. The polynucleotide of claim 19, wherein the polynucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 2-6.

24. The polynucleotide of claim 19, wherein at least one uridine nucleotide is replaced with a N^methylpseudouridine nucleotide.

25. The polynucleotide of claim 19, wherein all uridine nucleotides are replaced with N1- methylpseudouridine nucleotides.

26. The polynucleotide of claim 19, wherein at least one uridine nucleotide is replaced with a 5-methoxyuridine nucleotide.

27. The polynucleotide of claim 19, wherein all uridine nucleotides are replaced with 5- methoxyuridine nucleotides.

28. The polynucleotide of claim 19, wherein the polynucleotide further comprises a 5’-cap, a 5’ untranslated region, a 3’ untranslated region, and / or a tail region.

29. The polynucleotide of claim 28, wherein the 5’-cap comprises N7-Methyl-Gppp(2’-O- Methyl-A).

30. The polynucleotide of claim 28, wherein the 5’ untranslated region comprises or consists of the nucleobase sequence of SEQ ID NO: 8.

31. The polynucleotide of claim 28, wherein the 3’ untranslated region comprises or consists of the nucleobase sequence of SEQ ID NO: 10.

32. The polynucleotide of claim 28, wherein the tail region is a polyA tail region.

33. The polynucleotide of claim 32, wherein the polyA tail region is 60 to 220 adenosine nucleotides (SEQ ID NO: 20).

34. The polynucleotide of claim 33, wherein the polyA tail region is about 80 nucleotides in length.

35. The polynucleotide of claim 19, wherein the polynucleotide comprises the nucleobase sequence of SEQ ID NO: 19.

36. A composition comprising one or more polynucleotides of any of claims 1-35, and a pharmaceutically acceptable carrier.

37. The composition of claim 36, wherein the carrier comprises a transfection reagent, a lipid nanoparticle, or a liposome.

38. The composition of claim 37, wherein the carrier is a lipid nanoparticle.

39. The composition of claim 38, wherein the lipid nanoparticle comprises a cationic lipid selected from ATX-002, ATX-081, ATX-095, or ATX-126.

40. A composition of any of claims 36-39 for use in medical therapy.

41. A composition of any of claims 36-39 for use in the treatment of a human or animal body.

42. A composition of any of claims 36-39 for use in the treatment of a disease or disorder associated with reduced activity of the BSEP protein in a subject in need thereof.

43. The composition for use of claim 42, wherein the disease is progressive familial intrahepatic cholestasis type 2 (PFIC2).

44. The use of a composition of any of claims 36-39 for preparing or manufacturing a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of the BSEP protein in a subject in need thereof.

45. The use of claim 44, wherein the disease is progressive familial intrahepatic cholestasis type 2 (PFIC2).

46. A method for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with reduced activity of BSEP protein in a subject in need thereof, the method comprising administering to the subject a composition of any of claims 36-39.

47. The method of claim 46, wherein the disease is PFIC2.

48. The method of any of claims 46-47, wherein the administration is intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, nasal, or inhalation.

49. The method of any of claims 46-48, wherein the administration is once daily, weekly, every two weeks, monthly, every two months, quarterly, or yearly.

50. The method of any of claims 46-49, wherein the administration comprises a dose of from 0.01 to 10 mg / kg of the composition.

51. The method of any of claims 46-50, wherein the composition is administered at a dose of about 0.1, 0.3, 0.5, 1, 3, 5, or about 10 mg / kg.

52. The method of any of claims 46-51, wherein the administration increases expression of BSEP.

53. A kit for expressing a BSEP protein in vivo, the kit comprising a 0.1 to 500 mg dose of one or more polynucleotides of any of claims 1-35 and a device for administering the dose.

54. The kit of claim 53, wherein the device is an injection needle, an intravenous needle, or an inhalation device.

55. A polynucleotide comprising a nucleobase sequence that is less than 95% identical to the wild type BSEP coding sequence over the full length BSEP coding sequence of SEQ ID NO: 1, and wherein the polynucleotide comprises a nucleobase sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 2-6.

56. A polynucleotide comprising a nucleobase sequence that is less than 95% identical to the wild type BSEP coding sequence over the full length BSEP coding sequence of SEQ ID NO: 1, and wherein the polynucleotide comprises a nucleobase sequence that is at least 97% identical to a sequence selected from SEQ ID NOs: 2-6.

57. A polynucleotide comprising a nucleobase sequence that is less than 95% identical to the wild type BSEP coding sequence over the full length BSEP coding sequence of SEQ ID NO: 1, and wherein the polynucleotide comprises a nucleobase sequence that is at least 99% identical to a sequence selected from SEQ ID NOs: 2-6.

58. A polynucleotide comprising a nucleobase sequence that is less than 95% identical to the wild type BSEP coding sequence over the full length BSEP coding sequence of SEQ ID NO: 1, and wherein the polynucleotide comprises a nucleobase sequence that is at least 99.9% identical to a sequence selected from SEQ ID NOs: 2-6.

59. A polynucleotide comprising a nucleobase sequence that is at least 95% identical to SEQ ID NO: 2.

60. A polynucleotide comprising a nucleobase sequence that is at least 99% identical to SEQ ID NO: 2.

61. A polynucleotide comprising a nucleobase sequence that is at least 99.9% identical to SEQ ID NO: 2.

62. A polynucleotide comprising the nucleobase sequence of SEQ ID NO: 2.

63. A polynucleotide comprising a nucleobase sequence that is at least 95% identical to SEQ ID NO: 19.

64. A polynucleotide comprising a nucleobase sequence that is at least 99% identical to SEQ ID NO: 19.

65. A polynucleotide comprising the nucleobase sequence of SEQ ID NO: 19.

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