Nucleic acids encoding factor VIII polypeptides with reduced immunogenicity - Patent Application 20070123333

Abstract

The present disclosure provides codon-optimized Factor VIII sequences, vectors and host cells comprising the codon-optimized Factor VIII sequences, polypeptides encoded by the codon-optimized Factor VIII sequences, and methods of producing such polypeptides.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 250,575 (filed September 30, 2021), the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the sequence listing submitted electronically in an xml file (Name: 732714 SA9-486PC.xml; Size: 56,424 bytes; Creation Date: September 27, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] The main obstacle to providing low-cost recombinant FVIII protein to patients is the high cost of commercial production: FVIII protein is poorly expressed in heterologous expression systems, and expression is 2-3 fold lower than for proteins of similar size. (Lynch et al., Hum. Gene. Ther.; 4:259-72 (1993). The low expression of FVIII is due in part to the presence of cis-acting elements in the FVIII coding sequence that inhibit FVIII expression, including transcriptional silencer elements (Hoeben et al., Blood 85:2447-2454 (1995)), matrix attachment-like sequences (MARs) (Fallux et al., Mol. Cell. Biol. 16:4264-4272 (1996)), and transcription elongation inhibitor elements (Koeberl et al., Hum. Gene. Ther.; 6:469-479 (1995)). Thus, there is a need in the art for FVIII sequences that are efficiently expressed in heterologous systems. Summary of the Invention [Means for solving the problem]

[0004] Codon-optimized nucleic acid molecules that encode polypeptides having FVIII activity are disclosed.

[0005] In certain aspects, disclosed herein is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 11, wherein the nucleotide sequence encodes a polypeptide having factor VIII activity. In some embodiments, the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 11. In some embodiments, the nucleotide sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. Also disclosed herein is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11, wherein the nucleotide sequence encodes a polypeptide having factor VIII activity.

[0006] Also disclosed herein is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 58 to 4815 of SEQ ID NO: 11. In some embodiments, the isolated nucleic acid molecule of any one of claims 1 to 5, wherein the nucleotide sequence comprises nucleotides 58 to 4815 of SEQ ID NO: 11.

[0007] In certain aspects, disclosed herein is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 14, wherein the nucleotide sequence encodes a polypeptide having factor VIII activity. In some embodiments, the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the nucleotide sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. Also disclosed herein is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14, wherein the nucleotide sequence encodes a polypeptide having factor VIII activity.

[0008] In another aspect, disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII polypeptide, wherein the gene cassette comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. Also disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII polypeptide, wherein the gene cassette comprises the nucleotide sequence of SEQ ID NO: 16.

[0009] In another aspect, disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII polypeptide, the isolated nucleic acid molecule comprising: a nucleotide sequence encoding a FVIII protein comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:11 or SEQ ID NO:14, a promoter controlling transcription of the nucleotide sequence, and a transcription termination sequence.

[0010] In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is a mouse transthyretin (mTTR) promoter. In some embodiments, the promoter is a mTTR482 promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:9.

[0011] In some embodiments, the isolated nucleic acid molecule further comprises an enhancer element. In some embodiments, the enhancer element is an mTTR enhancer element. In some embodiments, the mTTR enhancer element comprises the nucleotide sequence of SEQ ID NO:8.

[0012] In some embodiments, the isolated nucleic acid molecule further comprises a synthetic enhancer sequence. In some embodiments, the synthetic enhancer sequence comprises the nucleotide sequence of SEQ ID NO:7.

[0013] In some embodiments, the nucleic acid molecule further comprises a polypurine track (PPT). In some embodiments, the PPT sequence comprises the nucleotide sequence of SEQ ID NO:6.

[0014] In some embodiments, the nucleic acid molecule further comprises a human CMV promoter region sequence. In some embodiments, the CMV promoter region sequence comprises the nucleotide sequence of SEQ ID NO:1.

[0015] In some embodiments, the nucleic acid molecule further comprises a 5' long terminal repeat (LTR) sequence. In some embodiments, the nucleic acid molecule further comprises a 3' LTR sequence.

[0016] In some embodiments, the nucleic acid molecule further comprises a stem-loop 4 sequence. In some embodiments, the stem-loop 4 sequence comprises the nucleotide sequence of SEQ ID NO:4.

[0017] In some embodiments, the nucleic acid molecule further comprises a primer binding site of SL123. In some embodiments, the primer binding site of SL123 comprises the nucleotide sequence of SEQ ID NO:3.

[0018] In some embodiments, the nucleic acid molecule further comprises a primer binding site of an RU5 region. In some embodiments, the RU5 region sequence comprises the nucleotide sequence of SEQ ID NO:2.

[0019] In another aspect, disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII polypeptide, the gene cassette comprising, from 5' to 3': a 5' long terminal repeat (LTR) sequence, a liver-specific modified mouse transthyretin (mTTR) promoter comprising a nucleotide sequence of SEQ ID NO:9, a nucleotide sequence encoding a FVIII protein comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:11 or SEQ ID NO:14, and a 3' LTR sequence.

[0020] In another aspect, disclosed herein is a vector comprising the nucleic acid molecule disclosed herein.

[0021] In another aspect, disclosed herein are host cells that contain the nucleic acid molecules disclosed herein. Also disclosed herein are polypeptides produced by the host cells.

[0022] In another aspect, disclosed herein is a method for producing a polypeptide having FVIII activity, comprising: culturing a host cell disclosed herein under conditions whereby a polypeptide having FVIII activity is produced; and recovering the polypeptide having FVIII activity.

[0023] In another aspect, disclosed herein is a pharmaceutical composition comprising a nucleic acid molecule disclosed herein. In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid molecule disclosed herein. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0024] In another aspect, also disclosed herein is a kit comprising a nucleic acid molecule disclosed herein and instructions for administering the nucleic acid molecule to a subject in need thereof.

[0025] In another aspect, the present specification discloses a method for increasing expression of a polypeptide having FVIII activity in a subject, the method comprising administering a nucleic acid molecule comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:16.

[0026] In another aspect, disclosed herein is a method of treating a bleeding disorder in a subject, comprising administering a nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:16. In some embodiments, the method of treating a bleeding disorder in a subject comprises administering a pharmaceutical composition disclosed herein. In some embodiments, the bleeding disorder is hemophilia A. [Brief description of the drawings]

[0027] [Figure 1] 1 is a graphical representation of the coBDDFVIII6-XTEN-3aa expression plasmid. [Figure 2A] FIG. 2B is a graphical representation of peak circulating FVIII levels in neonatal (2-day-old) HemA mice administered lentivirus expressing coBDDFVIII6-XTEN-3aa at doses of 1.5×109, 3.0×109, 6.0×109, or 1.3×1010 TU / kg via temporal vein injection, as measured by FVIII plasma activity (FIG. 2A) and FVIII plasma antigen levels (FIG. 2B) over approximately 25 weeks. [Figure 2B]FIG. 2B is a graphical representation of peak circulating FVIII levels in neonatal (2-day-old) HemA mice administered lentivirus expressing coBDDFVIII6-XTEN-3aa at doses of 1.5×109, 3.0×109, 6.0×109, or 1.3×1010 TU / kg via temporal vein injection, as measured by FVIII plasma activity (FIG. 2A) and FVIII plasma antigen levels (FIG. 2B) over approximately 25 weeks. [Diagram 3] FIG. 1 is a graphical representation of peak circulating FVIII levels in adult (16 week old) HemA mice administered lentivirus expressing coBDDFVIII6-XTEN-3aa at a dose of 1.3×1010 or 3.7×1010 TU / kg by tail vein injection, as measured by FVIII plasma activity for approximately 25 weeks. [Figure 4A-4B] 4A-4B are graphical representations of peak plasma levels of human FVIII activity (FIG. 4A) and human FVIII antigen levels (FIG. 4B) in male pigtailed macaques administered lentivirus expressing coBDDFVIII6-XTEN-3aa at 3×10 9 TU / kg or 6×10 9 TU / kg. FVIII plasma activity (FIG. 4A) and FVIII plasma antigen levels (FIG. 4B) are shown as averages across multiple time points. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure relates to codon-optimized nucleic acid molecules encoding polypeptides having factor VIII (FVIII) activity, vectors and host cells comprising the optimized nucleic acid molecules, polypeptides encoded by the optimized nucleic acid molecules, and methods of producing such polypeptides. The present disclosure also relates to a method of treating a bleeding disorder, such as hemophilia, comprising administering to a subject an optimized FVIII nucleic acid sequence, a vector comprising the optimized nucleic acid sequence, or a polypeptide encoded thereby.

[0029] The present disclosure fulfills an important need in the art by providing an optimized FVIII sequence that has increased expression in host cells, improved yields of FVIII protein in methods of producing recombinant FVIII, and potentially better therapeutic efficacy when used in gene therapy. In certain embodiments, the present disclosure describes an isolated nucleic acid molecule comprising a nucleotide sequence having sequence homology to the nucleotide sequence of SEQ ID NO: 11. In certain embodiments, the present disclosure describes an isolated nucleic acid molecule comprising a nucleotide sequence having sequence homology to the nucleotide sequence of SEQ ID NO: 14. In certain embodiments, the present disclosure describes an isolated nucleic acid molecule comprising a nucleotide sequence having sequence homology to the nucleotide sequence of SEQ ID NO: 16.

[0030] In order to provide a clear understanding of the specification and claims, the following definitions are set forth below.

[0031] definition It should be noted that the term "a" or "an" refers to one or more of that entity. For example, a nucleotide sequence preceded by "a" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0032] The term "about" is used herein to mean approximately, roughly, around, or in the region. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending its boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value by plus or minus 10 percent (above or below) the set forth value.

[0033] The term "isolated" for purposes of this disclosure refers to biological material (cells, polypeptides, polynucleotides, or fragments, variants, or derivatives thereof) that has been removed from its original environment (the environment in which the material naturally occurs). For example, a polynucleotide that is naturally present in a plant or animal is not isolated, but the same polynucleotide separated from the adjacent nucleic acids in which it naturally occurs is considered to be "isolated." No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells are considered to be isolated for purposes of this disclosure, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0034] "Nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine, "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine, "DNA molecules"), or any phosphoester analogs thereof (such as phosphorothioates and thioesters), either in single-stranded or double-stranded form. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA are also possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit the molecule to any particular tertiary form. Thus, the term includes double-stranded DNA found in, inter alia, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of showing the sequence only from the 5' to 3' end along the non-transcribed strand of DNA (i.e., the strand having sequence homology to mRNA). A "recombinant DNA molecule" is a DNA molecule that has been subjected to molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein.

[0035] As used herein, a "coding region" or "coding sequence" is a polynucleotide portion consisting of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid but can be considered part of the coding region. However, any flanking sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, etc.) are not part of the coding region. The boundaries of a coding region are typically defined by a start codon at the 5'-terminus, which encodes the amino-terminus of the resulting polypeptide, and a translation stop codon at the 3'-terminus, which encodes the carboxyl-terminus of the resulting polypeptide. Two or more coding regions can be present in one polynucleotide construct, e.g., in one vector, or in separate polynucleotide constructs, e.g., separate (different) vectors. That is, a vector can contain only one coding region or can contain two or more coding regions.

[0036] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once the growing protein chain begins to be transported through the rough endoplasmic reticulum. Signal peptides are generally fused to the N-terminus of a polypeptide and are known to those skilled in the art to be cleaved from the complete, or "full-length," polypeptide to create the secreted, or "mature," form of the polypeptide. In certain embodiments, the native signal peptide, or a functional derivative of that sequence that retains the ability to direct secretion of the polypeptide, is operably associated with the polypeptide. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0037] The term "downstream" refers to a nucleotide sequence that is 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence refers to a sequence that follows the transcription start point. For example, the translation start codon of a gene is downstream of the transcription start site.

[0038] The term "upstream" refers to a nucleotide sequence 5' to a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence relates to a coding region or sequence 5' to the transcription start site. For example, most promoters are upstream of the transcription start site.

[0039] As used herein, the terms "gene cassette," "expression cassette," and "gene expression cassette" are used interchangeably and refer to a DNA sequence capable of directing the expression of a particular polynucleotide sequence in a suitable host cell, comprising a promoter operably linked to a polynucleotide sequence of interest. A gene cassette may include nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, which affect transcription, RNA processing, stability, or translation of the associated coding region. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence. In some embodiments, a gene cassette comprises a polynucleotide encoding a gene product. In some embodiments, a gene cassette comprises a polynucleotide encoding a miRNA. In some embodiments, a gene cassette comprises a heterologous polynucleotide sequence. A polynucleotide encoding a product, e.g., a miRNA or a gene product (e.g., a polypeptide such as a therapeutic protein), may comprise a promoter and / or other expression (e.g., transcription or translation) control sequences operably associated with one or more coding regions. In an operable association, a coding region for a gene product is associated with one or more regulatory regions in such a manner that expression of the gene product, e.g., a polypeptide, is under the influence or control of the regulatory regions. For example, a coding region and a promoter are "operably associated" if, upon induction of promoter function, an mRNA encoding the gene product encoded by the coding region is transcribed, and if the nature of the association between the promoter and the coding region does not interfere with the ability of the promoter to induce expression of the gene product or to transcribe a DNA template. Other expression control elements besides promoters, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to induce expression of a gene product.

[0040] "Expression control sequence" refers to a regulatory nucleotide sequence (promoter, enhancer, terminator, etc.) that effects the expression of a coding sequence in a host cell. Expression control sequences generally encompass any regulatory nucleotide sequence that promotes efficient transcription and translation of a coding nucleic acid to which it is operably linked. Non-limiting examples of expression control sequences include promoters, enhancers, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. A variety of expression control sequences are known to those skilled in the art. These regions include expression control sequences that function in vertebrate cells, such as, but are not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter in conjunction with intron A), Simian Virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other expression control sequences include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling expression of genes in eukaryotic cells. Additional suitable expression control sequences include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Other expression control sequences include intron sequences, post-transcriptional regulatory elements, and polyadenylation signals. Additional exemplary expression control sequences are discussed elsewhere in this disclosure.

[0041] Similarly, a variety of translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly, internal ribosome entry sites, or IRES).

[0042] The term "expression" as used herein refers to the process of producing a gene product, e.g., an RNA or a polypeptide, from a polynucleotide. Expression includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of the mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcription product. Gene products as described herein further include nucleic acids that have been post-transcriptionally modified, e.g., polyadenylated or spliced, or polypeptides that have been post-translationally modified, e.g., methylated, glycosylated, lipidated, associated with other protein subunits, or proteolytic cleavage. The term "yield" as used herein refers to the amount of polypeptide produced by expression of a gene.

[0043] A "vector" refers to any vehicle for cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment can be attached, resulting in replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomously replicating unit in vivo, i.e., capable of replication under self-control. The term "vector" includes both viral and non-viral vehicles for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Many vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector having complementary cohesive termini.

[0044] Vectors can be engineered to encode a selectable marker or reporter that allows for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have incorporated and expressed other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include: genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamides, etc., and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0045] The term "selectable marker" refers to an identifying agent, usually an antibiotic or drug resistance gene, a colorimetric marker, an enzyme, a fluorescent marker, etc., that can be selected based on the function of the marker gene, i.e., antibiotic resistance, herbicide resistance, and can be used to track the inheritance of a nucleic acid of interest and / or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes that are known and used in the art include: genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc., and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0046] The term "reporter gene" refers to a nucleic acid that encodes an identifying factor that can be identified based on the action of the reporter gene, which is used to track the inheritance of the nucleic acid of interest, to identify cells or organisms that have inherited the nucleic acid of interest, and / or to measure induction or transcription of gene expression. Examples of reporter genes that are known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable marker genes can also be considered reporter genes.

[0047] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is 3' to the promoter sequence. A promoter may be derived entirely from a native gene, or may be composed of various elements derived from various promoters found in nature, or may even include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can induce expression of a gene in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types at most times is generally referred to as a "constitutive promoter". A promoter that causes a gene to be expressed in a particular cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter". A promoter that causes a gene to be expressed at a particular developmental or cell differentiation stage is generally referred to as a "developmental specific promoter" or "cell differentiation specific promoter". A promoter that is induced to express a gene when cells are exposed to or treated with a promoter-inducing agent, biomolecule, chemical, ligand, light, etc. is generally referred to as an "inducible promoter" or "regulatable promoter". It is further recognized that in most cases, the exact boundaries of regulatory sequences have not been completely defined, so that DNA fragments of different lengths may have the same promoter activity. Further exemplary promoters are discussed elsewhere in this disclosure.

[0048] A promoter sequence is typically linked at its 3' end to a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary for initiation of transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0049] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences from any source, linear, circular or supercoiled, single- or double-stranded DNA or RNA, in which many nucleotide sequences can be combined or recombined into unique constructs that can introduce into a cell promoter fragments and DNA sequences for selected gene products, along with appropriate 3' non-translated sequences.

[0050] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses, such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0051] "Cloning vector" refers to a "replicon," a unit length of nucleic acid that is sequentially replicated, such as a plasmid, phage, or cosmid, and that contains an origin of replication to which another nucleic acid segment can be attached so as to cause replication of the attached segment. A particular cloning vector is capable of replicating in one cell type, e.g., a bacterial cell, and expressing in another cell type, e.g., a eukaryotic cell. Cloning vectors typically contain one or more sequences that can be used for selection of cells that contain the vector and / or one or more multiple cloning sites for insertion of a nucleic acid sequence of interest.

[0052] The term "expression vector" refers to a vehicle designed to allow for the expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operative association with a regulatory region as described above.

[0053] Vectors are introduced into host cells by methods well known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or a DNA vector transporter.

[0054] "Culturing", "culturing" and "culture" as used herein refer to incubating cells under in vitro conditions that allow the cells to grow or divide or that keep the cells alive. "Cultured cells" as used herein refers to cells that have been propagated in vitro.

[0055] As used herein, the term "polypeptide" is intended to include a singular "polypeptide" as well as a plurality of "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or synonymously with any of the above terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but need not necessarily be translated from a specific nucleic acid sequence. Polypeptides can be produced in any manner, including by chemical synthesis.

[0056] The term "amino acid" includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y), and valine (Val or V). Amino acids other than conventional amino acids are also within the scope of the present disclosure, including norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs as described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-natural amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra can be used. Briefly, these procedures involve chemical activation of a suppressor tRNA bearing the non-natural amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of non-conventional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term "polar amino acid" includes amino acids with a net charge of zero, but with non-zero partial charges at different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids whose side chains can have a net charge other than 0 (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

[0057] The present disclosure also includes fragments or variants of the polypeptides, and any combination thereof. When referring to the polypeptide binding domains or binding molecules of the present disclosure, the term "fragment" or "variant" includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., the binding affinity of FcRn for an FcRn binding domain or Fc variant, the coagulation activity of an FVIII variant, or the FVIII binding activity of a VWF fragment). A fragment of a polypeptide includes proteolytic fragments as well as deletion fragments, but does not include the full-length native polypeptide (or the mature polypeptide), in addition to specific antibody fragments discussed elsewhere herein. A variant of a polypeptide binding domain or binding molecule of the present disclosure also includes the above fragments, as well as polypeptides in which the amino acid sequence has been altered by amino acid substitution, deletion, or insertion. A variant can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. A variant polypeptide can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0058] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Thus, if an amino acid in a polypeptide is replaced with another amino acid of the same side chain family, the substitution is considered conservative. In another embodiment, amino acid strings can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0059] The term "percent identity" is known in the art and is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence similarity between polypeptide sequences or polynucleotide sequences, as the case may be, as determined by the match between the sequences. "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity are designed to maximize the match between the sequences examined. Methods for determining identity are codified in publicly available computer programs.Sequence alignment and percent identity calculations can be performed using sequence analysis software such as the program Megalign from the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the programs from the GCG suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)) and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). Within the context of this application, it will be understood that unless otherwise specified, when sequence analysis software is used for analysis, the analysis results will be based on the "default values" of the program to which it refers. As used herein, "default values" shall mean any set of values ​​or parameters that are initially loaded into the software upon initialization. For purposes of determining the percent identity between the BDD optimized FVIII sequence of the present disclosure and a reference sequence, only nucleotides in the reference sequence that correspond to nucleotides in the BDD optimized FVIII sequence of the present disclosure are used to calculate the percent identity. For example, when comparing a full-length FVIII nucleotide sequence including the B domain with an optimized B-domain deleted (BDD) FVIII nucleotide sequence of the present disclosure, the alignment portion including the A1, A2, A3, C1, and C2 domains will be used to calculate the percent identity. Nucleotides in the B-domain coding portion of the full-length FVIII sequence (which are the large "gaps" in the alignment) are not considered to be non-matches. Additionally, when determining the percent identity between the optimized BDD FVIII sequence of the present disclosure or a specified portion thereof (e.g., nucleotides 2183-4474 and 4924-7006 of SEQ ID NO: 16) and a reference sequence, the percent identity will be calculated by dividing the number of matching nucleotides by the total number of nucleotides in the complete sequence of the optimized BDD-FVIII sequence or a specified portion thereof as set forth herein.

[0060] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, immediately upstream of a position at which a heterologous moiety can be inserted. An "insertion site" is designated by a number, which is the number of amino acids in mature native FVIII (SEQ ID NO: 18) that correspond to the insertion site immediately N-terminal to the insertion site. For example, the phrase "a3 contains a heterologous moiety at an insertion site corresponding to amino acid 1656 of SEQ ID NO: 24" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acids 1656 and 1657 of SEQ ID NO: 24.

[0061] The phrase "immediately downstream of an amino acid," as used herein, refers to the position immediately adjacent to the terminal carboxyl group of that amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position immediately adjacent to the terminal amine group of that amino acid.

[0062] The terms "inserted," "inserted into," "inserted into," or grammatically related terms, as used herein, refer to the position of a heterologous moiety in a recombinant FVIII polypeptide compared to the analogous position in native mature human FVIII (SEQ ID NO: 18).

[0063] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be represented by the time required for half of an administered dose to be removed from the circulating blood and / or other tissues of the animal. When a clearance curve of a given polypeptide is constructed as a function of time, it is usually a biphasic curve with a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibrium of an administered Fc polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The β-phase typically represents the catabolism of the polypeptide within the blood vessels. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, i.e., they have no α-phase and only one β-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of a polypeptide in the β-phase.

[0064] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence can be directly linked or juxtaposed to the second amino acid sequence or nucleotide sequence, or an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" not only means that the first amino acid sequence is fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes that the entire first amino acid sequence (or second amino acid sequence) is inserted into any two amino acids in the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. A linker can be a peptide or polypeptide (in the case of a polypeptide chain), or a nucleotide or a chain of nucleotides (in the case of a nucleotide chain), or any chemical moiety (in the case of both polypeptide and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0065] As used herein, the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" refers to a covalent, non-peptide or non-covalent bond. The association can be indicated by a colon, i.e. (:). In another embodiment, the association refers to a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with the thiol group of a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 and 229 in the EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, agostic bonds, bent bonds, dipole bonds, π back-donor bonds, double bonds, triple bonds, quadruple bonds, pentad bonds, sextad bonds, conjugation, hyperconjugation, aromaticity, haptic or antibonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, gold-gold aurophilicity, intercalation, stacking, entropic forces, or chemical polarity.

[0066] "Hemostasis," as used herein, means to stop or slow bleeding or hemorrhage, or to stop or slow the flow of blood through a blood vessel or body part.

[0067] "Hemostatic disorder" as used herein means a genetically inherited or acquired condition characterized by a tendency to bleed heavily, either spontaneously or due to trauma, due to a reduced or incapacitated ability to form fibrin clots. Examples of such disorders include hemophilia. The three major types are hemophilia A (deficiency of factor VIII), hemophilia B (deficiency of factor IX, i.e., "Christmas disease"), and hemophilia C (deficiency of factor XI, mild tendency to bleed). Other hemostatic disorders include, for example, von Willebrand's disease, deficiency of factor XI (deficiency of PTA), deficiency of factor XII, deficiency or structural abnormality of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, Bernard-Soulier syndrome (defect or deficiency of GPIb). Defects in the VWF receptor GPIb result in impaired primary clot formation (primary hemostasis) and increased tendency to bleed, which can lead to Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia). In acute and chronic liver failure, the liver may produce insufficient clotting factors, increasing the risk of bleeding.

[0068] The isolated nucleic acid molecule, isolated polypeptide, or vector comprising the isolated nucleic acid molecule of the present disclosure can be used prophylactically. As used herein, the term "prophylactic treatment" refers to administering the molecule before a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing or about to undergo surgery. The polynucleotide, polypeptide, or vector of the present disclosure can be administered before or after surgery as a prophylactic method. The polynucleotide, polypeptide, or vector of the present disclosure can be administered during or after surgery to control acute bleeding episodes. Surgery includes, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0069] The isolated nucleic acid molecule, isolated polypeptide, or vector of the present disclosure can also be used for on-demand treatment. The term "on-demand treatment" refers to administering the isolated nucleic acid molecule, isolated polypeptide, or vector in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, on-demand treatment can be administered to a subject once bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, on-demand treatment can be administered before an activity that increases the risk of bleeding, such as contact sports.

[0070] As used herein, the term "acute bleeding" refers to a bleeding episode, regardless of the underlying cause. For example, the subject may have trauma, uremia, inherited bleeding disorders (e.g., factor VII deficiency), platelet disorders, or resistance due to the development of antibodies against clotting factors.

[0071] "Treate", "treatment", or "treating" as used herein refers to, for example, reducing the severity of a disease or condition, shortening the course of a disease, ameliorating one or more symptoms associated with a disease or condition, providing a beneficial effect to a subject having a disease or condition (not necessarily curing the disease or condition), or preventing one or more symptoms associated with a disease or condition. In one embodiment, the term "treating" or "treatment" refers to maintaining FVIII trough levels at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL in a subject by administering an isolated nucleic acid molecule, isolated polypeptide, or vector of the disclosure. In another embodiment, treating or treatment means maintaining FVIII trough levels at about 1 to about 20 IU / dL, about 2 to about 20 IU / dL, about 3 to about 20 IU / dL, about 4 to about 20 IU / dL, about 5 to about 20 IU / dL, about 6 to about 20 IU / dL, about 7 to about 20 IU / dL, about 8 to about 20 IU / dL, about 9 to about 20 IU / dL, or about 10 to about 20 IU / dL. Treating a disease or condition or treating a disease or condition can also include maintaining FVIII activity in a subject at a level corresponding to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophilic subject. The minimum trough level required for treatment can be determined by one or more known methods and can be adjusted (raised or lowered) for each individual.

[0072] "Administering" as used herein means providing a subject with a pharma- ceutically acceptable nucleic acid molecule encoding factor VIII, a factor VIII polypeptide, or a vector comprising a nucleic acid molecule encoding factor VIII of the present disclosure via a pharma- ceutically acceptable route. The route of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Additional routes of administration include, e.g., subcutaneous, intraneural, intraocular, intrathecal, intramuscular, oral, nasal, and pulmonary administration. The nucleic acid molecule, polypeptide, and vector can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0073] As used herein, the phrase "subject in need thereof" includes a subject (such as a mammalian subject) that would benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure, for example, to improve hemostasis. In one embodiment, the subject includes, but is not limited to, an individual with hemophilia. In another embodiment, the subject includes, but is not limited to, an individual who has developed an FVIII inhibitor and therefore requires bypass therapy. The subject can be an adult or a minor (e.g., under the age of 12).

[0074] As used herein, the term "clotting factor" refers to a molecule, natural or recombinantly produced, or analog thereof, that prevents or shortens the duration of bleeding episodes in a subject. In other words, the factor refers to a molecule that has procoagulant activity, i.e., a molecule that is responsible for converting fibrinogen into a mesh-like, insoluble fibrin, causing blood to coagulate or clot. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., its zymogen form) that can be converted to an active form.

[0075] "Coagulation activity" as used herein means the ability to form a fibrin clot and / or participate in a cascade of biochemical reactions that reduce the severity, duration or frequency of hemorrhage or bleeding episodes.

[0076] As used herein, the terms "heterologous" or "exogenous" refer to a molecule that is not normally found in a given context, e.g., a cell or a polypeptide. For example, an exogenous or heterologous molecule can be introduced into a cell and is present only after engineering the cell, e.g., by transfection or other forms of genetic modification, or a heterologous amino acid sequence can be present in a protein where that sequence is not found in nature.

[0077] As used herein, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that is not found in nature in a given polynucleotide sequence. In one embodiment, the heterologous nucleotide sequence encodes a polypeptide that can extend the half-life of FVIII. In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of FVIII. In other embodiments, the heterologous nucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of FVIII without significantly affecting the biological activity or function of FVIII (e.g., its procoagulant activity). In some embodiments, FVIII is linked or joined to the polypeptide encoded by the heterologous nucleotide sequence by a linker.

[0078] A "reference nucleotide sequence," when used herein as a comparison sequence to a nucleotide sequence of the present disclosure, is a polynucleotide sequence that is essentially identical to the nucleotide sequence of the present disclosure, except that the portion corresponding to the FVIII sequence has not been optimized.

[0079] As used herein, the term "optimized" refers to a polynucleotide sequence that encodes a polypeptide and that has been mutated to enhance the properties of the polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding of regulatory proteins (such as general transcription factors), increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize the polynucleotide sequence include codon optimization, G / C content optimization, removal of repetitive sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis-acting elements that repress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences around the transcription start site that enhance transcription (such as Kozak consensus sequences), removal of sequences that may form stem-loop structures, removal of destabilizing sequences, removal of CpG motifs, and combinations of two or more thereof.

[0080] Polynucleotide sequence Certain aspects of the present disclosure relate to nucleic acid molecules comprising a genetic cassette encoding, for example, a therapeutic protein and / or a miRNA. In some embodiments, the genetic cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a clotting factor. In some embodiments, the genetic cassette encodes a miRNA. In some embodiments, the nucleic acid molecule further comprises at least one non-coding region. In certain embodiments, the at least one non-coding region comprises a promoter sequence, an intron, an expression control sequence, or any combination thereof.

[0081] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is codon-optimized. In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII driven by an mTTR promoter. In some embodiments, the gene cassette comprises a nucleotide sequence disclosed in International Application No. PCT / US2017 / 015879, the entirety of which is incorporated by reference. In some embodiments, the gene cassette is the gene cassette "hFVIIIco6XTEN" described in International Application No. PCT / US2017 / 015879. In some embodiments, the reference nucleotide sequence corresponds to the hFVIIIco6XTEN sequence disclosed in International Application No. PCT / US2017 / 015879.

[0082] In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding a B-domain deleted (BDD) codon-optimized human Factor VIII molecule. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the gene cassette comprises a nucleotide sequence encoding a coBDDFVIII6-3aa polypeptide.

[0083] In some embodiments, the gene cassette further comprises a nucleotide sequence encoding an XTEN polypeptide. In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding a B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) fused to a 144 amino acid XTEN polypeptide. In some embodiments, the gene cassette comprises a nucleotide sequence set forth as SEQ ID NO: 11. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the gene cassette comprises a nucleotide sequence encoding a coBDDFVIII6-XTEN-3aa polypeptide.

[0084] In some embodiments, the gene cassette comprises a nucleotide sequence set forth as SEQ ID NO: 16. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:16.

[0085] In some embodiments, the present disclosure relates to a codon-optimized nucleic acid molecule encoding a polypeptide having FVIII activity. In some embodiments, the polynucleotide encodes a full-length FVIII polypeptide. In other embodiments, the nucleic acid molecule encodes a B-domain deleted (BDD) FVIII polypeptide, in which all or part of the B-domain of FVIII is deleted. In a particular embodiment, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12 or a fragment thereof.

[0086] In some embodiments, the nucleic acid molecules of the present disclosure encode a FVIII polypeptide that includes a signal peptide or a fragment thereof. In other embodiments, the nucleic acid molecules encode a FVIII polypeptide that lacks a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 13.

[0087] In one embodiment, the gene cassette is a single stranded nucleic acid. In another embodiment, the gene cassette is a double stranded nucleic acid. In another embodiment, the gene cassette is a closed-end double stranded nucleic acid (ceDNA).

[0088] "Polypeptide having FVIII activity" as used herein means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term polypeptide having FVIII activity includes functional fragments, variants, analogs, or derivatives thereof that retain the function of full-length wild-type factor VIII in the coagulation pathway. "Polypeptide having FVIII activity" is used synonymously with FVIII protein, FVIII polypeptide, or FVIII. Examples of FVIII functions include the ability to activate coagulation, act as a cofactor for factor IX, or inhibit Ca.2+ The FVIII activity includes, but is not limited to, the ability to form a tenase complex with factor IX in the presence of FVIII and phospholipids, and then convert factor X to the activated form Xa. In one embodiment, the polypeptide having FVIII activity comprises two polypeptide chains, the first chain of which comprises a FVIII heavy chain and the second chain of which comprises a FVIII light chain. In another embodiment, the polypeptide having FVIII activity is a single-chain FVIII. The single-chain FVIII can comprise one or more mutations or substitutions at amino acid residues at positions 1645 and / or 1648, which correspond to the mature human FVIII sequence (SEQ ID NO: 19). See International Application No. PCT / US2012 / 045784, which is incorporated herein by reference in its entirety. The FVIII protein can be a human FVIII protein, a porcine FVIII protein, a canine FVIII protein, a rat FVIII protein, or a mouse FVIII protein. In addition, a comparison of human FVIII with FVIII from other species has identified conserved residues that are likely required for function. See, e.g., Cameron et al. (1998) Thromb. Haemost. 79:317-22; and U.S. Patent No. 6,251,632.

[0089] A number of tests are available to assess the FVIII activity of a polypeptide: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM® assay, prothrombin time (PT) test (also used to determine the INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether the abnormality is corrected when the patient's plasma is mixed with normal plasma), clotting factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0090] The aPTT test is a performance index that measures the efficacy of the "intrinsic" coagulation pathway (also called the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, e.g., FVIII or FIX. It is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0091] ROTEM® analysis provides information on the global dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. The various parameters of thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that affect their interactions. This assay can provide a complete view of secondary hemostasis.

[0092] A "B domain" of FVIII, as used herein, is a B domain known in the art and is defined by internal amino acid sequence identity and the site of proteolytic cleavage by thrombin, e.g., residues Ser741 to Arg1648 of full-length human FVIII (SEQ ID NO: 20). The other human FVIII domains are defined by the following amino acid residues: A1, amino acid residues Ala1 to Arg372; A2, amino acid residues Ser373 to Arg740; A3, amino acid residues Ser1690 to Ile2032; C1, amino acid residues Arg2033 to Asn2172; and C2, amino acid residues Ser2173 to Tyr2332. The sequence A3-C1-C2 includes residues Ser1690 to Tyr2332. The remaining sequence (residues Glu1649 to Arg1689) is commonly referred to as the FVIII light chain activation peptide. For porcine, murine and canine FVIII, the locations of all domain boundaries, including the B domain, are known in the art. An example of BDD FVIII is the recombinant BDD FVIII called REFACTO® (Wyeth Pharmaceuticals, Inc.). The "B domain deleted FVIII" is described in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,72 The nucleic acid sequence may have a complete or partial deletion as disclosed in US Pat. Nos. 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563 (each of which is incorporated herein by reference in its entirety). Other examples of B domain deleted FVIII are described in Hoeben RC, et al. (1990) J. Biol. Chem. 265(13):7318-7323; Meulien et al. (1988), Protein Eng. 2(4):301-6; Toole et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5939-5942; Eaton, et al. (1986) Biochemistry 25:8343-8347; (Sarver, et (1987) DNA 6:553-564; European Patent Publication No. 295597; and International Publication Nos. WO 91 / 09122, WO 88 / 00831, and WO 87 / 04187, each of which is incorporated herein by reference in its entirety. Each of the above deletions can be made in any FVIII sequence.

[0093] Codon Optimization In one embodiment, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleic acid sequence is codon-optimized. In some embodiments, the sequence encoding the polypeptide having FVIII activity is codon-optimized for expression in humans. In other embodiments, the sequence encoding the polypeptide having FVIII activity is codon-optimized for expression in mice.

[0094] The term "codon optimization," when referring to genes or coding regions of a nucleic acid molecule intended for transformation of various hosts, refers to modifying the codons of the genes or coding regions of the nucleic acid molecule to reflect the typical codon usage of the host organism without modifying the polypeptide encoded by the DNA. Such optimization includes replacing at least one, two or more, or a significant number of codons with one or more codons that are more frequently used in the genes of the host organism.

[0095] Deviations in the nucleotide sequence containing the codons that code for the amino acids of any polypeptide chain allow for variation in the coding sequence of the gene. Because each codon consists of three nucleotides and the nucleotides that make up DNA are restricted to four specific bases, there are 64 possible nucleotide combinations, 61 of which code for amino acids (the remaining three codons code for signals that stop translation). As a result, many amino acids are coded for by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six triplets, while tryptophan and methionine are coded for by only one triplet. This degeneracy allows for extensive variation in the base composition of DNA without altering the amino acid sequence of the protein coded for by the DNA.

[0096] Many organisms exhibit bias in the use of certain codons that code for the insertion of certain amino acids in the growing peptide chain. Codon preference or codon bias (differences in codon usage) between organisms is possible due to the degeneracy of the genetic code and is well documented among many organisms. Codon bias often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is believed to depend, among other things, on the properties of the codons being translated and on the availability of certain transfer RNA (tRNA) molecules. In cells, the predominance of selected tRNAs generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0097] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, relative codon usage has been calculated. Codon usage tables are available, for example, from the "Codon Usage Database" at www.kazusa.or.jp / codon / (accessed June 18, 2012). See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000).

[0098] Random assignment of optimized codon frequencies to encode a given polypeptide sequence can be performed manually by calculating the codon frequency for each amino acid and then randomly assigning those codons to the polypeptide sequence. In addition, various algorithms and computer software programs can be used to calculate optimal sequences.

[0099] Codon optimization may also include the removal of potential immunogenic sequences from the protein sequence encoded by the nucleotide sequence. In some embodiments, in silico methods are used to identify potential immunogenic sequences in a protein or nucleotide sequence. Non-limiting examples of these methods include identifying human leukocyte antigen (HLA) alleles (e.g., DR, DP, DQ) and identifying major histocompatibility complex class II (MHCII) binding sites in a given protein sequence. In some embodiments, public databases such as the Immune Epitope Database and Analysis Resources (IEDB) (http: / / www.iedb.org / ) can be used to identify potential immunogenic sequences (see, e.g., Zhang Q et al. Nucleic Acids Res (2008) 36: W513-8; Kim Y et al. Nucleic Acids Res (2012) 40: W525-30; Dhanda et al. Nucleic Acids Res (2019) 47: W502-W506). In some embodiments, the NetMHCIIpan3.0 method can be used to identify potentially immunogenic sequences, as described in Lamberth K, et al. Sci Transl Med. 2017;9(372):eaag1286. In some embodiments, the nucleotide sequence encoding the potentially immunogenic sequence is deleted.

[0100] Heterologous nucleotide sequences In some embodiments, the isolated nucleic acid molecule of the present disclosure further comprises a heterologous nucleotide sequence. In some embodiments, the isolated nucleic acid molecule of the present disclosure further comprises at least one heterologous nucleotide sequence. The heterologous nucleotide sequence can be linked to the optimized BDD-FVIII nucleotide sequence of the present disclosure at the 5' end, the 3' end, or inserted in the middle of the optimized BDD-FVIII nucleotide sequence. Thus, in some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is linked to the N-terminus or C-terminus of the FVIII amino acid sequence encoded by the nucleotide sequence, or inserted between two amino acids in the FVIII amino acid sequence. In some embodiments, the heterologous amino acid sequence can be inserted between two amino acids at one or more insertion sites. In some embodiments, a heterologous amino acid sequence can be inserted into a FVIII polypeptide encoded by a nucleic acid molecule of the present disclosure at any site disclosed in WO 2013 / 123457 A1, WO 2015 / 106052 A1, or U.S. Patent Application Publication No. 2015 / 0158929 A1, each of which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted within the B domain or a fragment thereof. In some embodiments, the heterologous amino acid sequence is inserted within FVIII immediately downstream of the amino acid corresponding to amino acid position 745 of wild-type mature human FVIII (SEQ ID NO: 19). In one particular embodiment, the FVIII comprises a deletion of amino acids corresponding to positions 746-1637 of wild-type mature human FVIII (SEQ ID NO: 19) and the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted immediately downstream of the amino acid corresponding to position 745 of wild-type mature human FVIII (SEQ ID NO: 19). The insertion sites referred to herein indicate amino acid positions corresponding to amino acid positions of wild-type mature human FVIII (SEQ ID NO: 19).

[0102] In some embodiments, the heterologous moiety is a peptide or polypeptide having either non-structural or structural features associated with increased in vivo half-life when incorporated into a protein of the present disclosure. Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, HAP sequences, XTEN sequences, transferrin or fragments thereof, PAS polypeptides, polyglycine linkers, polyserine linkers, albumin binding moieties, or any fragments, derivatives, variants, or combinations of these polypeptides.

[0103] In certain embodiments, the heterologous moiety improves one or more pharmacokinetic properties of the FVIII protein without significantly affecting the biological activity or function of the FVIII protein. In some embodiments, the heterologous moiety extends the in vivo and / or in vitro half-life of the FVIII protein of the present disclosure. The in vivo half-life of the FVIII protein can be determined by any method known to those skilled in the art, such as activity assays (colorimetric assays or one-stage clotting aPTT assays), ELISA, ROTEM®, etc.

[0104] In other embodiments, the heterologous moiety enhances the stability of the disclosed FVIII protein or fragment thereof (e.g., a fragment comprising a heterologous moiety following proteolytic cleavage of the FVIII protein). As used herein, the term "stability" refers to an art-recognized measure of the maintenance of one or more physical properties of a FVIII protein in response to environmental conditions (e.g., increased or decreased temperature). In certain aspects, the physical property can be the maintenance of the covalent structure of the FVIII protein (e.g., no proteolytic cleavage, no unwanted oxidation or deamidation). In other aspects, the physical property can also be the presence of the FVIII protein in a correctly folded state (e.g., no soluble or insoluble aggregation or precipitation). In one aspect, the stability of the FVIII protein is measured by assaying a biophysical property of the FVIII protein, such as thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to a protein, receptor, or ligand), and / or a combination thereof. In another aspect, the biochemical function is demonstrated by the binding affinity of an interaction. In one aspect, the measure of protein stability is thermal stability, i.e., resistance to heat stress.Stability can be measured using methods known in the art, such as HPLC (High Performance Liquid Chromatography), SEC (Size Exclusion Chromatography), DLS (Dynamic Light Scattering).Methods for measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), and heat stress assay.

[0105] In some embodiments, the heterologous moiety comprises one or more XTEN sequences, fragments, variants, or derivatives thereof. As used herein, "XTEN sequence" refers to an extended polypeptide having a non-naturally occurring, substantially non-repetitive sequence that is composed primarily of small hydrophilic amino acids and has little or no secondary or tertiary structure under physiological conditions. As a heterologous moiety, XTEN can act as a half-life extending moiety. In addition, XTEN can provide desirable properties, including, but not limited to, enhanced pharmacokinetic parameters and solubility properties. Other advantageous properties that may be conferred by the introduction of XTEN sequences include conformational flexibility, improved aqueous solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius.

[0106] XTEN can be of various lengths for insertion into or attachment to FVIII. In some embodiments, XTEN sequences useful in the present disclosure are peptides or polypeptides having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the XTEN is a peptide or polypeptide having more than about 20 to about 3000 amino acid residues, more than 30 to about 2500 residues, more than 40 to about 2000 residues, more than 50 to about 1500 residues, more than 60 to about 1000 residues, more than 70 to about 900 residues, more than 80 to about 800 residues, more than 90 to about 700 residues, more than 100 to about 600 residues, more than 110 to about 500 residues, or more than 120 to about 400 residues. In a particular embodiment, the XTEN comprises an amino acid sequence longer than 42 amino acids and shorter than 144 amino acids in length.

[0107] The XTEN sequences of the present disclosure can include one or more sequence motifs of 5-14 (e.g., 9-14) amino acid residues, or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence motif, which motif comprises, consists essentially of, or consists of 4-6 amino acids (e.g., 5 amino acids) selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See U.S. Patent Application Publication No. 2010-0239554A1.

[0108] Examples of XTEN sequences that can be used as heterologous moieties in the chimeric proteins of the present disclosure are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1. or in International Publication Nos. 2010091122A1, 2010144502A2, 2010144508A1, 2011028228A1, 2011028229A1, or 2011028344A2 (each of which is incorporated by reference in its entirety).

[0109] One or more XTEN sequences can be inserted at the C-terminus or N-terminus of the amino acid sequence encoded by the nucleotide sequence, or between two amino acids in the amino acid sequence encoded by the nucleotide sequence. For example, XTEN can be inserted between two amino acids at one or more insertion sites. Examples of sites within FVIII that are acceptable for XTEN insertion sites can be found, for example, in WO 2013 / 123457 A1 or US 2015 / 0158929 A1, which are incorporated herein by reference in their entireties.

[0110] In certain embodiments, the heterologous moiety is a peptide linker.

[0111] As used herein, the term "peptide linker" or "linker moiety" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) that connects two domains in the linear amino acid sequence of a polypeptide chain.

[0112] In some embodiments, a heterologous nucleotide sequence encoding a peptide linker can be inserted between the optimized FVIII polynucleotide sequence of the present disclosure and the heterologous nucleotide sequence encoding one of the above heterologous moieties, such as albumin. The peptide linker can provide flexibility to the chimeric polypeptide molecule. Although the linker is typically not cleaved, such cleavage may be desirable. In one embodiment, these linkers are not removed during processing.

[0113] A type of linker that can be present in the chimeric proteins of the present disclosure is a protease-cleavable linker that contains a cleavage site (i.e., a protease cleavage site substrate, e.g., Factor XIa, Factor Xa, or thrombin cleavage site) and can include additional linkers at either the N-terminus or C-terminus or both of the cleavage site. These cleavable linkers, when incorporated into the constructs of the present disclosure, result in chimeric molecules with heterologous cleavage sites.

[0114] In one embodiment, a FVIII polypeptide encoded by a nucleic acid molecule of the present disclosure comprises two or more Fc domains or moieties linked via a cscFc linker to form an Fc region contained in a single polypeptide chain. The cscFc linker is adjacent to at least one intracellular processing site, i.e., a site that is cleaved by an intracellular enzyme. Cleavage of the polypeptide at the at least one intracellular processing site results in a polypeptide comprising at least two polypeptide chains.

[0115] Other peptide linkers can optionally be used in the constructs of the present disclosure, for example to connect the FVIII protein to the Fc region. Some exemplary linkers that can be used in the context of the present disclosure include, for example, polypeptides that include a GlySer amino acid, which are described in more detail below.

[0116] In one embodiment, the peptide linker is synthetic, i.e., non-naturally occurring. In one embodiment, the peptide linker comprises a peptide (or polypeptide) (which may or may not be naturally occurring) that comprises an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids that are not linked or genetically fused in nature. For example, in one embodiment, the peptide linker may comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., containing mutations such as additions, substitutions, or deletions). In another embodiment, the peptide linker may comprise a non-naturally occurring amino acid. In another embodiment, the peptide linker may comprise a naturally occurring amino acid that is present in a linear sequence that is not found in nature. In yet another embodiment, the peptide linker may comprise a naturally occurring polypeptide sequence.

[0117] In another embodiment, the peptide linker comprises or consists of a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. In certain embodiments, the gly-ser linker can be inserted between two other sequences of the peptide linker. In other embodiments, the gly-ser linker is attached to one or both ends of another sequence of the peptide linker. In yet other embodiments, two or more gly-ser linkers are incorporated in tandem into the peptide linker. In one embodiment, the peptide linker of the present disclosure comprises at least a portion of the upstream hinge region (e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule), at least a portion of the middle hinge region (e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule), and a series of gly / ser amino acid residues.

[0118] The peptide linkers of the present disclosure are at least one amino acid long and can be of various lengths. In one embodiment, the peptide linkers of the present disclosure are about 1 to about 50 amino acids long. As used in this context, the term "about" refers to + / - 2 amino acid residues. Since the linker length must be a positive integer, a length of about 1 to about 50 amino acids long means a length of 1 to 3 to 48 to 52 amino acids long. In another embodiment, the peptide linkers of the present disclosure are about 10 to about 20 amino acids long. In another embodiment, the peptide linkers of the present disclosure are about 15 to about 50 amino acids long. In another embodiment, the peptide linkers of the present disclosure are about 20 to about 45 amino acids long. In another embodiment, the peptide linkers of the present disclosure are about 15 to about 35 or about 20 to about 30 amino acids long. In another embodiment, the peptide linker of the present disclosure is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or 2000 amino acids in length. In one embodiment, the peptide linker of the present disclosure is 20 or 30 amino acids in length.

[0119] In some embodiments, the peptide linker may comprise at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In other embodiments, the peptide linker may comprise at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In some embodiments, the peptide linker may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The peptide linker may contain 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids.

[0120] Peptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells so that the polypeptide produced is stably produced.

[0121] Expression control sequences In some embodiments, the nucleic acid molecule or vector of the present disclosure further comprises at least one expression control sequence. For example, the isolated nucleic acid molecule of the present disclosure can be operably linked to at least one expression control sequence. The expression control sequence can be, for example, a promoter sequence or a promoter-enhancer combination.

[0122] Mammalian constitutive promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of skill in the art. Promoters useful as gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced in the presence of certain metal ions to promote transcription and translation. Other inducible promoters are known to those of skill in the art.

[0123] In one embodiment, the disclosure includes expressing the transgene under the control of a tissue-specific promoter and / or enhancer. In another embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes. In a particular embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes, sinusoidal cells, and / or endothelial cells. In a particular embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in endothelial cells. In a particular embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in muscle cells, the central nervous system, the eye, the liver, the heart, or any combination thereof. Examples of liver-specific promoters include, but are not limited to, the mouse transthyretin promoter (mTTR), the native human factor VIII promoter, the human alpha 1-antitrypsin promoter (hAAT), the human albumin minimal promoter, and the mouse albumin promoter. In some embodiments, the nucleic acid molecule disclosed herein comprises the mTTR promoter. The mTTR promoter is described in Costa et al. (1986) Mol. Cell. Biol. 6:4697. The FVIII promoter is described in Figueiredo and Brownlee, 1995, J. Biol. Chem. 270:11828-11838. In some embodiments, the promoter is selected from a liver-specific promoter (e.g., α1-antitrypsin (AAT)), a muscle-specific promoter (e.g., muscle creatine kinase (MCK), myosin heavy chain alpha (αMHC), myoglobin (MB), and desmin (DES)), a synthetic promoter (e.g., SPc5-12, 2R5Sc5-12, dMCK, and tMCK), or any combination thereof.

[0124] In some embodiments, transgene expression is targeted to the liver. In certain embodiments, transgene expression is targeted to hepatocytes. In other embodiments, transgene expression is targeted to endothelial cells. In one particular embodiment, transgene expression is targeted to any tissue that naturally expresses endogenous FVIII. In some embodiments, transgene expression is targeted to the central nervous system. In certain embodiments, transgene expression is targeted to neurons. In some embodiments, transgene expression is targeted to afferent neurons. In some embodiments, transgene expression is targeted to efferent neurons. In some embodiments, transgene expression is targeted to interneuronal cells. In some embodiments, transgene expression is targeted to glial cells. In some embodiments, transgene expression is targeted to astrocytes. In some embodiments, transgene expression is targeted to oligodendrocytes. In some embodiments, transgene expression is targeted to microglia. In some embodiments, transgene expression is targeted to ependymal cells. In some embodiments, transgene expression is targeted to Schwann cells. In some embodiments, transgene expression is targeted to satellite cells. In some embodiments, transgene expression is targeted to muscle cells. In some embodiments, transgene expression is targeted to smooth muscle. In some embodiments, transgene expression is targeted to cardiac muscle. In some embodiments, transgene expression is targeted to skeletal muscle. In some embodiments, transgene expression is targeted to the eye. In some embodiments, transgene expression is targeted to photoreceptor cells. In some embodiments, transgene expression is targeted to retinal ganglion cells.

[0125] Other promoters useful in the nucleic acid molecules disclosed herein include the mouse transthyretin promoter (mTTR), the native human factor VIII promoter, the human alpha 1-antitrypsin promoter (hAAT), the human albumin minimal promoter, the mouse albumin promoter, the tristetraprolin (TTP; also known as ZFP36) promoter, the CASI promoter, the CAG promoter, the cytomegalovirus (CMV) promoter, the alpha 1-antitrypsin (AAT) promoter, the muscle creatine kinase (MCK) promoter, the myosin heavy chain alpha (αMHC) promoter, the myoglobin (MB) promoter, the desmin (DES) promoter, the SPc5-12 promoter, the 2R5Sc5-12 promoter, the dMCK promoter, and the tMCK promoter, the phosphoglycerate kinase (PGK) promoter, or any combination thereof.

[0126] In some embodiments, the nucleic acid molecule disclosed herein comprises a transthyretin (TTR) promoter. In some embodiments, the promoter is a mouse transthyretin (mTTR) promoter. Non-limiting examples of mTTR promoters include the mTTR202 promoter, the mTTR202opt promoter, and the mTTR482 promoter, as disclosed in US Patent Application Publication No. 2019 / 0048362, the entire contents of which are incorporated herein by reference. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter mTTR482. Examples of the mTTR482 promoter are described in Kyostio-Moore et al. (2016) Mol Ther Methods Clin Dev. 3:16006, and Nambiar B. et al. (2017) Hum Gene Ther Methods, 28(1):23-28. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter comprising the nucleic acid sequence of SEQ ID NO:9.

[0127] One or more enhancer elements can be used to further increase expression levels to achieve therapeutic efficacy. One or more enhancers can be provided alone or together with one or more promoter elements. Typically, the expression control sequence includes multiple enhancer elements and tissue-specific promoters. In one embodiment, the enhancer includes one or more copies of the α1-microglobulin / bikunin enhancer (Rouet et al. (1992) J. Biol. Chem. 267:20765-20773; Rouet et al. (1995), Nucleic Acids Res. 23:395-404; Rouet et al. (1998) Biochem. J. 334:577-584; Ill et al. (1997) Blood Coagulation Fibrinolysis 8:S23-S30). In some embodiments, the enhancer is derived from liver-specific transcription factor binding sites such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, and Enh1 includes HNF1, (sense)-HNF3, (sense)-HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4 (antisense). In some embodiments, the enhancer is an mTTR482 enhancer comprising the nucleic acid sequence of SEQ ID NO:8.

[0128] In some embodiments, the enhancer comprises one or two modified prothrombin enhancers (pPrT2), one or two alpha 1-microbikunin enhancers (A1MB2), a modified mouse albumin enhancer (mEalb), Hepatitis B virus enhancer II (HE11), or a CRM8 enhancer. In some embodiments, the enhancer is a synthetic enhancer. In some embodiments, the enhancer is a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO:7.

[0129] In some embodiments, the nucleic acid molecules disclosed herein comprise an intron or an intron sequence. In some embodiments, the intron sequence is a naturally occurring intron sequence. In some embodiments, the intron sequence is a synthetic sequence. In some embodiments, the intron sequence is derived from a naturally occurring intron sequence. In some embodiments, the intron sequence is a hybrid synthetic intron or a chimeric intron. In some embodiments, the intron sequence is a chimeric intron consisting of a chicken beta-actin / rabbit beta-globin intron, modified to remove five existing ATG sequences to reduce false translation initiation. In certain embodiments, the intron sequence comprises an SV40 small T intron.

[0130] In some embodiments, the nucleic acid molecules disclosed herein comprise one or more DNA nuclear targeting sequences (DTSs). The DTSs facilitate the translocation of DNA molecules containing such sequences into the nucleus. In certain embodiments, the DTSs comprise SV40 enhancer sequences. In certain embodiments, the DTSs comprise c-Myc enhancer sequences. In some embodiments, the nucleic acid molecules comprise a DTS located between the first and second ITRs. In some embodiments, the nucleic acid molecules comprise a DTS located 3' to the first ITR and 5' to the transgene (e.g., FVIII protein). In some embodiments, the nucleic acid molecules comprise a DTS located 3' to the transgene and 5' to the second ITR on the nucleic acid molecule.

[0131] In some embodiments, the nucleic acid molecules disclosed herein comprise a Toll-like receptor 9 (TLR9) inhibitory sequence. Exemplary TLR9 inhibitory sequences are described, for example, in Trieu et al. (2006) Crit Rev Immunol. 26(6):527-44; Ashman et al. Int'l Immunology 23(3):203-14.

[0132] Vector Some embodiments of the present disclosure relate to vectors comprising one or more codon-optimized nucleic acid molecules encoding a polypeptide having FVIII activity, as described herein, host cells comprising the vectors, and methods of treating bleeding disorders using the vectors.The present disclosure fills an important need in the art by providing vectors comprising optimized FVIII sequences that have increased expression in a subject, potentially enhancing therapeutic efficacy when used in gene therapy.

[0133] Vectors suitable for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In one embodiment, the vector is a viral vector.

[0134] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, the necessary elements for replication and translation upon introduction into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0135] The expression vector of the present disclosure comprises an optimized polynucleotide encoding the BDD FVIII protein described herein. In one embodiment, the optimized coding sequence of the BDD FVIII protein is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to retain its function. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked when induction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and when the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence is considered to be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0136] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus; and RNA viruses such as retroviruses. Other vectors well known in the art can be readily employed. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes are replaced with genes of interest. In one embodiment, the virus is adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-deficient and can infect a wide variety of cell types and species.

[0137] One or more of the different AAV vector sequences from nearly any serotype can be used in accordance with the present disclosure. The selection of a particular AAV vector sequence will be guided by known parameters such as the desired tropism, required vector yield, etc. In general, AAV serotypes have genomic sequences that share a significant degree of homology at the amino acid and nucleic acid levels, confer a related set of genetic functions, produce related virions, and replicate and assemble in a similar fashion. For an overview of the genomic sequences of various AAV serotypes and their genomic similarities, see, for example, GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chlorini et al. (1997) J. Vir. 71:6823-33; Srivastava et al. (1983) J. Vir. 45:555-64; Chlorini et al. (1999) J. Vir. 73:1309-1319; Rutledge et al. (1998), J. Vir. 72:309-319; or Wu et al. (2000) J. Vir. 74:8635-47. AAV serotypes 1, 2, 3, 4, and 5 are exemplary sources of AAV nucleotide sequences for use in the context of this disclosure. For certain disclosed applications, AAV6, AAV7, AAV8, or AAV9, or newly developed AAV-like particles obtained, for example, by capsid shuffling methods and AAV capsid libraries, or newly designed, developed, or evolved ITR libraries, are also suitable. See Dalkara et al. (2013), Sci. Transl. Med. 5(189): 189ra76; Kotterman MA (2014) Nat. Rev. Genet. 15(7): 455.

[0138] Other vectors include plasmid vectors. Plasmid vectors are described in detail in the art and are well known to those skilled in the art. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly useful for delivering genes to cells in vivo, as they cannot replicate or integrate into the host genome. However, these plasmids with a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial vendors include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1 (catalog number V79020); pcDNA3.1 / hygro (catalog number V87020); pcDNA4 / myc-His (catalog number V86320); and pBudCE4.1 (catalog number V53220), all from Invitrogen (Carlsbad, Calif.). Other plasmids will be known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0139] In certain embodiments, it will be useful to include one or more miRNA target sequences in the vector, for example, operably linked to the optimized FVIII transgene. More than one copy of the miRNA target sequence in the vector can increase the effectiveness of the vector system. For example, a vector expressing two or more transgenes can have those transgenes under the control of two or more miRNA target sequences, which can be the same or different. The miRNA target sequences can be in tandem, although other arrangements are also included. The transgene expression cassette containing the miRNA target sequence can be inserted in the vector in an antisense orientation. Examples of miRNA target sequences are described in WO 2007 / 000668, WO 2004 / 094642, WO 2010 / 055413, or WO 2010 / 125471, which are incorporated herein by reference in their entirety. However, in certain other embodiments, the vector will not contain any miRNA target sequences. The choice of whether to include miRNA target sequences (and how many miRNA target sequences to include) will be guided by known parameters such as the intended tissue target, the required expression level, and the like.

[0140] Lentiviral Vectors Lentiviruses include members of the bovine lentivirus group, the equine lentivirus group, the feline lentivirus group, the ovine caprine lentivirus group, and the primate lentivirus group. The development of lentivirus vectors for gene therapy is reviewed in Klimatcheva et al. (1999) Frontiers in Bioscience 4:481-496. The design and use of lentivirus vectors suitable for gene therapy are described, for example, in U.S. Patent Nos. 6,207,455 and 6,615,782. Examples of lentiviruses include, but are not limited to, HIV-1, HIV-2, HIV-1 / HIV-2 pseudotyped, HIV-1 / SIV, FIV, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus, and bovine immunodeficiency virus.

[0141] In some embodiments, the lentiviral vector of the present disclosure is a "third generation" lentiviral vector. As used herein, the term "third generation" lentiviral vector refers to a lentiviral packaging system that has characteristics of a second generation vector system and further lacks a functional tat gene, e.g., the tat gene is deleted or inactivated. Typically, the gene encoding rev is provided on a separate expression construct. See, e.g., Dull et al. (1998) J.Virol. 72:8463-8471. As used herein, a "second generation" lentiviral vector system refers to a lentiviral packaging system that lacks functional accessory genes, e.g., the accessory genes vif, vpr, vpu, and nef are deleted or inactivated. See, e.g., Zufferey et al. (1997) Nat.Biotechnol. 15:871-875. As used herein, a "packaging system" refers to a set of viral constructs that encode the viral proteins involved in packaging the recombinant virus. Typically, the packaging system construct is ultimately incorporated into a packaging cell.

[0142] In some embodiments, the third generation lentiviral vector of the present disclosure is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotyped lentiviral vector. In some embodiments, the lentiviral vector comprises a hepatocyte-specific promoter for transgene expression. In some embodiments, the hepatocyte-specific promoter is an enhanced transthyretin promoter. In some embodiments, the lentiviral vector comprises one or more target sequences of miR-142 to reduce immune responses to the transgene product. In some embodiments, incorporating one or more target sequences of miR-142 into the lentiviral vector of the present disclosure allows for a desired transgene expression profile. For example, incorporating one or more target sequences of miR-142 can suppress transgene expression in intravascular and extravascular hematopoietic cell lineages, while maintaining transgene expression in non-hematopoietic cells. No carcinogenesis has been detected in tumor-prone mice treated with the lentiviral vector system of the present disclosure. See Brown et al. (2007) Blood 110:4144-52, Brown at al. (2006) Nat. Ned. 12:585-91, and Cantore et al. (2015) Sci. Transl. Med. 7(277):277ra28.

[0143] The lentiviral vector of the present disclosure comprises a codon-optimized polynucleotide encoding the BDD FVIII protein described herein. In one embodiment, the optimized coding sequence of the BDD FVIII protein is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to retain its function. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked when induction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and when the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence is considered to be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

[0144] A schematic diagram of an exemplary lentiviral vector embodiment disclosed herein is presented as Figure 1. Additional information on exemplary lentiviral vector production embodiments can be found in Example 2. Further discussion of retroviral vector design for gene therapy is provided in Poletti & Mavilio, Viruses. 2021(13):1526.

[0145] In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting liver cells (e.g., hepatocytes). Lentiviral genomes and proviral DNA typically contain three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins, the pol gene encodes the RNA-dependent DNA polymerase (reverse transcriptase), protease, and integrase, and the env gene encodes the viral envelope glycoproteins. The 5'LTR and 3'LTR act to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2 and / or SIV).

[0146] Adjacent to the 5'LTR are sequences required for reverse transcription of the genome (tRNA primer binding site) and efficient encapsidation of viral RNA into particles (Psi site). If the sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA.

[0147] In some embodiments, the lentiviral vector comprises a primer binding site (PBS) of stem loop 123 (SL123). In some embodiments, the PBS comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the lentiviral vector comprises a Psi stem loop 4 (SL4) sequence. In some embodiments, the lentiviral vector comprises the nucleotide sequence of SEQ ID NO: 4. Further discussion of Psi and related sequences can be found in Kim et al. PLoS ONE 2012.7(11):e50148.

[0148] However, the resulting mutants are still capable of directing the synthesis of all virion proteins. The present disclosure provides a method for producing a recombinant lentivirus capable of infecting non-dividing cells, comprising transfecting a suitable host cell with two or more vectors carrying the packaging functions, i.e., gag, pol and env, and rev and tat. As disclosed herein below, in certain applications, vectors lacking a functional tat gene are desirable. Thus, for example, a first vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env, to produce a packaging cell. A vector providing a heterologous gene (defined herein as a transfer vector) is introduced into the packaging cell, resulting in a producer cell that releases infectious viral particles carrying the foreign gene of interest.

[0149] According to the above configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphotropic envelope protein that allows transduction of cells of human and other species.

[0150] Examples of env genes from retroviruses include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus env genes, influenza env genes, etc., may also be used. In some embodiments, the viral env nucleic acid sequence is operably associated with regulatory sequences described elsewhere herein.

[0151] In certain embodiments, the lentiviral vector is deleted of the HIV virulence genes env, vif, vpr, vpu, and nef without compromising the ability of the vector to transduce non-dividing cells. In some embodiments, the lentiviral vector comprises a deletion of the U3 region of the 3'LTR. The deletion of the U3 region can be a complete deletion or a partial deletion.

[0152] In some embodiments, a lentiviral vector of the disclosure comprising a FVIII nucleotide sequence as described herein can be transfected into a cell with (a) a first nucleotide sequence comprising a gag gene, a pol gene, or a gag gene and a pol gene, and (b) a second nucleotide sequence comprising a heterologous env gene, the lentiviral vector lacking a functional tat gene. In other embodiments, the cell is further transfected with a fourth nucleotide sequence comprising a rev gene. In certain embodiments, the lentiviral vector lacks a functional gene selected from vif, vpr, vpu, vpx, and nef, or a combination thereof.

[0153] In certain embodiments, the lentiviral vector of the present disclosure comprises one or more nucleotide sequences encoding a gag protein, a Rev response element, a central polypurine track (cPPT), or any combination thereof.

[0154] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that improve targeting and / or activity of the lentiviral vector or the encoded FVIII polypeptide. The one or more polypeptides can be encoded by the lentiviral vector or can be incorporated during budding of the lentiviral vector from the host cell. During lentivirus production, viral particles bud from the producer host cell. During the budding process, the viral particles acquire a lipid coat, which originates from the lipid membrane of the host cell. As a result, the lipid coat of the viral particle can include membrane-associated polypeptides that were previously present on the surface of the host cell.

[0155] In some embodiments, the lentiviral vector expresses one or more polypeptides on its surface that inhibit immune responses to the lentiviral vector after administration to a human subject. In some embodiments, the surface of the lentiviral vector comprises one or more CD47 molecules. CD47 is a "self-marker" protein that is ubiquitously expressed on human cells. Surface expression of CD47 inhibits macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 with SIRPα expressed by macrophages. Cells that express high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo.

[0156] In some embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is generated in a cell line with a high CD47 expression level. In certain embodiments, the lentiviral vector is generated in a CD47high cell, the cell having a high expression of CD47 on the cell membrane. In certain embodiments, the lentiviral vector is generated in a CD47highHEK293T cell, the HEK293T having a high expression of CD47 on the cell membrane. In some embodiments, the HEK293T cell is modified to increase the expression of CD47 compared to unmodified HEK293T cell. In certain embodiments, the CD47 is human CD47.

[0157] In some embodiments, lentiviral vectors have little or no surface expression of major histocompatibility complex class I (MHC-I). Surface expressed MHC-I presents peptide fragments of "non-self" proteins from within the cell, e.g., protein fragments indicative of infection, and promotes an immune response against the cell. ... low In some embodiments, the lentiviral vector is produced in a cell that has reduced expression of MHC-I on the cell membrane. free ", "MHC-1 neg " or "MHC negative" cells, which lack expression of MHC-I.

[0158] In certain embodiments, the lentiviral vector comprises a lipid coat that contains a high concentration of CD47 polypeptide and lacks MHC-I polypeptide. low Cell lines, e.g., CD47high / MHC-I low In some embodiments, the lentiviral vector is produced in a CD47high / MHC-Ifree cell line, e.g., CD47high / MHC-I free Produced in the HEK293T cell line.

[0159] Examples of lentiviral vectors are disclosed in U.S. Pat. No. 9,050,269, as well as WO 9931251, WO 9712622, WO 9817815, WO 9817816, and WO 9818934, which are incorporated by reference in their entireties.

[0160] Inverted terminal repeat (ITR) sequences In some embodiments, the nucleic acid sequences disclosed herein comprise an inverted terminal repeat (ITR) sequence. As used herein, an "inverted terminal repeat" (or "ITR") refers to a nucleic acid sequence located at either the 5' or 3' end of a single stranded nucleic acid sequence, comprising a set of nucleotides (an initial sequence) followed downstream by its reverse complement, i.e., a palindromic sequence. The intervening nucleotide sequence between the initial sequence and the reverse complement can be of any length, including zero. In one embodiment, an ITR useful in the present disclosure comprises one or more "palindromic sequences." An ITR can have any number of functions. In some embodiments, the ITRs described herein form a hairpin structure. In some embodiments, the ITRs form a T-shaped hairpin structure. In some embodiments, the ITRs form a non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. In some embodiments, the ITRs promote long-term persistence of a nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITRs promote persistent persistence of a nucleic acid molecule in the nucleus of a cell (e.g., throughout the life of the cell). In some embodiments, the ITRs enhance the stability of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITRs enhance the retention of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITRs enhance the persistence of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITRs inhibit or prevent the degradation of the nucleic acid molecule in the nucleus of the cell.

[0161] Thus, an "ITR" as used herein can fold back on itself and form a double-stranded segment. For example, the sequence GATCXXXXGATC includes an initial sequence GATC and its complementary strand (3'CTAG5') that form a double helix when folded. In some embodiments, an ITR includes a continuous palindromic sequence (e.g., GATCGATC) between the initial sequence and the reverse complementary strand. In some embodiments, an ITR includes an interrupted palindromic sequence (e.g., GATCXXXXGATC) between the initial sequence and the reverse complementary strand. In some embodiments, the complementary sections of the continuous or interrupted palindromic sequence interact with each other to form a "hairpin loop" structure. As used herein, a "hairpin loop" structure occurs when at least two complementary sequences on a single-stranded nucleotide molecule base pairs to form a double-stranded section. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop.

[0162] In the present disclosure, at least one ITR is an ITR of a non-adenovirus-associated virus (non-AAV). In certain embodiments, the ITR is an ITR of a non-AAV member of the Parvoviridae family of viruses. In some embodiments, the ITR is an ITR of a non-AAV member of the Dependovirus or Erythrovirus genera.

[0163] In some embodiments, the ITRs in the nucleic acid molecules as described herein may be transcriptionally activating ITRs. The transcriptionally activating ITRs may comprise all or part of a wild-type ITR that has been transcriptionally activated by the inclusion of at least one transcriptionally active element. A variety of transcriptionally active elements are suitable for use in this context. In some embodiments, the transcriptionally active elements are constitutive transcriptionally active elements. Constitutive transcriptionally active elements provide a sustained level of gene transcription and are preferred when it is desired that the transgene be continuously expressed. In other embodiments, the transcriptionally active elements are inducible transcriptionally active elements. Inducible transcriptionally active elements generally exhibit low activity in the absence of an inducer (or inducing conditions) and are upregulated in the presence of an inducer (or a switch to inducing conditions). Inducible transcriptionally active elements may be preferred when expression is desired only at certain times or in certain locations, or when it is desired to titrate expression levels using an inducing agent. Transcriptionally active elements may be tissue specific; i.e., they are only active in certain tissues or cell types.

[0164] Transcriptionally active elements can be incorporated into the ITRs in a variety of ways. In some embodiments, the transcriptionally active element is incorporated 5' of any portion of the ITR or 3' of any portion of the ITR. In other embodiments, the transcriptionally active element of the transcriptionally activating ITR is between two ITR sequences. If the transcriptionally active element contains two or more elements that must be spaced apart, the elements can alternate portions of the ITR. In some embodiments, the hairpin structure of the ITR is deleted and replaced with an inverted repeat of the transcription element. This latter arrangement forms a hairpin that mimics the deleted portion of the structure. There can be multiple tandem transcriptionally active elements in the transcriptionally activating ITR, which can be adjacent or separated. Additionally, protein binding sites (e.g., Rep binding sites) can be introduced into the transcriptionally active element of the transcriptionally activating ITR. The transcriptionally active element can include any sequence that allows for controlled transcription of DNA by RNA polymerase to form RNA, and can include, for example, the transcriptionally active elements defined below.

[0165] Transcriptionally activating ITRs provide both transcriptional activation and ITR functions to a nucleic acid molecule of relatively limited nucleotide sequence length, which effectively maximizes the length of the transgene that can be carried and expressed from the nucleic acid molecule. Incorporation of transcriptionally active elements into ITRs can be accomplished in a variety of ways. Comparison of ITR sequences and sequence requirements of transcriptionally active elements can provide insight into how to encode elements within the ITR. For example, transcriptional activity can be added to an ITR through the introduction of specific alterations in the ITR sequence that duplicate functional elements of the transcriptionally active element. Numerous techniques exist in the art for efficiently adding, deleting, and / or modifying specific nucleotide sequences at specific sites (see, for example, Deng and Nickoloff (1992) Anal. Biochem. 200:81-88). Another method of creating transcriptionally activating ITRs includes the introduction of restriction sites at desired positions in the ITR. Additionally, multiple transcriptionally active elements can be incorporated into a transcriptionally activating ITR using methods known in the art.

[0166] By way of illustration, transcriptionally activating ITRs can be generated by the inclusion of one or more transcriptionally active elements, such as a TATA box, a GC box, a CCAAT box, an Sp1 site, an Inr region, a CRE (cAMP regulatory element) site, an ATF-1 / CRE site, an APBβ box, an APBα box, a CArG box, a CCAC box, or any other element involved in transcription as known in the art.

[0167] host cell The disclosure also provides a host cell comprising a nucleic acid molecule or vector of the disclosure. As used herein, the term "transformation" refers broadly to the introduction of DNA into a recipient host cell, which is used to refer to an introduction that alters the genotype and, therefore, the recipient cell.

[0168] "Host cell" refers to a cell that has been constructed using recombinant DNA techniques and transformed with a vector encoding at least one heterologous gene. The host cells of the present disclosure are preferably of mammalian origin, and most preferably of human or murine origin. Those skilled in the art are capable of selectively determining the particular host cell line that is most suitable for the purpose. Exemplary host cell lines include, but are not limited to, CHO, DG44 and DUXB11 (Chinese hamster ovary line, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1C1BPT (bovine endothelial cells), RAJI (human lymphocytes), PER.C6®, NS0, CAP, BHK21 and HEK293 (human kidney). In a particular embodiment, the host cell is selected from the group consisting of: CHO cells, HEK293 cells, BHK21 cells, PER.C6® cells, NS0 cells, and CAP cells. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or from the public literature.

[0169] The isolated nucleic acid molecule or vector of the present disclosure can be introduced into a host cell by various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of the plasmid into the host can be by electroporation. The transformed cells are cultured under conditions suitable for the production of light and heavy chains and assayed for synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0170] The host cells comprising the isolated nucleic acid molecule or vector of the present disclosure are cultured in an appropriate culture medium. As used herein, the term "appropriate culture medium" refers to a medium that contains nutrients necessary for cell growth. The nutrients necessary for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain calf serum or fetal calf serum (FCS). In one embodiment, the medium is substantially free of IgG. The culture medium generally selects for cells containing the DNA construct, for example, by drug selection or by a deficiency of an essential nutrient that is complemented by a selectable marker on the DNA construct or a selectable marker co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CDoptiCHO (Invitrogen, Carlsbad, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.) Selection of an appropriate medium for the particular cell line used is within the level of ordinary skill in the art.

[0171] In some embodiments, suitable host cells for use in the present invention are of insect origin. In some embodiments, suitable insect host cells include, for example, cell lines isolated from Spodoptera frugiperda (Sf) or cell lines isolated from Trichoplusia ni (Tni). A person skilled in the art can easily determine the suitability of any Sf or Tni cell line. Exemplary insect host cells include, but are not limited to, Sf9 cells, Sf21 cells, and High Five™ cells. Exemplary insect host cells also include, but are not limited to, any Sf or Tni cell line that is free of adventitious virus contamination, such as, but not limited to, Sf-rhabdovirus negative (Sf-RVN) and Tn-nodavirus negative (Tn-NVN) cells. Other suitable host insect cells are known to those skilled in the art. In one particular embodiment, the insect host cell is an Sf9 cell.

[0172] An aspect of the present disclosure is a method for cloning a nucleic acid molecule described herein, comprising inserting a nucleic acid molecule capable of adopting a complex secondary structure into a suitable vector and introducing the resulting vector into a suitable bacterial host strain. As is known in the art, complex secondary structures of nucleic acids (e.g., long palindromic regions) can be unstable and difficult to clone into bacterial host strains. For example, a nucleic acid molecule comprising a first ITR and a second ITR (e.g., a non-AAV parvovirus ITR, e.g., HBoV1 ITR) of the present disclosure can be difficult to clone using conventional methodologies. Long DNA palindromes inhibit DNA replication and are unstable in the genomes of E. coli, Bacillus, Streptococcus, Streptomyces, S. cerevisiae, mice, and humans. These effects result from the formation of hairpin or cruciform structures by intrastrand base pairing. In E. coli, inhibition of DNA replication can be significantly overcome in SbcC or SbcD mutants. SbcD is the nuclease subunit and SbcC is the ATPase subunit of the SbcCD complex. The E. coli SbcCD complex is an exonuclease complex responsible for preventing the replication of long palindromes. The SbcCD complex is the core of ATP-dependent double-stranded DNA exonuclease activity and ATP-dependent single-stranded DNA endonuclease activity. SbcCD can recognize DNA palindromes and collapse the replication fork by attacking the resulting hairpin structure.

[0173] In certain embodiments, a suitable bacterial host strain cannot degrade cruciform DNA structures. In certain embodiments, a suitable bacterial host strain comprises a disruption in the SbcCD complex. In some embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene and / or the SbcD gene. In certain embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene. Various bacterial host strains comprising a gene disruption in the SbcC gene are known in the art. For example, but not limited to, bacterial host strain PMC103 comprises the genotypes sbcC, recD, mcrA, ΔmcrBCF; bacterial host strain PMC107 comprises the genotypes recBC, recJ, sbcBC, mcrA, ΔmcrBCF; bacterial host strain SURE comprises the genotypes recB, recJ, sbcC, mcrA, ΔmcrBCF, umuC, uvrC. Thus, in some embodiments, the method of cloning a nucleic acid molecule described herein comprises inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into host strain PMC103, PMC107, or SURE. In certain embodiments, the method of cloning a nucleic acid molecule described herein comprises inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into host strain PMC103.

[0174] Suitable vectors are known in the art and described elsewhere herein. In certain embodiments, the vector suitable for use in the cloning method of the present disclosure is a low copy vector. In certain embodiments, the vector suitable for use in the cloning method of the present disclosure is pBR322.

[0175] Polypeptide production The present disclosure also provides a polypeptide encoded by a nucleic acid molecule of the present disclosure. In other embodiments, the polypeptide of the present disclosure is encoded by a vector comprising an isolated nucleic acid molecule of the present disclosure. In yet other embodiments, the polypeptide of the present disclosure is produced by a host cell comprising an isolated nucleic acid molecule of the present disclosure.

[0176] A variety of methods are available for recombinantly producing FVIII proteins from the optimized nucleic acid molecules of the present disclosure. A polynucleotide of the desired sequence can be generated by de novo solid-phase DNA synthesis or PCR mutagenesis of a previously prepared polynucleotide. Oligonucleotide-mediated mutagenesis is one method for making substitutions, insertions, deletions, or modifications (e.g., modified codons) in a nucleotide sequence. For example, the starting DNA is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize the entire second complementary strand of the template in which the oligonucleotide primer is incorporated. In one embodiment, genetic recombination, such as primer-based PCR mutagenesis, is sufficient to incorporate modifications to generate a polynucleotide of the present disclosure, as defined herein.

[0177] For production of recombinant proteins, the optimized polynucleotide sequences of the present disclosure encoding a FVIII protein are inserted into an appropriate expression vehicle, i.e., a vector that contains the necessary elements for transcription and translation of the inserted coding sequence, or, in the case of an RNA viral vector, the necessary elements for replication and translation.

[0178] The polynucleotide sequence of the present disclosure is inserted into a vector in the proper reading frame. The expression vector is then transfected into a suitable target cell that will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. 1978, Cell 14:725) and electroporation (Neumann et al. 1982, EMBO, J. 1:841). Various host-expression vector systems can be used to express the FVIII protein described herein in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including a mammalian cell (e.g., HEK293 cell, PER.C6® cell, CHO cell, BHK cell, Cos cell, HeLa cell). The polynucleotide sequence of the present disclosure can also encode a signal sequence that allows the FVIII protein to be secreted. Those skilled in the art will appreciate that when the FVIII protein is translated, the signal sequence is cleaved by the cell to form the mature protein. A variety of signal sequences are known in the art, such as the native Factor VII signal sequence, the native Factor IX signal sequence, and the mouse IgK light chain signal sequence. Alternatively, if no signal sequence is included, the FVIII protein can be recovered by lysing the cells.

[0179] The FVIII protein of the present disclosure can be synthesized in a transgenic animal, such as a rodent, goat, sheep, pig, or cow. The term "transgenic animal" refers to a non-human animal that has a foreign gene integrated into its genome. This gene is present in the germ line tissues and is thus passed from parent to offspring. The exogenous gene is introduced into a single cell embryo (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438). Methods for producing transgenic animals, including transgenic animals that produce immunoglobulin molecules, are known in the art (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; ​​McKnight et al. 1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie et al. 1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology 59:831; Robl et al. 2003, Theriogenology 59:107; Malassagne et al. 2003, Xenotransplantation 10(3):267).

[0180] The expression vector can encode a tag that allows for easy purification or identification of the recombinantly produced protein. Examples include, but are not limited to, the vector pUR278 (Ruther et al. 1983, EMBO J. 2:1791), into which the coding sequence of the FVIII protein described herein can be ligated in frame with the lac Z coding region to produce a hybrid protein, and pGEX vectors can be used to express proteins with glutathione S-transferase (GST) tags. These proteins are usually soluble and can be easily purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vector contains a cleavage site (e.g., PreCission Protease (Pharmacia, Peapack, NJ)) to facilitate removal of the tag after purification.

[0181] For the purposes of this disclosure, numerous expression vector systems can be used. These expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. Expression vectors can include expression control sequences, including but not limited to promoters (e.g., naturally associated or heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors can also use DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV), cytomegalovirus (CMV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites.

[0182] Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362). Cells into which the desired DNA has integrated into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can confer prototrophy to an auxotrophic host, confer biocide (e.g., antibiotic) resistance or heavy metal (such as copper) resistance. Selectable marker genes can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation.

[0183] An example of a useful vector for expressing optimized FVIII sequences is NEOSPLA (U.S. Pat. No. 6,159,730). This vector contains the cytomegalovirus promoter / enhancer, mouse beta globin major promoter, SV40 origin of replication, bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, dihydrofolate reductase gene, and leader sequence. This vector has been shown to result in very high antibody expression levels when incorporated into variable and constant region genes and transfected into cells followed by selection in G418-containing medium and methotrexate amplification. A vector system is also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system results in high expression levels (e.g., >30 pg / cell / day). Other exemplary vector systems are disclosed, for example, in US Pat. No. 6,413,777.

[0184] In other embodiments, the polypeptides of the present disclosure can be expressed using polycistronic constructs. In these expression systems, multiple target gene products (such as multiple polypeptides of multimeric binding proteins) can be produced from one polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to produce relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein.

[0185] More generally, once a vector or DNA sequence encoding a polypeptide is prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be performed by various techniques well known to those skilled in the art, as discussed above. The transformed cells are grown under conditions suitable for the production of FVIII polypeptides and assayed for the synthesis of FVIII polypeptides. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0186] In describing processes for isolating polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the polypeptide, unless clearly specified otherwise. In other words, recovery of the polypeptide from the "cells" can mean recovery from either spun down whole cells, or from the cell culture medium containing both the medium and suspended cells.

[0187] Since the isolated nucleic acid of the present disclosure is optimized for expression in human cells, the host cell line used for protein expression is preferably a line of mammalian origin, most preferably a line of human or mouse origin. Exemplary host cell lines are described above. In one embodiment of the method for producing a polypeptide having FVIII activity, the host cell is a HEK293 cell. In another embodiment of the method for producing a polypeptide having FVIII activity, the host cell is a CHO cell.

[0188] Genes encoding the polypeptides of the present disclosure can also be expressed in non-mammalian cells, such as bacterial, yeast or plant cells. In this regard, it will be appreciated that various non-mammalian unicellular microorganisms, such as bacteria, can also be transformed, i.e., grown in culture or fermentation. Bacteria that can be transformed include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella, the Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the polypeptides typically become part of inclusion bodies, which must be isolated and purified before assembly into functional molecules.

[0189] Alternatively, the optimized nucleotide sequences of the present disclosure can be incorporated into a transgene for introduction into the genome of a transgenic animal and then expression in the milk of the transgenic animal (see, e.g., U.S. Patent No. 5,741,957 to Deboer et al., U.S. Patent No. 5,304,489 to Rosen, and U.S. Patent No. 5,849,992 to Meade et al.). A suitable transgene comprises a polypeptide coding sequence operably linked to the promoter and enhancer of a mammary gland specific gene, such as casein or beta-lactoglobulin.

[0190] In vitro production allows for scale-up to obtain large quantities of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogenous suspension culture in airlift or continuous stirred reactors, or culturing cells immobilized or entrapped, for example, in hollow fibers, in microcapsules, on agarose microbeads or on ceramic cartridges. If necessary and / or desired, for example, after selective biosynthesis of a synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE cellulose chromatography or (immuno) affinity chromatography. An affinity tag sequence (e.g., His(6) tag) can optionally be attached to or included in the polypeptide sequence to facilitate downstream purification.

[0191] Once expressed, the FVIII protein can be purified according to standard procedures in the art, including ammonium sulfate fractionation, affinity column chromatography, HPLC purification, gel electrophoresis, etc. (See generally, Scopes, Protein Purification, Springer-Verlag, NY, (1982)). For pharmaceutical uses, substantially pure proteins of at least about 90-95% homogeneity are preferred, with 98-99% or more homogeneity being most preferred.

[0192] Pharmaceutical Compositions A composition comprising an isolated nucleic acid molecule of the present disclosure, a polypeptide having FVIII activity encoded by the nucleic acid molecule, a vector or a host cell can include a suitable pharma- ceutically acceptable carrier, for example, the composition can include excipients and / or auxiliary agents that facilitate processing of the active compound into a preparation designed for delivery to the site of action.

[0193] The pharmaceutical composition can be formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) by bolus injection. The formulation for injection can be supplied, for example, in ampoules or in multi-dose containers in unit dosage form with added preservatives. The composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents (such as suspending, stabilizing, and / or dispersing agents). Alternatively, the active ingredient can be in powder form, constituted with a suitable vehicle, for example pyrogen-free water.

[0194] Suitable formulations for parenteral administration also include aqueous solutions of the active compound in water-soluble form, e.g., water-soluble salts. In addition, suspensions of the active compound as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty acids, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, including, e.g., sodium carboxymethylcellulose, sorbitol, and dextran. Optionally, the suspension can also contain stabilizers. Liposomes can also be used to encapsulate the molecules of the present disclosure for delivery to cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the composition includes an isotonicity agent, e.g., sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride. In other embodiments, the composition contains pharma- ceutically acceptable substances (such as wetting agents) or minor amounts of auxiliary substances (such as wetting agents, emulsifying agents, preservatives, or buffers) that enhance the shelf life or effectiveness of the active ingredient.

[0195] The compositions of the present disclosure can be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the method of administration and therapeutic application.

[0196] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of ingredients as required above, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0197] The active ingredient can be formulated in controlled release preparations or devices.Examples of such preparations and devices include implants, skin patches and microencapsulated delivery systems.Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid.The preparation method of such preparations and devices is known in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0198] Injectable depot formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the polymer used, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions.

[0199] The composition may incorporate a supplementary active compound. In one embodiment, the chimeric protein of the present disclosure is formulated with another coagulation factor, or its variant, fragment, analog, or derivative. For example, the coagulation factor may include, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF) or activated form of any of these. The hemostatic coagulation factor may also include an antifibrinolytic agent, such as ε-aminocaproic acid, tranexamic acid.

[0200] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in dosage unit form. See, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

[0201] In addition to the active compound, liquid dosage forms may contain inactive ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0202] Non-limiting examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering agents, and wetting or emulsifying agents.

[0203] For oral administration, the pharmaceutical composition can take the form of a tablet or capsule prepared by conventional means. The composition can also be prepared as a liquid, for example, a syrup or suspension. The liquid can contain a suspending agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifying agent (lecithin or gum arabic), a non-aqueous vehicle (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and a preservative (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparation can also contain flavorings, colorings, and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

[0204] For buccal administration, the compositions may take the form of tablets or lozenges following conventional protocols.

[0205] For administration by inhalation, the compound for use according to the present disclosure is conveniently delivered in the form of a nebulized aerosol with or without excipients, or in the form of an aerosol spray from a pressurized pack or nebulizer, optionally with a propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0206] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0207] In one embodiment, the pharmaceutical composition comprises a polypeptide having factor VIII activity, an optimized nucleic acid molecule encoding a polypeptide having factor VIII activity, a vector comprising the nucleic acid molecule, or a host cell comprising the vector, and a pharma- ceutically acceptable carrier. In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. Parenteral administration can be intravenous or subcutaneous administration.

[0208] Treatment method In some aspects, the present disclosure relates to a method of treating a disease or condition in a subject in need thereof comprising administering a nucleic acid molecule, vector, polypeptide, or pharmaceutical composition disclosed herein.

[0209] In some embodiments, the disclosure relates to a method for increasing expression of a polypeptide having FVIII activity in a subject, hi some embodiments, the method comprises administering a nucleic acid molecule comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:16.

[0210] In some embodiments, the present disclosure relates to methods of treating bleeding disorders, hi some embodiments, the present disclosure relates to methods of treating hemophilia A.

[0211] The isolated nucleic acid molecule, vector, or polypeptide can be administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, orally, sublingually, buccal, sublingually, nasally, rectally, vaginally, or via the pulmonary route. The isolated nucleic acid molecule, vector, or polypeptide can also be administered intraneurally, intraocularly, and intrathecally. The coagulation factor protein can be implanted in or connected to a biopolymer solid support that allows for sustained release of the chimeric protein to the desired site.

[0212] In one embodiment, the route of administration of the isolated nucleic acid molecule, vector, or polypeptide is parenteral. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. In some embodiments, the isolated nucleic acid molecule, vector, or polypeptide is administered intravenously. Although all of these administration forms are clearly contemplated within the scope of the present disclosure, the administration form will be an injection solution, particularly an intravenous injection, an intraarterial injection, or an infusion solution.

[0213] The effective dose of the composition of the present disclosure for treating a condition will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. To optimize safety and efficacy, treatment dosages can be adjusted using routine methods known to those skilled in the art.

[0214] The nucleic acid molecules, vectors, or polypeptides of the disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).

[0215] As used herein, administration of an isolated nucleic acid molecule, vector, or polypeptide of the present disclosure in conjunction or in combination with an adjunctive therapy refers to sequential, simultaneous, concurrent, parallel, concomitant, or simultaneous administration or application of the therapy and the disclosed polypeptide. Those skilled in the art will understand that the time of administration or application of various components of a combined therapeutic regimen may be adjusted to enhance the overall effect of the treatment. It is believed that those skilled in the art (e.g., physicians) can easily determine an effective combined therapeutic regimen based on the selected adjunctive therapy and the teachings of the present specification without undue experimentation.

[0216] It will further be understood that the isolated nucleic acid molecules, vectors, or polypeptides of the present disclosure may be used in conjunction or in combination with one or more agents (e.g., to provide a combined therapeutic regimen). Exemplary agents that may be combined with the polypeptides or polynucleotides of the present disclosure include agents that represent the current standard of care for the particular disorder being treated. Such agents may be chemical or biological in nature. The term "biological" or "biological agent" refers to any pharmacologic active agent produced from a living organism and / or its products that is intended for use as a therapeutic agent.

[0217] The amounts of agents used in combination with the polynucleotides or polypeptides of the present disclosure may vary from subject to subject or may be administered according to what is known in the art. See, e.g., Bruce A Chabner et al., Antineoplastic Agents ((Joel G. Hardman et al., eds., 9th ed. 1996), in Goodman & Gilman's The Pharmacological Basis of Therapeutics 1233-1287. In another embodiment, amounts of such agents are administered consistent with standard medical practice.

[0218] In one embodiment, also disclosed herein is a kit comprising a nucleic acid molecule disclosed herein and instructions for administering the nucleic acid molecule to a subject in need thereof. In another embodiment, disclosed herein is a baculovirus system for the production of the nucleic acid molecule provided in the present invention. The nucleic acid molecule is produced in insect cells. In another embodiment, a nanoparticle delivery system for an expression construct is provided. The expression construct comprises a nucleic acid molecule disclosed herein.

[0219] Gene therapy Somatic cell gene therapy is being explored as a potential treatment for bleeding disorders, particularly hemophilia A. Gene therapy is a particularly attractive treatment for hemophilia because it has the potential to cure the disease through continuous endogenous production of FVIII after a single administration of a vector encoding FVIII. Hemophilia A is well suited to a gene replacement approach because its clinical pathology is entirely due to the lack of a single gene product (FVIII) that circulates in minute amounts (200 ng / ml) in plasma.

[0220] Lentiviruses have attracted attention as gene delivery vehicles due to their large capacity and ability to maintain transgene expression through integration. Lentiviruses have been evaluated in numerous ex vivo cell therapy clinical programs and have shown promising efficacy and safety profiles.

[0221] The present disclosure fulfills an important need in the art by providing lentiviral vectors comprising codon-optimized FVIII sequences that have increased expression in a subject and potentially enhance therapeutic efficacy when used in gene therapy.Embodiments of the present disclosure are directed to lentiviral vectors comprising one or more codon-optimized nucleic acid molecules encoding a polypeptide having FVIII activity, as described herein, host cells (e.g., hepatocytes) comprising the lentiviral vectors, and methods of using the disclosed lentiviral vectors (e.g., treatment for bleeding disorders using the lentiviral vectors disclosed herein).

[0222] In general, the therapeutic methods disclosed herein involve administration of a lentiviral vector comprising a nucleic acid molecule comprising at least one codon-optimized nucleic acid sequence encoding a FVIII coagulation factor, the nucleic acid sequence encoding the FVIII coagulation factor being operably linked to a suitable expression control sequence, which in some embodiments is incorporated into a lentiviral vector (e.g., a replication-deficient lentiviral vector).

[0223] The present disclosure provides a method of treating a bleeding disorder (e.g., hemophilia A) in a subject in need thereof, comprising administering to a subject a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, the method comprising administering to a subject a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, the ... 10 Transduction units / kg (TU / kg) or less, (10 9 TU / kg or less, or 10 8Methods are provided that include administering to a subject at least one dose of SEQ ID NO: 11 (e.g., 100 mg / mL or less TU / kg) in a single dose of SEQ ID NO: 11 (e.g., 100 mg / mL or less TU / kg). In some embodiments, the nucleotide sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the nucleotide sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14.

[0224] In some embodiments, the lentiviral vector is administered as a single dose or multiple doses. In some embodiments, the lentiviral vector dose is administered at once or divided into multiple partial doses, for example, two partial doses, three partial doses, four partial doses, five partial doses, six partial doses, or more than six partial doses. In some embodiments, two or more lentiviral vectors are administered.

[0225] In some embodiments, the dose of the lentiviral vector is repeatedly administered at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, the lentiviral vector is administered via intravenous injection.

[0226] In some embodiments, the subject is a pediatric subject, while in other aspects, the subject is an adult subject.

[0227] In some embodiments, the lentiviral vector comprises at least one tissue-specific promoter, i.e., a promoter that is believed to regulate the expression of a polypeptide having FVIII activity in a specific tissue or cell type. In some embodiments, the tissue-specific promoter in the lentiviral vector selectively enhances the expression of a polypeptide having FVIII activity in target liver cells. In some embodiments, the tissue-specific promoter selectively enhances the expression of a polypeptide having FVIII activity in target liver cells comprises the mTTR promoter. In some embodiments, the target liver cells are hepatocytes.

[0228] Since lentiviral vectors can transduce all liver cell types, expression of a transgene (e.g., FVIII) in different cell types can be controlled using different promoters in the lentiviral vector. Thus, lentiviral vectors can contain specific promoters that will control expression of a FVIII transgene in different tissues or cell types, such as different liver tissues or cell types. Thus, in some embodiments, lentiviral vectors can contain an endothelial-specific promoter that will control expression of a FVIII transgene in liver endothelial tissues, or a hepatocyte-specific promoter that will control expression of a FVIII transgene in hepatocytes, or both.

[0229] In some embodiments, the lentiviral vector comprises one or more tissue-specific promoters that control expression of the FVIII transgene in tissues other than the liver. In some embodiments, the isolated nucleic acid molecule is stably integrated into the genome of a target cell or tissue, for example, the genome of a hepatocyte or the genome of a liver endothelial cell.

[0230] In some embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity in a lentiviral vector of the present disclosure comprises, consists of, or consists essentially of coBDDFVIII-3aa (SEQ ID NO: 14).

[0231] In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity in a lentiviral vector of the present disclosure comprises, consists of, or consists essentially of coBDDFVIII6-XTEN-3aa (SEQ ID NO:11).

[0232] In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity in a lentiviral vector of the present disclosure comprises, consists of, or consists essentially of SEQ ID NO:16.

[0233] The lentiviral vectors disclosed herein can be administered in vivo in a mammal, e.g., a human patient at low doses (e.g., 10 to 20 mg / kg) using a gene therapy approach for the treatment of a bleeding disease or disorder selected from the group consisting of hemorrhagic coagulopathy, hemarthrosis, muscle bleeds, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath, which may be therapeutically beneficial. 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A.

[0234] In some embodiments, the target cells (e.g., hepatocytes) are administered at a low dose (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8In certain embodiments, target cells (e.g., hepatocytes) are treated in vitro with about 3.0×10 TU / kg or less of a lentiviral vector disclosed herein prior to administration to the patient. 9 In yet another embodiment, cells from a patient (e.g., hepatocytes) are treated in vitro with TU / kg of the lentiviral vector disclosed herein. In yet another embodiment, cells from a patient (e.g., hepatocytes) are treated in vitro with a low dose (e.g., 10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 The mice are treated ex vivo with a lentiviral vector disclosed herein (maximum 100 TU / kg or less).

[0235] In some embodiments, the lentiviral vectors disclosed herein (e.g., 10 TU / kg or less, 10 9 TU / kg or less, or 10 8 In some embodiments, the plasma FVIII activity after administration of the antibody (administered at or below TU / kg) is increased by at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% relative to physiologically normal circulating FVIII levels.

[0236] The present disclosure also provides a method of treating, preventing, or ameliorating a hemostatic disorder (e.g., a bleeding disorder such as hemophilia A) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the lentiviral vector is administered to a subject in an amount of at least 5×10 10 TU / kg or less, 10 9 TU / kg or less, or 10 8Also provided are methods administered in at least one dose of TU / kg or less.

[0237] The treatment, amelioration, and prevention by the lentiviral vector of the present disclosure can be a bypass therapy. The subject undergoing the bypass therapy already has an inhibitor against a clotting factor, such as FVIII, or is predisposed to developing a clotting factor inhibitor.

[0238] The lentiviral vectors of the present disclosure treat or prevent hemostatic disorders by promoting the formation of fibrin clots. The polypeptides having FVIII activity encoded by the nucleic acid molecules of the present disclosure can activate members of the coagulation cascade. The coagulation factors can be involved in the extrinsic pathway, the intrinsic pathway, or both.

[0239] The lentiviral vectors of the present disclosure can be used to treat hemostatic disorders known to be treatable by FVIII. Hemostatic disorders that can be treated using the methods of the present disclosure include, but are not limited to, hemophilia A, hemophilia B, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, and deficiency or structural abnormality of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, hemarthrosis, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath.

[0240] Compositions for administration to a subject include lentiviral vectors (for gene therapy applications) that contain a nucleic acid molecule that includes an optimized nucleotide sequence of the present disclosure that encodes a FVIII coagulation factor, as well as FVIII polypeptide molecules. In some embodiments, the composition for administration is a cell that has been contacted in vivo, in vitro, or ex vivo with a lentiviral vector of the present disclosure.

[0241] In some embodiments, the hemostatic disorder is a genetic disorder. In one embodiment, the subject has hemophilia A. In other embodiments, the hemostatic disorder is the result of a deficiency of FVIII. In other embodiments, the hemostatic disorder can be the result of a defective FVIII clotting factor.

[0242] In another embodiment, the hemostatic disorder may be an acquired disorder. The acquired disorder may be due to an underlying secondary disease or condition. Non-related conditions include, but are not limited to, cancer, autoimmune disease, or pregnancy. The acquired disorder may be due to old age or to drug therapy (e.g., cancer chemotherapy) to treat the underlying secondary disorder.

[0243] The present disclosure also relates to a method for treating a subject who does not have a hemostatic disorder or a secondary disease or condition that results in the acquisition of a hemostatic disorder.Accordingly, the present disclosure relates to a method for treating a subject who needs a general hemostatic agent, comprising administering a therapeutically effective amount of the lentiviral vector of the present disclosure.For example, in one embodiment, the subject who needs a general hemostatic agent is undergoing or about to undergo surgery.The lentiviral vector of the present disclosure can be administered before or after surgery as a prophylactic agent.

[0244] The lentiviral vectors of the present disclosure can be administered during or after surgery, including but not limited to liver transplantation, liver resection, or stem cell transplantation, to control acute bleeding episodes.

[0245] In another embodiment, the lentiviral vectors of the present disclosure can be used to treat subjects who do not have a hemostatic disorder and have an acute bleeding episode. The acute bleeding episode can result from severe trauma, such as surgery, a car accident, a wound, a gunshot wound, or any other traumatic event that results in uncontrollable bleeding.

[0246] Lentiviral vectors can be used to prophylactically treat subjects with hemostatic disorders. Lentiviral vectors can also be used to treat acute bleeding episodes in subjects with hemostatic disorders.

[0247] In another embodiment, administration of the lentiviral vector disclosed herein and / or subsequent expression of the FVIII protein does not induce an immune response in the subject. In some embodiments, the immune response includes the development of antibodies against FVIII. In some embodiments, the immune response includes cytokine secretion. In some embodiments, the immune response includes activation of B cells, T cells, or both B and T cells. In some embodiments, the immune response is an inhibitory immune response in the subject that reduces the activity of the FVIII protein compared to the activity of FVIII in a subject that has not developed an immune response. In certain embodiments, expression of the FVIII protein by administering the lentiviral vector of the present disclosure prevents an inhibitory immune response against the FVIII protein or the FVIII protein expressed from the isolated nucleic acid molecule or lentiviral vector.

[0248] In some embodiments, the lentiviral vector of the present disclosure is administered in combination with at least one other agent that promotes hemostasis. The other agent that promotes hemostasis is a therapeutic agent that has been demonstrated to have clotting activity. By way of example, the hemostatic agent includes, but is not limited to, factor V, factor VII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, or fibrinogen, or any of the above in an activated form. The clotting factor or hemostatic agent can also include an antifibrinolytic agent, such as epsilon-aminocaproic acid, tranexamic acid.

[0249] In one embodiment of the present disclosure, the composition (e.g., lentiviral vector) is one in which FVIII is present in an activatable form when administered to a subject. Such an activatable molecule can be activated in vivo at the site of clotting after administration to a subject.

[0250] The lentiviral vectors of the present disclosure can be administered intravenously, subcutaneously, intramuscularly, or through any mucosal surface, for example, orally, sublingually, buccal, sublingually, nasally, rectally, vaginally, or via the pulmonary route. The lentiviral vector can be implanted in or connected to a biopolymer solid support that allows for the slow release of the vector to the desired site.

[0251] In one embodiment, the route of administration of the lentiviral vector is parenteral. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intravenous forms of parenteral administration are preferred. All of these forms of administration are clearly contemplated within the scope of the present disclosure, but the form of administration will be an injectable solution, particularly a solution for intravenous injection, intraarterial injection, or infusion. Typically, suitable injectable pharmaceutical compositions can include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, in other methods consistent with the teachings herein, the lentiviral vector can be delivered directly to the site of harmful cell populations, thereby increasing the exposure of affected tissues to the therapeutic agent.

[0252] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the present disclosure, pharma- ceutically acceptable carriers include, but are not limited to, 0.01-0.1M, preferably 0.05M, phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0253] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that it can be easily injected. It should be stable under the conditions of manufacture and storage, and preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0254] Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0255] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the polypeptide alone or in combination with other active agents) in the required amount in an appropriate solvent, together with one or a combination of ingredients listed herein, as needed, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterilized solution that has been sterilized by filtration. The preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Furthermore, the preparations can be packaged and sold in the form of a kit. Such articles of manufacture would preferably have a label or package insert indicating that the accompanying composition is useful for treating a subject suffering from or predisposed to a coagulation disorder.

[0256] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0257] The effective dose of the composition of the present disclosure for treating a condition will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. To optimize safety and efficacy, treatment dosages can be adjusted using routine methods known to those skilled in the art.

[0258] Lentiviral vectors can be administered as a single dose or multiple doses, and multiple doses can be administered consecutively or at specific time intervals. In vitro assays can be used to determine optimal dose ranges and / or administration schedules. In vitro assays for measuring clotting factor activity are known in the art. In addition, effective doses can be estimated by extrapolating dose-response curves obtained from animal models, such as hemophilic dogs (Mount et al. 2002, Blood 99(8):2670).

[0259] Intermediate doses within the above ranges are also considered to be within the scope of the present disclosure.Such doses can be administered to subjects daily, every other day, weekly, or according to any other schedule that is determined by empirical analysis.Exemplary treatment requires administration of multiple doses over a long period of time, for example, at least 6 months.

[0260] The lentiviral vector of the present disclosure can be administered on multiple occasions. The interval between single doses can be daily, weekly, monthly, or yearly. The interval can also be irregular as indicated by measuring the blood level of modified polypeptide or antigen in the patient. The dosage and frequency of the lentiviral vector of the present disclosure depends on the half-life of the FVIII polypeptide encoded by the transgene in the patient.

[0261] The dosage and frequency of administration of the lentiviral vector of the present disclosure may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a composition containing the lentiviral vector of the present disclosure is administered to a patient who is not yet in a disease state to enhance the patient's resistance or minimize the effects of the disease. Such an amount is defined as a "prophylactically effective dose." A relatively low dosage is administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives.

[0262] The lentiviral vectors of the present disclosure can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).

[0263] As used herein, administration of the lentiviral vector of the present disclosure in conjunction with or in combination with an adjunctive therapy refers to sequential, simultaneous, concurrent, parallel, concomitant, or simultaneous administration or application of the therapy and the disclosed polypeptide. Those skilled in the art will understand that the time of administration or application of various components of a combined therapeutic regimen may be adjusted to enhance the overall effect of the treatment. It is believed that those skilled in the art (e.g., physicians) can easily determine an effective combined therapeutic regimen based on the selected adjunctive therapy and the teachings of the present specification without undue experimentation.

[0264] It will further be understood that the lentiviral vectors of the present disclosure may be used in conjunction or in combination with one or more agents (e.g., to provide a combined therapeutic regimen). Exemplary agents that may be combined with the lentiviral vectors of the present disclosure include agents that represent the current standard of care for the particular disorder being treated. Such agents may be chemical or biological in nature. The term "biological" or "biological agent" refers to any pharmacologic active agent produced from a living organism and / or its products that is intended for use as a therapeutic agent.

[0265] The amount of drugs used in combination with the lentiviral vectors of the present disclosure may vary from subject to subject or may be administered according to what is known in the art. See, e.g., Chabner et al., Pharmacological Basis of Therapeutics 1233-1287 (Joel G. Hardman et al., eds., 9th ed. 1996). In another embodiment, amounts of such drugs are administered that are consistent with standard medical practice.

[0266] In certain embodiments, the lentiviral vector of the present disclosure is administered in combination with an immunosuppressant, anti-allergic, or anti-inflammatory agent. These agents generally refer to substances that act to suppress or mask the immune system of the subject being treated herein. These agents include substances that suppress cytokine production, downregulate or suppress the expression of self-antigens, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines; azathioprine; cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde; anti-idiotypic antibodies against MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticoids, e.g., prednisone, methylprednisolone, and dexamethasone; anti-interferon-gamma, -beta, or -alpha antibodies, anti-tumor necrosis factor- ... Examples of the agent include cytokine or cytokine receptor antagonists, including anti-IL-β antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; xenogeneic antilymphocyte globulins; pan-T antibodies; soluble peptides containing LFA-3 binding domains; streptokinase; TGF-β; streptodornase; FK506; RS-61443; deoxyspergualin; and rapamycin. In certain embodiments, the agent is an antihistamine. As used herein, an "antihistamine" is an agent that antagonizes the physiological action of histamine. Examples of antihistamines include chlorpheniramine, diphenhydramine, promethazine, cromolyn sodium, astemizole, azatadine maleate, bropheniramine maleate, carbinoxamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, dexbrompheniramine maleate, dexchlorpheniramine maleate, dimenhydrinate, diphenhydramine hydrochloride, doxylamine succinate, fexofendazine hydrochloride, terfenadine hydrochloride, hydroxyzine hydrochloride, loratidine, meclizine hydrochloride, tripelennamine citrate, tripelennamine hydrochloride, and triprolidine hydrochloride.

[0267] Immunosuppressants, anti-allergic agents, or anti-inflammatory agents may be incorporated into the lentiviral vector administration regimen. For example, administration of the immunosuppressant or anti-inflammatory agent may begin prior to administration of the disclosed lentiviral vectors and may be followed by one or more subsequent administrations. In certain embodiments, the immunosuppressant or anti-inflammatory agent is administered as a premedication for the lentiviral vector.

[0268] As previously discussed, the lentiviral vector of the present disclosure can be administered in a pharmacologic effective amount for the in vivo treatment of coagulation disorders. In this regard, it will be understood that the lentiviral vector of the present disclosure can be formulated to aid administration and promote the stability of the active agent. Preferably, the pharmaceutical composition according to the present disclosure comprises a pharmacologic acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc. Of course, the pharmaceutical composition of the present disclosure can be administered in a single or multiple doses to provide a pharmacologic effective amount of the polypeptide.

[0269] Many tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM® assay, prothrombin time (PT) test (also used to determine the INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing tests (if the patient's plasma is mixed with normal plasma, will the abnormality be corrected?), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), multifaceted platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0270] The aPTT test is a performance index that measures the efficacy of the "intrinsic" coagulation pathway (also called the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, e.g., FVIII or FIX. It is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0271] ROTEM® analysis provides information on the global dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. The various parameters of thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that affect their interactions. This assay can provide a complete view of secondary hemostasis.

[0272] All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0273] All publications, patents, and patent applications mentioned in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0274] Having generally described the present disclosure, a better understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0275] Example 1: Generation of the optimized coBDDFVIII6-XTEN-3aa transgene It was hypothesized that transgene expression levels could be increased by codon-optimizing the coding sequence of the target host. Previous studies have demonstrated higher levels of FVIII expression using a codon-optimized FVIIIco6XTEN gene cassette. This gene cassette contains a codon-optimized cDNA encoding B-domain deleted human factor VIII (BDDcoFVIII) fused with the XTEN144 peptide in the B-domain of FVIII.

[0276] To further improve target specificity and reduce immunogenicity, in silico antigenicity analysis was used to evaluate and minimize the risk of introducing neo-epitopes into the FVIIIco6XTEN protein. Various representative human leukocyte antigen (HLA) alleles (e.g., DR, DP, DQ) were evaluated using the open source immune epitope database and analysis resource (IEDB) and recommended prediction methods to determine major histocompatibility complex class II (MHCII) binding to the chimeric protein. Additional analysis also used the NetMHCIIpan3.0 method using HLA-DR alleles (representative of North American or Japanese populations) (see Lamberth K, et al. Sci Transl Med. 2017;9(372):eaag1286,).

[0277] Half-maximal inhibitory concentration (IC 50 Peptides with IC values ​​of <50 nM, <500 nM, and <5000 nM were considered to have high affinity (high immunogenicity risk), moderate affinity, and low affinity for MHCII, respectively. 50 HLA-DR alleles were assessed using cutoffs: IC 50 Values ​​>500 nM were not considered to have significant immunogenic potential.

[0278] The GAP residues located at the FVIII-XTEN junction of the chimeric protein were identified as potentially immunogenic. Applicant identified these GAP residues as encoded by nucleotide sequences corresponding to the XhoI restriction enzyme site originally introduced to facilitate cloning. The nine nucleotides encoding the GAP residues were deleted from the coding sequence of the FVIII protein. It was determined that the deletion of these nucleotides and the corresponding GAP residues in the translated protein eliminated the potential for immunogenicity at the FVIII-XTEN junction.

[0279] This resulted in the final FVIII nucleotide sequence encoding the chimeric FVIII protein tested herein, which is referred to as "coBDDFVIII6-XTEN-3aa." The nucleotide sequence encoding coBDDFVIII6-XTEN-3aa is disclosed as SEQ ID NO: 11. The amino acid sequence of coBDDFVIII6-XTEN-3aa is disclosed as SEQ ID NO: 12 (see Table 1 for further sequence information).

[0280] Example 2: Generation of a gene expression cassette encoding coBDDFVIII-XTEN-3aa A gene expression cassette was designed to carry the coBDDFVIII-XTEN-3aa transgene under the control of a hepatocyte-specific promoter for in vivo expression. The gene expression cassette is flanked by 5' and 3' long terminal repeat (LTR) sequences that facilitate integration of the transfer plasmid sequences into the host genome.

[0281] The LTR coding element includes a chimeric 5'LTR (SEQ ID NO:1) fused to a heterologous human cytomegalovirus (CMV) early gene promoter region, a self-inactivating (SIN) deletion of the enhancer / promoter sequence in the U3 region of the 3'LTR (annotated as SEQ ID NO:X_"dU3RU5"), and the R and U5 regions that allow Tat binding (SEQ ID NO:2_RU5 region).

[0282] The transfer plasmid maintains the cis-acting viral sequences required for encapsidation, reverse transcription, and integration into the host cell genome: packaging signal (Psi, Ψ), primer binding site (PBS) of SL123 (SEQ ID NO: 3), stem loop 4 (SL4) (SEQ ID NO: 4), polypurine tract (PPT) (SEQ ID NO: 6) required for reverse transcription, an intron with donor and acceptor splice sites, and Rev response element (RRE) (SEQ ID NO: 5) required for Rev-mediated nuclear transport of the complete unspliced ​​genome transcript. In addition, the plasmid also encodes four tandem copies of the complementary sequence of hematopoietic specific microRNA, miR-142-3pT (SEQ ID NO: 10), which is integrated into the 3'UTR while maintaining transgene expression in hematopoietic antigen presenting cells in non-hematopoietic cells (Brown et al. Nature 12:585-591 (2006)).

[0283] The gene cassette contains a codon-optimized cDNA encoding B-domain deleted human factor VIII (BDDcoFVIII) fused to the XTEN144 peptide in which three amino acid residues (Gly-Ala-Pro) at the FVIII / XTEN junction have been removed to avoid a potential MHCII binding site (XTEN-3aa) (see Example 1). This transgene, also called coBDDFVIII6-XTEN-3aa, is regulated by a liver-specific modified mouse transthyretin (mTTR) promoter (SEQ ID NO: 9) with two upstream enhancer sequences, an mTTR enhancer element (SEQ ID NO: 8), and a synthetic enhancer (SEQ ID NO: 7).

[0284] A graphical representation of the plasmid containing the gene expression cassette encoding coBDDFVIII6-XTEN-3aa is shown in FIG.

[0285] In the following examples, the in vivo function of the coBDDFVIII-XTEN-3aa transgene is tested in multiple animal models.

[0286] Example 3: Long-term dose response of LV-coBDDFVIII6-XTEN-3aa treatment in neonatal HemA mice To evaluate the efficacy of using a lentiviral system to express coBDDFVIII6-XTEN-3aa and generate FVIII activity in the pediatric HemA model, neonatal (2-day-old) HemA mice were administered approximately 1.5 × 10 LV-coBDDFVIII6-XTEN-3aa via temporal vein injection. 9 , 3.0×10 9 , 6×10 9 , or 1.3 × 10 10 TU / kg was administered. Circulating FVIII activity was measured by a FVIII chromogenic assay. Circulating FVIII protein was measured by a human FVIII-specific ELISA assay.

[0287] Sustained long-term FVIII expression was observed in a dose-dependent manner for all mice treated with LV-coBDDFVIII6-XTEN-3aa. After lentiviral vector treatment, FVIII activity levels in mice treated with LV-coBDDFVIII6-XTEN-3aa remained relatively stable until the end of the study at 25 weeks (Figure 2A). The highest FVIII activity, approximately 50%, was observed with 1.3 × 10 10 This was observed in mice receiving the lentiviral dose of 100 TU / kg. Consistent with the FVIII activity data, circulating FVIII protein levels remained relatively stable for all lentiviral doses until the end of the study (Figure 2B).

[0288] These data demonstrate that coBDDFVIII6-XTEN-3aa delivered using a lentiviral system can generate therapeutic FVIII levels in neonatal HemA mice.

[0289] These data support the potential therapeutic benefit of using LV-coBDDFVIII6-XTEN-3aa for the treatment of pediatric HemA patients.

[0290] Example 4: Long-term dose response of LV-coBDDFVIII6-XTEN-3aa treatment in adult HemA mice To evaluate the efficacy of using a lentiviral system to express coBDDFVIII6-XTEN-3aa and generate FVIII activity in the adult HemA model, adult (16-week-old) HemA mice were administered approximately 1.3 × 10 LV-coBDDFVIII6-XTEN-3aa via temporal vein injection. 10 or 3.7 x 10 10 TU / kg was administered. Circulating FVIII activity was measured by a FVIII chromogenic assay. Circulating FVIII protein was measured by a human FVIII-specific ELISA assay.

[0291] Sustained long-term FVIII expression was observed in both dose groups in a dose-dependent manner. After lentiviral vector treatment, FVIII activity levels in all mice receiving LV-coBDDFVIII6-XTEN-3aa remained relatively stable until at least the end of the study at week 20 (Figure 3). 10 Mice receiving 1.3 × 10 TU / kg had FVIII activity of approximately 50% of normal for the duration of the study. 10 Mice receiving TU / kg had FVIII activity 5-7% or greater of normal.

[0292] These data indicate that coBDDFVIII6-XTEN-3aa delivered using a lentiviral system can generate therapeutic FVIII levels in adult HemA mice.

[0293] These data support the potential therapeutic benefit of using LV-coBDDFVIII6-XTEN-3aa for the treatment of adult HemA patients. These data also suggest that the therapeutic benefit of using LV-coBDDFVIII6-XTEN-3aa may be achieved with relatively low doses of LV.

[0294] Example 5: Long-term dose response of LV-coBDDFVIII6-XTEN-3aa treatment in non-human primates To evaluate the efficacy of expressing coBDDFVIII6-XTEN-3aa using the lentiviral system to generate FVIII activity in non-human primates, 10 male pigtailed macaques (body weight 3.5-4.3 kg) were transfected with CD47high / MHC-I free Patients were treated with LV-coFVIII-6 or LV-coFVIII-6-XTEN produced from 293T cells by intravenous (IV) infusion at an infusion rate of 1.5 mL / min. The dose for LV-coBDDFVIII6-XTEN-3aa was 1×10 9 Or 3 x 10 9 The doses were TU / kg. Animals were treated daily with intramuscular injections of SOLU-MEDROL® (methylprednisolone) at a dose of 10 mg / kg from 1 day prior to LV treatment through day 7 to control anti-human FVIII antibody formation. Animals were also treated with an IV injection of Polaramine (dexchlorpheniramine) at a dose of 4 mg / kg 30 minutes prior to LV treatment to control potential allergic reactions.

[0295] Plasma samples were collected on days 0, 1, 3, 7, 14, 21, 28, 45, and 60 after LV treatment and analyzed for human FVIII activity and FVIII antigen levels. Circulating FVIII activity was measured by a FVIII chromogenic assay. Circulating FVIII protein was measured by a human FVIII-specific ELISA assay. FVIII activity and FVIII antigen levels for each LV treatment group were averaged across post-treatment time points.

[0296] 1×10 9 and 3 x 10 9 The mean FVIII activity levels in the TU / kg treatment groups were approximately 20% and 75% of normal, respectively (Figure 4A). 9 Or 3 x 10 9 The mean FVIII antigen levels in the TU / kg treatment groups were approximately 31 ng / mL or approximately 140 ng / mL, respectively (Figure 4B).

[0297] These data demonstrate that LV-coBDDFVIII6-XTEN-3aa is capable of producing therapeutic levels of human FVIII in non-human primates.

[0298] array

[0299] [Table 1]

[0300] [Table 2]

[0301] [Table 3]

[0302] [Table 4]

[0303] [Table 5]

[0304] [Table 6]

[0305] [Table 7]

[0306] [Table 8]

[0307] [Table 9]

[0308]

Table 10

[0309]

Table 11

[0310]

Table 12

[0311]

Table 13

[0312]

Table 14

[0313]

Table 15

[0314]

Table 16

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 14, wherein the nucleotide sequence encodes a polypeptide having factor VIII (FVIII) activity.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to nucleotides 58 to 4815 of SEQ ID NO:

11.

3. 1. An isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII (FVIII) polypeptide: i) a nucleic acid molecule according to claim 1; ii) a promoter controlling the transcription of said nucleotide sequence; iii) a transcription termination sequence; and An isolated nucleic acid molecule comprising:

4. the promoter is a liver-specific promoter; the promoter is a mouse transthyretin (mTTR) promoter; the promoter is the mTTR482 promoter; and / or The promoter comprises the nucleotide sequence of SEQ ID NO:

9. The isolated nucleic acid molecule of claim 3.

5. 4. The isolated nucleic acid molecule of claim 3, further comprising an enhancer element, optionally wherein the enhancer element is an mTTR enhancer element and / or the mTTR enhancer element comprises the nucleotide sequence of SEQ ID NO:

8.

6. (a) a synthetic enhancer sequence, optionally wherein said synthetic enhancer sequence comprises the nucleotide sequence of SEQ ID NO:7; (b) Polyprint Track (PPT), optionally wherein the PPT sequence is the nucleic acid sequence of SEQ ID NO:

6. Contains a nucleotide sequence; (c) a human CMV promoter region sequence, optionally wherein the CMV promoter region sequence comprises the nucleotide sequence of SEQ ID NO:1; (d) 5' or 3' long terminal repeat (LTR) sequences; (e) a stem-loop 4 sequence, optionally wherein said stem-loop 4 sequence comprises the nucleotide sequence of SEQ ID NO: 4; (f) a primer binding site of SL123, optionally wherein said primer binding site of SL123 comprises the nucleotide sequence of SEQ ID NO:3; and / or (g) a primer binding site of the RU5 region, optionally wherein the RU5 region sequence comprises the nucleotide sequence of SEQ ID NO:2; 4. The isolated nucleic acid molecule of claim 3, further comprising:

7. The gene cassette comprises, from 5' to 3': (a) 5′ long terminal repeat (LTR) sequence; (b) a liver-specific modified mouse transthyretin (mTTR) promoter comprising the nucleotide sequence of SEQ ID NO: 9; (c) a nucleotide sequence encoding a FVIII protein comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 14; and (d) comprising a 3' LTR sequence; The isolated nucleic acid molecule of claim 3.

8. 4. The isolated nucleic acid molecule of claim 3, wherein the gene cassette comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO:

16.

9. A vector comprising the nucleic acid molecule of any one of claims 1 to 8.

10. A host cell comprising the nucleic acid molecule of any one of claims 1 to 8 or the vector of claim 9.

11. A polypeptide produced by the host cell of claim 10.

12. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

13. A kit comprising the nucleic acid molecule of any one of claims 1 to 8 and instructions for administering said nucleic acid molecule to a subject in need thereof.

14. 13. Use of the isolated nucleic acid molecule of any one of claims 1 to 8 or the pharmaceutical composition of claim 12 for treating a bleeding disorder in a subject, optionally wherein the bleeding disorder is hemophilia A.

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