Optimized Factor VIII Gene

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 making such polypeptides.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 236,225, filed August 23, 2021, which is incorporated by reference in its entirety.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the sequence listing submitted electronically in XML format (Name: SA9-484_SeqListing.xml; Size: 117,240 bytes; Creation Date: August 22, 2022) are incorporated by reference in their entirety into this specification. [Background technology]

[0003] The main obstacle to providing patients with inexpensive recombinant FVIII protein is the high commercial price. In heterologous expression systems, FVIII protein is poorly expressed, 2-3 orders of magnitude lower than proteins of similar size (Non-Patent Document 1). Poor expression of FVIII is due in part to the presence of cis-acting elements in the FVIII coding sequence that inhibit FVIII expression, such as transcriptional silencer elements (Non-Patent Document 2), matrix attachment region-like sequences (MARs) (Non-Patent Document 3), and transcriptional elongation inhibitor elements (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lynch et al., Hum. Gene. Ther., 4:259-72 (1993). [Non-Patent Document 2] Hoeben et al., Blood, 85:2447–2454 (1995) [Non-Patent Document 3] Fallux et al., Mol. Cell. Biol., 16:4264~4272 (1996) [Non-Patent Document 4] Koeberl et al., Hum. Gene. Ther., 6:469–479 (1995) Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for FVIII sequences that are efficiently expressed in heterologous systems. [Means for solving the problem]

[0006] Codon-optimized nucleic acid molecules that encode polypeptides with FVIII activity are disclosed.

[0007] In certain aspects herein, an isolated nucleic acid molecule is disclosed that comprises a nucleotide sequence that is at least about 85% identical to SEQ ID NO:9, wherein the nucleotide sequence encodes a polypeptide with factor VIII (FVIII) activity. In some embodiments, the nucleotide sequence is at least 90% identical to SEQ ID NO:9. In some embodiments, the nucleotide sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:9. In some embodiments, the nucleotide sequence is at least 50% identical to SEQ ID NO:9.

[0008] Also disclosed herein is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:9, wherein the nucleotide sequence encodes a polypeptide with Factor VIII activity.

[0009] Also disclosed herein are isolated nucleic acid molecules comprising a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleotides 58-4824 of SEQ ID NO: 9. In some embodiments, the isolated nucleic acid molecule comprises nucleotides 58-4824 of SEQ ID NO:9.

[0010] In certain aspects herein, an isolated nucleic acid molecule is disclosed that comprises a nucleotide sequence that is at least about 85% identical to SEQ ID NO: 33, wherein the nucleotide sequence encodes a polypeptide with factor VIII (FVIII) activity. In some embodiments, the nucleotide sequence is at least 90% identical to SEQ ID NO: 33. In some embodiments, the nucleotide sequence is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, the nucleotide sequence is at least 50% identical to SEQ ID NO: 33.

[0011] In some embodiments, the isolated nucleic acid molecule disclosed herein further comprises a nucleotide sequence encoding a signal peptide. In some embodiments, the nucleotide sequence encoding the signal peptide comprises the amino acid sequence of SEQ ID NO:11.

[0012] In some embodiments, the isolated nucleic acid molecules disclosed herein are codon-optimized to contain fewer CpG motifs than SEQ ID NO: 32. In some embodiments, the isolated nucleic acid molecules disclosed herein have one or more CpG motifs deleted compared to SEQ ID NO: 32.

[0013] In another aspect herein, an isolated nucleic acid molecule is disclosed that comprises a gene cassette expressing a Factor VIII (FVIII) polypeptide, the gene cassette comprising a nucleotide sequence that is at least about 85% identical to SEQ ID NO: 14. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 90% identical to SEQ ID NO: 14. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the nucleotide sequence is at least 50% identical to SEQ ID NO: 14.

[0014] Also disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a factor VIII (FVIII) polypeptide, wherein the gene cassette comprises the nucleotide sequence of SEQ ID NO:14.

[0015] In another aspect herein, an isolated nucleic acid molecule is disclosed that comprises a gene cassette expressing a factor VIII (FVIII) polypeptide, the gene cassette comprising a nucleotide sequence that is at least about 85% identical to SEQ ID NO: 35. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 90% identical to SEQ ID NO: 35. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, the nucleotide sequence is at least 50% identical to SEQ ID NO: 35.

[0016] Also disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII (FVIII) polypeptide, wherein the gene cassette comprises the nucleotide sequence of SEQ ID NO:35.

[0017] In another aspect of the present specification, an isolated nucleic acid molecule is disclosed that comprises a gene cassette expressing a Factor VIII (FVIII) polypeptide, the gene cassette comprising a nucleotide sequence encoding a FVIII protein, the nucleotide sequence comprising a nucleic acid sequence that is at least 85% identical to SEQ ID NO:9 or SEQ ID NO:33; a promoter that controls transcription of the nucleotide sequence; and a transcription termination sequence.

[0018] 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: 16.

[0019] In some embodiments, the transcription termination sequence is a polyadenylation (polyA) sequence. In some embodiments, the transcription termination sequence is a bovine growth hormone polyadenylation (bGHpA) signal sequence. In some embodiments, the transcription termination sequence comprises the nucleotide sequence of SEQ ID NO: 19.

[0020] In some embodiments, the isolated nucleic acid molecule further comprises an enhancer element. In some embodiments, the enhancer element is an A1MB2 enhancer element. In some embodiments, the A1MB2 enhancer element comprises the nucleotide sequence of SEQ ID NO: 15.

[0021] In some embodiments, the isolated nucleic acid molecule further comprises an intron sequence. In some embodiments, the intron sequence is a chimeric intron, a hybrid intron, or a synthetic intron. In some embodiments, the intron sequence comprises the nucleotide sequence of SEQ ID NO: 17.

[0022] In some embodiments, the isolated nucleic acid molecule further comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element comprises a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE comprises the nucleotide sequence of SEQ ID NO: 18.

[0023] In another aspect herein, an isolated nucleic acid molecule is disclosed that includes a gene cassette expressing a factor VIII (FVIII) polypeptide, a first inverted terminal repeat (ITR) and a second ITR flanking the gene cassette. In some embodiments, the first ITR and / or the second ITR are derived from a member of the Parvoviridae family. In some embodiments, the first ITR and / or the second ITR are derived from a human bocavirus (HBoV1), human erythrovirus (B19), goose parvovirus (GPV), or variants thereof. In some embodiments, the first ITR and / or the second ITR comprise a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:1, 2, or 21-30. In some embodiments, the first ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:1, and the second ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:2. In some embodiments, the first ITR comprises a polynucleotide sequence that is at least about 50% identical to SEQ ID NO:1, and the second ITR comprises a polynucleotide sequence that is at least about 50% identical to SEQ ID NO:2. In some embodiments, the first ITR comprises the polynucleotide sequence of SEQ ID NO:1, and the second ITR comprises the polynucleotide sequence of SEQ ID NO:2.

[0024] In another aspect of the present specification, an isolated nucleic acid molecule is disclosed that comprises a gene cassette expressing a factor VIII (FVIII) polypeptide, the gene cassette comprising, from 5' to 3', an A1MB2 enhancer element comprising the nucleotide sequence of SEQ ID NO: 15; a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR) comprising the nucleotide sequence of SEQ ID NO: 16; a chimeric intron comprising the nucleotide sequence of SEQ ID NO: 17; a nucleotide sequence encoding a FVIII protein comprising a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 9 or SEQ ID NO: 33; a woodchuck post-transcriptional regulatory element (WPRE) comprising the nucleotide sequence of SEQ ID NO: 18; and a bovine growth hormone polyadenylation (bGHpA) signal comprising the nucleotide sequence of SEQ ID NO: 19.

[0025] In another aspect of the present specification, a vector is disclosed that includes the nucleic acid molecule disclosed herein.

[0026] In another aspect of the present specification, a host cell is disclosed that comprises the nucleic acid molecule disclosed herein. Also disclosed herein is a polypeptide produced by the host cell. In some embodiments, the host cell is an insect cell.

[0027] In another aspect of the present specification, a baculovirus system for producing the nucleic acid molecules disclosed herein is disclosed. In some aspects, the nucleic acid molecules are produced in insect cells.

[0028] In another aspect of the present specification, a pharmaceutical composition is disclosed that comprises the nucleic acid molecule disclosed herein.In some embodiments, the pharmaceutical composition comprises a vector that comprises the nucleic acid molecule disclosed herein.In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0029] In another aspect of the present specification, a kit is disclosed that includes a nucleic acid molecule disclosed herein and instructions for administering the nucleic acid molecule to a subject in need thereof.

[0030] In another aspect of the present specification, a method for producing a polypeptide with FVIII activity is disclosed, the method comprising culturing a host cell disclosed herein under conditions whereby a polypeptide with FVIII activity is produced, and recovering the polypeptide with FVIII activity.

[0031] In another aspect herein, a method of increasing expression of a polypeptide with FVIII activity in a subject is disclosed, comprising administering a nucleic acid molecule comprising a nucleotide sequence at least about 85% identical to SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35.

[0032] In another aspect herein, a method of treating a bleeding disorder in a subject is disclosed, comprising administering a nucleic acid molecule comprising a nucleotide sequence at least about 85% identical to SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35.

[0033] In another aspect herein, a method of treating a bleeding disorder in a subject is disclosed, comprising administering a pharmaceutical composition comprising a nucleotide sequence at least about 85% identical to SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35.

[0034] In another aspect herein, a method of treating hemophilia A in a subject is disclosed, comprising administering a pharmaceutical composition comprising a nucleotide sequence at least about 85% identical to SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35. [Brief description of the drawings]

[0035] [Figure 1]1 is a linear schematic map for a human FVIIIXTEN expression construct according to an embodiment of the present invention. The V1.0 cassette contains codon-optimized cDNA clone 6 encoding B-domain deleted human factor VIII (BDD-FVIIIco6) (see US Publication No. 20190185543) fused with XTEN 144 peptide (FVIIIco6XTEN) under the control of a tristetraprolin (TTP) promoter, an intron, a woodchuck post-transcriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal. The V2.0 cassette (SEQ ID NO: 14) contains a codon-optimized cDNA, with further CpG motifs removed, encoding B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) fused with XTEN 144 peptide (FVIIIXTEN) under the control of a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) with enhancer element (A1MB2), a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal. The V3.0 cassette (SEQ ID NO:35) contains a codon-optimized cDNA encoding B-domain deleted (BDD) codon-optimized human Factor VIII (Co-BDD-FVIII) fused with XTEN 144 peptide (FVIIIXTEN) under the control of the liver-specific alpha 1 antitrypsin (A1AT) promoter, a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal, with additional CpG motifs removed. The FVIIIXTEN expression cassette is flanked by parvovirus ITRs. [Diagram 2]FIG. 1 shows a schematic representation of the approach used to generate ssDNA in which a FVIIIXTEN expression cassette flanked by parvoviral ITRs is digested with restriction enzymes that recognize ITR-related sequences and result in blunt-ended DNA, and the double-stranded DNA products of the digestion (FVIII expression cassette and plasmid backbone) are heat denatured at 95° C. (denaturation) followed by cooling at 4° C. (renaturation) to allow the palindromic ITR sequences to fold. The resulting ss(ssDNA)FVIIIXTEN was used for systemic delivery via hydrodynamic tail vein injection in HemA mice. [Diagram 3] 1 is a graphical representation of plasma FVIII activity levels measured by Chromogenix Coatest® SP Factor VIII chromogenic assay. Blood samples were collected at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected with 800 μg / kg of V1.0 or single-stranded V2.0 ss(ssDNA)FVIIIXTEN flanked by B19 ITR via fluid tail vein injection. Error bars represent standard deviation. [Figure 4] 1 is a graphical representation of plasma FVIII activity levels measured by Chromogenix Coatest® SP Factor VIII chromogenic assay. Plasma samples were collected at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected with 200, 800, or 1600 μg / kg of single-stranded V2.0 ss (ssDNA) FVIIIXTEN flanked by ITRs of human bocavirus (HBoV1), human erythrovirus (B19), goose parvovirus (GPV), or their mutants, or combinations thereof, as indicated, via fluid tail vein injection. Two hybrid ITR sets were also examined (5′B19-3′GPV and 5′GPV-3′B19). Error bars represent standard deviation. The ITR sequences and their mutants were described in previous US patent application Ser. No. 63 / 069,114. [Figure 5A]5A shows the agarose gel electrophoresis images of purified ceFVIIIXTEN (ceDNA) with AAV2 ITR or HBoV1 ITR obtained from continuous elution electrophoresis as described in US patent application Ser. No. 63 / 069,073. Purity is shown in comparison with starting material (SM), and arrows indicate the DNA bands that correspond to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 5B] 5A and 5B show the purified ceFVIIIXTEN (ceDNA) obtained from the baculovirus system and their in vivo efficacy. FIG. 5B shows a graphical representation of plasma FVIII activity levels measured by Chromogenix Coatest® SP Factor VIII chromogenic assay. Plasma samples were collected at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected with 80, 40, or 12 μg / kg ceFVIIIXTEN (ceDNA) flanked by AAV2 ITRs or HBoV1 ITRs as indicated via fluid tail vein injection. Error bars represent standard deviation. The ITR sequences and their variants were described in previous US patent application Ser. No. 63 / 069,073. [Figure 6A] Figure 6 shows the study of the liver-specific mTTR promoter and the human A1AT promoter driving expression of FVIIIXTEN in HBoV1 ITR constructs. Figure 6A shows a schematic diagram for a FVIIIXTEN expression cassette with either the liver-specific mTTR (SEQ ID NO: 3) or the A1AT promoter flanked by HBoV1 WT ITRs. [Figure 6B]Figure 6B shows the liver-specific mTTR promoter and human A1AT promoter driving expression of FVIIIXTEN in HBoV1 ITR constructs. Figure 6B shows the agarose gel electrophoresis image of single-stranded DNA (ssDNA) FVIIIXTEN HBoV1 generated by restriction enzyme digestion as described. [Figure 6C] Figure 6 shows the liver-specific mTTR promoter and human A1AT promoter driving the expression of FVIIIXTEN in HBoV1 ITR constructs. Figure 6C shows the FVIII expression levels normalized to the percentage of normal in mice injected with the mTTR promoter construct or the A1AT promoter construct depicted in Figure 6A. Error bars represent standard deviation. [Figure 7A] Figure 7 shows the results of a study on purified ceFVIIIXTEN AAV2 (ceDNA) species obtained from the baculovirus system. Figure 7A depicts an agarose gel electrophoresis image showing full-length (8.3 kb) and truncated (6.0 kb) species of purified ceFVIIIXTEN (ceDNA) with AAV2 WT ITR obtained from continuous elution electrophoresis. [Figure 7B] Figure 7B shows the results of a study on purified ceFVIIIXTENAAV2 (ceDNA) species obtained from the baculovirus system. Figure 7B shows next-generation sequencing (NGS) analysis of 8.3 kb full-length ceFVIIIXTEN (upper panel) and 6.0 kb truncated ceFVIIIXTEN (lower panel) with AAV2 WT ITRs. [Figure 7C] Figure 7 shows the results of a study on purified ceFVIIIXTENAAV2 (ceDNA) molecular species obtained from the baculovirus system. Figure 7C shows FVIII expression levels normalized to percent normal in mice injected with full-length or truncated ceFVIIIXTEN AAV2 constructs at 80 or 40 μg / kg. Error bars represent standard deviation. [Figure 8A]Figure 8A shows the agarose gel electrophoresis images of purified ceFVIIIXTEN (ceDNA) with AAV2 ITR or HBoV1 ITR obtained from continuous elution electrophoresis as described in US Patent Application No. 63 / 069,073. Purity is shown in comparison with starting material (SM), and arrows indicate the DNA bands that correspond to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 8B] Figure 8B shows the purified ceFVIIIXTEN (ceDNA) obtained from the baculovirus system and its efficacy in vivo. Figure 8B shows the FVIII expression levels normalized to percent normal in mice injected with 80 or 40 μg per kg of ceFVIIIXTEN (ceDNA) flanked by AAV2 ITR or HBoV1 ITR as indicated. Error bars represent standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure describes a codon-optimized gene encoding a polypeptide with Factor VIII (FVIII) activity. The present disclosure is directed to a codon-optimized nucleic acid molecule encoding a polypeptide with Factor VIII activity, a vector, and a host cell comprising the optimized nucleic acid molecule, a polypeptide encoded by the optimized nucleic acid molecule, and a method for producing such a polypeptide. The present disclosure is also directed to a method for treating a bleeding disorder, such as hemophilia, comprising administering to a subject an optimized Factor VIII nucleic acid sequence, a vector comprising the optimized nucleic acid sequence, or a peptide encoded thereby.

[0037] The present disclosure fulfills an important need in the art by providing an optimized FVIII sequence that supports increased expression in host cells, improved yield of FVIII protein in methods of making recombinant FVIII, and potentially results in increased 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:9.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:33.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:35.In some embodiments, the gene cassette further comprises a nucleotide sequence encoding an XTEN polypeptide.

[0038] For a clear understanding of the specification and claims, the following definitions are provided below.

[0039] definition It is noted that the term "a" entity or "an" entity refers to one or more of that entity: for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0040] The term "about" is used herein to mean approximately, in the region of, roughly, or in the vicinity thereof. When used in conjunction with a numerical range, the term "about" modifies the range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10 percent upward or downward (high or low).

[0041] For purposes of this disclosure, the term "isolated" refers to a biological material (cell, polypeptide, polynucleotide, or fragment, variant, or derivative thereof) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide occurring in a natural state 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 natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0042] "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 molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded form or within a double-stranded helix. DNA-DNA helices, DNA-RNA helices, and RNA-RNA helices that are double-stranded are also possible. The term nucleic acid molecule, and in particular the term 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 is described herein, following conventional practice, in the 5' to 3' direction, showing only the sequence 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 undergone 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 described herein.

[0043] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons that can be translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid but is considered part of the coding region, whereas any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end that codes for the amino terminus of the resulting polypeptide and a translation stop codon at the 3' end that codes for the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present within a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Thus, a single vector may contain only a single coding region or may include two or more coding regions.

[0044] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once the growing protein chain is triggered to export across the rough endoplasmic reticulum. Those skilled in the art are aware that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of this sequence retains the ability to direct the secretion of a polypeptide operatively associated therewith. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA), or mouse β-glucuronidase signal peptide, or a functional derivative thereof, is used.

[0045] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0046] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' of a coding region or at the origin of transcription. For example, most promoters are located upstream of the transcription start site.

[0047] As used herein, the term "gene cassette" refers 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 the polynucleotide sequence of interest. A gene cassette may be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region and may include nucleotide sequences that affect transcription, RNA processing, stability, or translation of the associated coding region. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal sequence and a transcription termination sequence will typically be located 3' to the coding sequence. In some embodiments, a gene cassette comprises a polynucleotide that encodes a gene product. In some embodiments, a gene cassette comprises a polynucleotide that encodes 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 sequence operably associated with one or more coding regions. When in operative association, a coding region of a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a manner that expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA that encodes the gene product encoded by the coding region, and the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product or the ability of the DNA template to be transcribed. Other expression control sequences other than promoters, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct expression of a gene product.

[0048] "Expression control sequence" refers to a regulatory nucleotide sequence, such as a promoter, enhancer, or terminator, that results in the expression of a coding sequence in a host cell. Expression control sequences generally encompass any regulatory nucleotide sequence that facilitates efficient transcription and translation of an operably linked coding nucleic acid. 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 include expression control sequences that function in vertebrate cells, such as, but are not limited to, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter with intron A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other expression control sequences include expression control sequences 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 gene expression in eukaryotic cells. Additional suitable expression control sequences include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters induced 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.

[0049] Likewise, various translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation / termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites, or IRES).

[0050] As used herein, the term "expression" refers to the process by which a polynucleotide results in a gene product, e.g., an RNA or a polypeptide. "Expression" includes, without limitation, the 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 the translation of an mRNA into a polypeptide. Expression results in a "gene product." As used herein, a gene product can be a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage. As used herein, the term "yield" refers to the amount of polypeptide resulting from expression of a gene.

[0051] "Vector" refers to any vehicle for cloning and / or introduction of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment is attached to effect replication of the attached segment. "Replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes viral and non-viral vehicles for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Numerous 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 is accomplished by ligating a suitable polynucleotide fragment into a selected vector with complementary cohesive termini.

[0052] Vectors are 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 incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialaphos, sulfonamides, and the like; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, and the like. 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), and the like. A selectable marker is also considered to be a reporter.

[0053] The term "selectable marker" refers to an identifying factor, typically an antibiotic resistance gene or a chemical resistance gene, that allows selection based on the effect of the marker gene, i.e., resistance to antibiotics, resistance to herbicides, colorimetric markers, enzymes, fluorescent markers, etc., where the effect is used to trace the inheritance of the nucleic acid of interest and / or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialaphos, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0054] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that allows identification based on the effect of the reporter gene, where the effect is used to trace 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 of gene expression or transcription of the gene. Examples of reporter genes 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 are also considered reporter genes.

[0055] "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 located 3' to the promoter sequence. Promoters are composed of different elements in their entirety from a natural gene or from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters may direct the expression of a gene in different tissues or cell types, may direct the expression of a gene at different developmental stages, or may direct the expression of a gene 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 specific 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 specific stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter". A promoter that is induced to express a gene after exposure or treatment of cells with a drug, biomolecule, chemical, ligand, light, etc. that induces the promoter 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 are not fully defined, so that DNA fragments of different lengths may have the same promoter activity. Additional exemplary promoters are discussed elsewhere in this disclosure.

[0056] A promoter sequence is typically bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to incorporate the minimum number of bases or elements necessary to induce transcription at a detectable level 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.

[0057] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences of single- or double-stranded DNA or RNA, linear, circular, or supercoiled, from any source, in which multiple nucleotide sequences are joined or recombined into unique constructs that can introduce promoter fragments and DNA sequences for selected gene products, along with appropriate 3' untranslated sequences, into cells.

[0058] Eukaryotic viral vectors that may 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, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0059] "Cloning vector" refers to a "replicon", a unit length of sequentially replicated nucleic acid, such as a plasmid, phage, or cosmid, to which another nucleic acid segment is attached to effect replication of the attached segment, and which contains an origin of replication. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another cell, e.g., eukaryotic cells. Cloning vectors typically contain one or more sequences used for the insertion of a nucleic acid sequence of interest into the vector and / or for the selection of cells that contain one or more multiple cloning sites.

[0060] 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 operable association with a regulatory region, as described above.

[0061] 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.

[0062] As used herein, "culture," "to culture," and "culturing" refer to incubating cells under in vitro conditions that allow the cells to grow or divide, or to maintaining the cells in a viable state. As used herein, "cultured cells" refers to cells that have been propagated in vitro.

[0063] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "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, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modified products of a polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. The polypeptides may be produced in any manner, including by chemical synthesis.

[0064] 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). Unconventional amino acids are also within the scope of the present disclosure, including norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym., 202:301-336 (1991). To generate such unnatural amino acid residues, the procedures of Noren et al., Science, 244:182 (1989); and Ellman et al., supra, are used. Briefly, these procedures involve chemical activation of a suppressor tRNA with the unnatural amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of unconventional amino acids can also be accomplished using peptide chemistry reactions known in the art. As used herein, the term "polar amino acid" includes amino acids that have a net charge of zero, but have nonzero partial charges at different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids may participate in hydrophobic and electrostatic interactions. As used herein, the term "charged amino acids" includes amino acids that may have a non-zero net charge on their side chains (e.g., R, K, H, E, D). These amino acids may participate in hydrophobic and electrostatic interactions.

[0065] The present disclosure also includes fragments or variants of the polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to the polypeptide-binding domains or polypeptide-binding molecules of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for FcRn binding domains or Fc variants, coagulation activity for FVIII variants, or FVIII binding activity for VWF fragments). Polypeptide fragments include proteolytic fragments as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide-binding domains or polypeptide-binding molecules of the present disclosure include the fragments described above, and also include polypeptides in which the amino acid sequence is altered due to amino acid substitution, deletion, or insertion. Variants may be naturally occurring variants or non-naturally occurring variants. Non-naturally occurring variants are generated using mutagenesis methods known in the art. Variant polypeptides can contain conservative amino acid substitutions, deletions, or additions, or can contain non-conservative amino acid substitutions, deletions, or additions.

[0066] "Conservative amino acid substitution" refers to the amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, a family of amino acid residues with similar side chains is defined, including 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), beta-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 from the same side chain family, the substitution is considered to be conservative. In another embodiment, a stretch of amino acids is conservatively replaced with a structurally similar stretch of side chain family members that differs in order and / or composition.

[0067] The term "percent identity," as known in the art, 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 relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" is 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 Heijne, G., ed.), Academic Press (1987); and "Sequence Analysis Primer" (Gribskov, M. and Devereux, J., eds.), Stockton Press, New York (1991). Preferred methods of determining identity are designed to give the best match between the sequences tested. Methods of determining identity are codified in publicly available computer programs.Sequence alignment and percent identity calculations are performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing software package (DNASTAR, Inc., Madison, WI), the GCG program package (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). In the context of this application, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein, "default values" refers to any set of values ​​or parameters that were originally loaded by the software when it was first initialized. For the purpose of determining the percent identity between the BDD optimized FVIII sequence of the present disclosure and a reference sequence, only the nucleotides in the reference sequence that correspond to the 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 containing B domain with the BDD optimized FVIII nucleotide sequence of the present disclosure, the portion of the alignment that includes the A1, A2, A3, C1 and C2 domains will be used to calculate the percent identity.Nucleotides in the portion of the full-length FVIII sequence that encodes the B domain (resulting in a large "gap" in the alignment) will not be counted as mismatches.Additionally, in determining the percent identity between the optimized BDD FVIII sequence of the present disclosure, or a designated portion thereof (e.g., nucleotides 2183-4474 and 4924-7006 of SEQ ID NO: 14), and a reference sequence, the percent identity is calculated by dividing the number of matched nucleotides by the total number of nucleotides in the complete sequence of the optimized BDD-FVIII sequence, or a designated portion thereof, as listed herein.

[0068] 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 the position at which a heterologous moiety is inserted. An "insertion site" is designated as a number that corresponds to the number of the amino acid in mature native FVIII (SEQ ID NO:20) to which the insertion site corresponds, immediately N-terminal to the insertion position. 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 acid 1656 and amino acid 1657 of SEQ ID NO:20.

[0069] As used herein, the phrase "immediately downstream of an amino acid" refers to the position immediately adjacent to the terminal carboxyl group of the amino acid. Similarly, "immediately upstream of an amino acid" refers to the position immediately adjacent to the terminal amine group of the amino acid.

[0070] As used herein, the terms "inserted," "inserted into," "inserted into," or grammatically similar terms refer to the position of a heterologous moiety in a recombinant FVIII polypeptide relative to the analogous position in native mature human FVIII (SEQ ID NO: 20).

[0071] The term "half-life" as used herein refers to the biological half-life of a particular polypeptide in vivo. Half-life is represented by the time required for half of the amount administered to a subject to be cleared from the circulation and / or other tissues in an animal. When a clearance curve for a given polypeptide is constructed as a function of time, the curve is usually biphasic, with a rapid α-phase and a slow β-phase. The α-phase typically represents the equilibrium of the administered Fc polypeptide in 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 in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, so they do not have an alpha phase, but only a single beta phase. Thus, in certain embodiments, the term "half-life" as used herein refers to the half-life of a polypeptide in the β-phase.

[0072] As used herein, "linked" refers to a first amino acid sequence or a first nucleotide sequence that is covalently or non-covalently connected to a second amino acid sequence or a second nucleotide sequence, respectively. The first amino acid sequence or the first nucleotide sequence may be directly connected or juxtaposed to the second amino acid sequence or the second nucleotide sequence, or alternatively, an intervening sequence may covalently connect the first sequence to the second sequence. The term "linked" does not only mean the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of any two amino acids within the second amino acid sequence (or within the first amino acid sequence) of the entire first amino acid sequence (or the entire second amino acid sequence), respectively. In one embodiment, the first amino acid sequence is linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence is linked to the second amino acid sequence by a phosphodiester bond or a linker. A linker can be a peptide or polypeptide (for a polypeptide chain), a nucleotide or a chain of nucleotides (for a nucleotide chain), or any chemical moiety (for both polypeptide and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0073] 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 bond, a non-peptide bond, or a non-covalent bond. The association is indicated by a colon, i.e., (:). In another embodiment, the term refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or disulfide bridge with the thiol group on the 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 (226 or 229; EU numbering system) using the Kabat numbering system. Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, agostic bonds, bent bonds, dipolar bonds, pi back-donor bonds, double bonds, triple bonds, quadruple bonds, pentad bonds, sextad bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-pi 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, aurophilic, intercalation, stacking, entropic forces, or chemical polarity.

[0074] As used herein, "hemostasis" means the stopping or slowing of bleeding or hemorrhage; or the stopping or slowing of blood flow through a blood vessel or body part.

[0075] As used herein, "hemostatic disorder" refers to a congenital or acquired condition characterized by a tendency to bleed spontaneously or as a result of trauma due to an impaired ability to form a fibrin clot or an inability to form a fibrin clot. Examples of such disorders include hemophilia. The three major forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease") and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, Bernard-Soulier syndrome, which is a deficiency or deficiency of GPIb. GPIb, the receptor for vWF, can be defective, leading to lack of primary clot formation (primary hemostasis) and increased bleeding tendency, as well as Glanzmann / Naegeli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of clotting factors by the liver; this can increase the risk of bleeding.

[0076] The isolated nucleic acid molecule, isolated polypeptide, or vector comprising the isolated nucleic acid molecule of the present disclosure is used prophylactically. As used herein, the term "prophylactic treatment" refers to administration of 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 is administered as a prophylactic agent before or after surgery. The polynucleotide, polypeptide, or vector of the present disclosure is administered before or after surgery to control an acute bleeding episode. The surgery may include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0077] 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 the administration of 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, the on-demand treatment is administered to a subject when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the on-demand treatment is administered before an activity that increases the risk of bleeding, such as contact athletics.

[0078] As used herein, the term "acute bleeding" refers to a bleeding episode regardless of the underlying cause.For example, a subject may have trauma, uremia, inherited bleeding disorder (e.g., factor VII deficiency), platelet disorder, or tolerance due to the development of antibodies against clotting factors.

[0079] As used herein, "treat", "treatment", or "treating" refers to, for example, lessening the severity of a disease or condition; shortening the duration of the course of a disease; ameliorating one or more symptoms associated with a disease or condition; imparting a beneficial effect to a subject with a disease or condition, without 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 a trough level of FVIII in a subject 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 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 between 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 or managing a disease or condition may also include maintaining FVIII activity in a subject at a level equivalent 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 is determined by one or more known methods and adjusted (increased or decreased) for each patient.

[0080] As used herein, "administering" refers to administering to a subject a pharma- ceutically acceptable factor VIII-encoding nucleic acid molecule, factor VIII polypeptide, or vector comprising the factor VIII-encoding nucleic acid molecule of the present disclosure via a pharma- ceutically acceptable route. The route of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Further routes of administration include, e.g., subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The nucleic acid molecules, polypeptides, and vectors are administered as part of a pharmaceutical composition that includes at least one excipient.

[0081] As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who will benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure, for example, by improving 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 a FVIII inhibitor and therefore requires bypass therapy. The subject may be an adult or a juvenile (e.g., under the age of 12).

[0082] As used herein, the term "clotting factor" refers to a naturally occurring or recombinantly produced molecule or analog thereof that prevents or reduces the persistence of bleeding episodes in a subject. In other words, the term "clotting factor" refers to a molecule that has procoagulant activity, i.e., a molecule that contributes to the conversion of fibrinogen into a mesh of insoluble fibrin, which causes blood to coagulate or clot. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., in its zymogen form) that is capable of being converted to an active form.

[0083] As used herein, "clotting activity" means the ability to participate in the cascade of biochemical reactions that lead to the formation of a fibrin clot and / or to reduce the severity, duration, or frequency of bleeding or bleeding episodes.

[0084] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., within a cell or polypeptide. For example, an exogenous or heterologous molecule is introduced into a cell and is present only after manipulation of the cell, e.g., by transfection or other form of genetic engineering, whereas a heterologous amino acid sequence may be present within a protein where it is not found in nature.

[0085] As used herein, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not naturally occur with 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 its biological activity or function (e.g., its procoagulant activity). In some embodiments, FVIII is linked or connected to the polypeptide encoded by the heterologous nucleotide sequence by a linker.

[0086] As used herein for comparison with the nucleotide sequence of the present disclosure, a "reference nucleotide sequence" 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 is not optimized. In some embodiments, the reference nucleotide sequence for the nucleic acid molecules disclosed herein is SEQ ID NO:32.

[0087] As used herein, the term "optimized" in relation to a nucleotide sequence refers to a polynucleotide sequence that codes for a polypeptide, where the polynucleotide sequence is mutated to enhance the properties of the polynucleotide sequence. In some embodiments, the optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding to 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 changes that can be made to a polynucleotide sequence to optimize the nucleotide sequence include codon optimization, G / C content optimization, removal of repeat sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis-activating elements that suppress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences near the transcription start site that enhance transcription, such as Kozak consensus sequences, removal of sequences that can form stem-loop structures, removal of destabilizing sequences, removal of CpG motifs, and combinations of two or more of these.

[0088] Polynucleotide sequence Certain aspects of the present disclosure aim to overcome the deficiencies of AAV vectors for gene therapy. In particular, some aspects of the present disclosure are directed to a nucleic acid molecule comprising a gene cassette, for example, encoding a therapeutic protein and / or a therapeutic miRNA. In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a clotting factor. In some embodiments, the gene 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, a regulatory element, a 3'UTR poly(A) sequence, or any combination thereof. In some embodiments, the regulatory element is a post-transcriptional regulatory element.

[0089] 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 ceDNA.

[0090] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a FVIII polypeptide, where 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 and a synthetic intron. In some embodiments, the gene cassette comprises a nucleotide sequence disclosed in International Application No. PCT / US2017 / 015879, which is incorporated by reference in its entirety. In some embodiments, the gene cassette is "hFVIIIco6XTEN", a gene cassette described in PCT / US2017 / 015879. In some embodiments, the gene cassette comprises SEQ ID NO: 32.

[0091] In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) fused with an XTEN 144 peptide. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:9. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:14. In some embodiments, the gene cassette has a nucleotide sequence of SEQ ID NO:14. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:33. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:35. In some embodiments, the gene cassette further comprises a nucleotide sequence encoding an XTEN polypeptide.

[0092] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII driven by an mTTR promoter and a synthetic intron. In some embodiments, the gene cassette further comprises a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the gene cassette further comprises a bovine growth hormone polyadenylation (bGHpA) signal.

[0093] In some embodiments, the present disclosure is directed to a codon-optimized nucleic acid molecule that encodes a polypeptide with 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 with SEQ ID NO: 10 or a fragment thereof.

[0094] In some embodiments, the nucleic acid molecule of the present disclosure encodes a FVIII polypeptide comprising a signal peptide or a fragment thereof.In other embodiments, the nucleic acid molecule encodes a FVIII polypeptide lacking a signal peptide.In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:11.

[0095] As used herein, "polypeptide with FVIII activity" refers to a functional FVIII polypeptide under its normal role in coagulation, unless otherwise specified. The term "polypeptide with 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 with FVIII activity" is used interchangeably 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 bind Ca2 + and phospholipids, and then the tenase complex converts factor X to its activated form, Xa. In one embodiment, the polypeptide with FVIII activity comprises two polypeptide chains, the first chain having a FVIII heavy chain and the second chain having a FVIII light chain. In another embodiment, the polypeptide with FVIII activity is a single-chain FVIII. The single-chain FVIII may contain one or more mutations or substitutions at amino acid residues 1645 and / or 1648, which correspond to the mature human FVIII sequence (SEQ ID NO: 20). See International Application No. PCT / US2012 / 045784, which is incorporated herein by reference in its entirety. The FVIII protein may be a human, porcine, canine, rat, or murine FVIII protein. In addition, comparison between human-derived FVIII and FVIII from other species has identified conserved residues that are likely required for function. See, e.g., Cameron et al. (1985), Thromb. Haemost., 79:317-22; and U.S. Patent No. 6,251,632.

[0096] Numerous 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 International Normalized Ratio (INR)), fibrinogen test (often via the Clauss method), platelet count, platelet function test (often via PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), clotting factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., prothrombin mutation G20210A, which is the factor V Leiden mutation), dilute Russell's snake venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0097] The aPTT test is a performance indicator that measures the efficacy of both the "intrinsic" pathway (also called the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, such as FVIII or FIX. The aPTT test is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0098] ROTEM® analysis provides information about the overall dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. In thromboelastometry, the different parameters depend on many factors that affect the activity of the plasma coagulation system, platelet function, fibrinolysis, or their interactions. This assay can provide a complete picture about secondary hemostasis.

[0099] As used herein, the "B domain" of FVIII is identical to the B domains known in the art, 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: residues Ala1 to Arg372; A2: residues Ser373 to Arg740; A3: residues Ser1690 to Ile2032; C1: residues Arg2033 to Asn2172; C2: residues Ser2173 to Tyr2332. The sequence of 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. The location of the boundaries for all of the domains, including the B domain, for porcine, murine, and canine FVIII are also known in the art. An example of a BDD FVIII is REFACTO® recombinant BDD FVIII (Wyeth Pharmaceuticals, Inc.).

[0100] A "B domain deleted FVIII" may have a complete deletion or a partial deletion as disclosed in U.S. Pat. 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, 6,048,720, 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 disclosed 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 al. (1987), DNA, 6:553-564; European Patent 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 foregoing deletions may be made within any FVIII sequence.

[0101] Codon Optimization In one embodiment, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, the nucleic acid sequence being codon-optimized.In another embodiment, the starting nucleic acid sequence encoding the polypeptide with FVIII activity and being subjected to codon optimization is SEQ ID NO: 32.In some embodiments, the sequence encoding the polypeptide with FVIII activity is codon-optimized for expression in humans.In other embodiments, the sequence encoding the polypeptide with FVIII activity is codon-optimized for expression in mice.

[0102] The term "codon optimized" when referring to the coding region of a gene or nucleic acid molecule for transformation of various hosts refers to the alteration of codons in the coding region of a gene or nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization involves the replacement of at least one codon, or more than one codon, or a significant number of codons, with one or more codons that are more frequently used in the genes of the organism.

[0103] Deviations in the nucleotide sequence, including the codons that code for the amino acids of any polypeptide chain, allow for variation in the sequence that codes for a gene. Because each codon consists of three nucleotides and the nucleotides that comprise DNA are limited 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 terminate translation). As a result, many amino acids are designated 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 the base composition of DNA to vary over a wide range without changing the amino acid sequence of the protein coded for by the DNA.

[0104] Many organisms exhibit biases in the use of certain codons that code for the insertion of certain amino acids in the growing peptide chain. Codon bias, the difference in codon preference or codon usage between organisms, is caused by 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 is believed to depend, among other things, on the properties of the codon being translated and on the availability of certain transfer RNA (tRNA) molecules. The dominance of tRNAs selected in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes are fine-tuned for optimal gene expression in a given organism based on codon optimization.

[0105] Given the large number of gene sequences available for a wide variety of animal, plant, and microbial species, the relative frequency of codon usage has been calculated. Codon usage tables are available, for example, in the "Codon Usage Database" available at www.kazusa.or.jp / codon / (accessed June 18, 2012). See Nakamura, Y. et al., Nucl. Acids Res., 28:292 (2000).

[0106] The assignment of random codons at optimized frequencies to code for a given polypeptide sequence is done manually by calculating the codon frequencies for each amino acid and then randomly assigning the codons to the polypeptide sequence. In addition, a variety of algorithms and computer software programs are used to calculate optimal sequences.

[0107] In other embodiments, the nucleic acid molecules disclosed herein are further optimized by removing one or more CpG motifs and / or methylating at least one CpG motif. As used herein, "CpG motif" refers to a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine. The term "CpG motif" encompasses both methylated and unmethylated CpG dinucleotides. Unmethylated CpG motifs are common in bacterial and viral nucleic acids (e.g., plasmid DNA), but are repressed and largely methylated in vertebrate DNA. Thus, unmethylated CpG motifs prime the mammalian host to mount a rapid inflammatory response. Klinman et al. (1996), PNAS, 93:2879-2883. Exemplary methods of CpG removal are described in Yew, NS et al. (2002), Mol Ther., 5(6):731-738; and International Application No. PCT / US2001 / 010309. In some embodiments, the nucleic acid molecules disclosed herein are modified to contain a small number of CpG motifs (i.e., "CpG-reduced" or "CpG-deleted"). In one embodiment, a CpG motif located within a codon triplet for a selected amino acid is changed to a codon triplet for the same amino acid that lacks the CpG motif. In some embodiments, the nucleic acid molecules disclosed herein are optimized to reduce innate immune responses.

[0108] In some embodiments herein, nucleic acid molecules are disclosed that include a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:9.

[0109] In some embodiments herein, nucleic acid molecules are disclosed that include a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:33.

[0110] In some embodiments herein, nucleic acid molecules are disclosed that include a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:14.

[0111] In some embodiments herein, nucleic acid molecules are disclosed that include a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:35.

[0112] 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 may be linked to the optimized BDD-FVIII nucleotide sequence of the present disclosure at the 5' end, 3' end, or may be inserted into the middle of the optimized BDD-FVIII nucleotide sequence. Thus, in some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence may be linked to the N-terminus or C-terminus of the FVIII amino acid sequence encoded by the nucleotide sequence, or may be inserted between two amino acids in the FVIII amino acid sequence. In some embodiments, the heterologous amino acid sequence is inserted between two amino acids at one or more insertion sites. In some embodiments, the heterologous amino acid sequence is inserted into a FVIII polypeptide encoded by a nucleic acid molecule of the present disclosure at any site disclosed in International Publication No. WO2013 / 123457A1, WO2015 / 106052A1, or U.S. Publication No. 2015 / 0158929A1, each of which is incorporated by reference in its entirety.

[0113] In some embodiments, the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted within the B domain or within 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 745 of wild-type mature human FVIII (SEQ ID NO:20). In one particular embodiment, the FVIII comprises a deletion of amino acids 746-1637 (SEQ ID NO:20), which corresponds to wild-type mature human FVIII, and the heterologous amino acid sequence encoded by the heterologous nucleotide sequence is inserted immediately downstream of amino acid 745, which corresponds to wild-type mature human FVIII (SEQ ID NO:20). The insertion sites of FVIII referred to herein refer to amino acid positions corresponding to amino acid positions of wild-type mature human FVIII (SEQ ID NO:20).

[0114] In some embodiments, the heterologous moiety is a peptide or polypeptide with non-structural or structural features associated with increased half-life in vivo when incorporated into the 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 sequence, XTEN sequence, transferrin or fragments thereof, PAS polypeptide, polyglycine linker, polyserine linker, albumin binding moiety, or any fragment, derivative, variant, or combination of these polypeptides. In a particular embodiment, the heterologous amino acid sequence is an immunoglobulin constant region or portion thereof, transferrin, albumin, or PAS sequence. In other related aspects, the heterologous moiety may include a conjugation site (e.g., the amino acid cysteine) for a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these elements. In some aspects, the heterologous moiety comprises cysteine, an amino acid that serves as an attachment site for a non-polypeptide moiety, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these elements.

[0115] In certain embodiments, the heterologous moiety improves one or more pharmacokinetic properties of the FVIII protein without significantly affecting its biological activity or function. 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 is determined by any method known to those skilled in the art, such as activity assay (colorimetric assay or one-step clotting aPTT assay), ELISA, ROTEM™, etc.

[0116] In other embodiments, the heterologous moiety increases 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., the absence of proteolytic cleavage, undesired oxidation or deamidation). In other aspects, the physical property can also be the presence of the FVIII protein in a properly folded state (e.g., the absence of soluble or insoluble aggregates or precipitates). In one aspect, the stability of the FVIII protein is measured by assaying a biophysical property of the FVIII protein, such as thermal stability, unfolding profile with pH, ​​stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to proteins, receptors, or ligands), and / or a combination thereof. In another embodiment, the biochemical function is supported by the binding affinity of the interaction. In one embodiment, the measure of protein stability is thermal stability, i.e., resistance to heat stress. Stability is 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.

[0117] In some embodiments, the heterologous moiety comprises one or more XTEN sequences, fragments, variants, or derivatives thereof. As used herein, "XTEN sequence" refers to a polypeptide that is extended in length by 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 is used as a half-life extending moiety. In addition, XTEN can provide desirable properties, including but not limited to enhanced pharmacokinetic parameters and solubility characteristics. Other advantageous properties that are conferred by introducing XTEN sequences include enhanced conformational flexibility, enhanced water solubility, increased protease resistance, reduced immunogenicity, reduced binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius.

[0118] XTEN vary in length for insertion or linkage 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, XTEN is a peptide or polypeptide having from about 20 to about 3000 amino acid residues, from about 30 to about 2500 residues, from about 40 to about 2000 residues, from about 50 to about 1500 residues, from about 60 to about 1000 residues, from about 70 to about 900 residues, from about 80 to about 800 residues, from about 90 to about 700 residues, from about 100 to about 600 residues, from about 110 to about 500 residues, or from about 120 to about 400 residues. In one particular embodiment, XTEN comprises an amino acid sequence longer than 42 amino acids in length and shorter than 144 amino acids in length.

[0119] The XTEN sequences of the present disclosure may 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, where the 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), glutamine (E), and proline (P). See US2010-0239554A1.

[0120] For examples of XTEN sequences used as heterologous moieties in the chimeric proteins of the present disclosure, see, e.g., U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, and 2011 / 0077199A1, or 2011 / 0172146A1, or International Publication Nos. WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, or WO2011028344A2.

[0121] One or more XTEN sequences may be inserted at the C-terminus or N-terminus of the amino acid sequence encoded by the nucleotide sequence, or may be inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence.For example, XTEN is inserted between two amino acids at one or more insertion sites.Examples of acceptable sites for XTEN insertion in FVIII can be found, for example, in International Publication No. WO2013 / 123457A1, or US Publication No. 2015 / 0158929A1, which are incorporated herein by reference in their entirety.

[0122] In certain embodiments, the heterologous moiety is a peptide linker.

[0123] 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 within the linear amino acid sequence of a polypeptide chain.

[0124] In some embodiments, a heterologous nucleotide sequence encoding a peptide linker is inserted between the optimized FVIII polynucleotide sequence of the present disclosure and the heterologous nucleotide sequence encoding one of the heterologous moieties described above, such as albumin. The peptide linker can provide flexibility to the chimeric polypeptide molecule. The linker is typically not cleaved, although such cleavage may be desired. In one embodiment, these linkers are not removed during processing.

[0125] The types of linkers present in the chimeric proteins of the present disclosure are protease-cleavable linkers that contain a cleavage site (i.e., a substrate that is a protease cleavage site, e.g., Factor XIa, Factor Xa, or a thrombin cleavage site), which may contain additional linkers at the N-terminus or C-terminus, or both. These cleavable linkers, when incorporated into the constructs of the present disclosure, result in chimeric molecules with heterologous cleavage sites.

[0126] In one embodiment, the FVIII polypeptide encoded by the nucleic acid molecule of the present disclosure comprises two or more Fc domains or Fc moieties linked via a cscFc linker to form an Fc region integrated into a single polypeptide chain. The cscFc linker is flanked by 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.

[0127] Other peptide linkers are also optionally used within the constructs of the present disclosure, for example, connecting the FVIII protein to the Fc region. Some exemplary linkers used in the context of the present disclosure include, for example, polypeptides that include the amino acid GlySer, which is described in more detail below.

[0128] In one embodiment, the peptide linker is a synthetic peptide linker, i.e., a non-naturally occurring peptide linker. 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 to which it is not naturally 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 (e.g., including mutations such as additions, substitutions, or deletions) of a naturally occurring polypeptide. 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 occurs in a linear sequence that does not occur in nature. In yet another embodiment, the peptide linker may comprise a naturally occurring polypeptide sequence.

[0129] 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 is inserted between two other sequences of the peptide linker. In other embodiments, the gly-ser linker is joined at 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 within the peptide linker. In one embodiment, the peptide linker of the present disclosure comprises at least a portion of the upper 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.

[0130] The peptide linkers of the present disclosure are at least one amino acid long and can be peptide linkers of varying 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 linker length is a positive integer, a length of about 1 to about 50 amino acids means a length of 1 to 3 to 48 to 52 amino acids. 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.

[0131] 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 1,000 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.

[0132] The peptide linker is introduced into the polypeptide sequence using techniques known in the art. The modification is confirmed by DNA sequence analysis. The plasmid DNA is used to transform host cells for stable production of the produced polypeptide.

[0133] 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 is 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.

[0134] Constitutive mammalian 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 derived from cytomegalovirus (CMV), simian viruses (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 skilled in the art. Promoters useful for the gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0135] In one embodiment, the disclosure includes expression of a 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 certain embodiments, 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 certain embodiments, 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., alpha 1 antitrypsin (AAT) promoter), a muscle-specific promoter (e.g., muscle creatine kinase (MCK) promoter, myosin heavy chain alpha (αMHC) promoter, myoglobin (MB) promoter, and desmin (DES) promoter), a synthetic promoter (e.g., SPc5-12 promoter, 2R5Sc5-12 promoter, dMCK promoter, and tMCK promoter), or any combination thereof.

[0136] 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 a 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 interneurons. 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 tissue. 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.

[0137] Other promoters that are useful within 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.

[0138] 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 mTTR202 promoter, mTTR202opt promoter, and mTTR482 promoter, which are disclosed in US Publication No. US2019 / 0048362, which is incorporated herein by reference in its entirety. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter. In some embodiments, the promoter is a mTTR482 promoter, which is a liver-specific modified mouse transthyretin (mTTR) promoter. Examples of mTTR482 promoters 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:16.

[0139] To achieve therapeutic efficacy, the expression level is further enhanced using one or more enhancer elements. One or more enhancers may be administered alone or in conjunction 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 alpha-1-microglobin / 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, including HNF1, (sense)-HNF3, (sense)-HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4 (antisense), along with Enh1.

[0140] In some embodiments, the enhancer element comprises one or two modified prothrombin enhancers (pPrT2), one or two alpha 1 microbikunin enhancers (A1MB2), modified mouse albumin enhancer (mEalb), Hepatitis B virus enhancer II (HE11), or CRM8 enhancer. In some embodiments, the A1MB2 enhancer is an enhancer disclosed in International Application No. PCT / US2019 / 055917. In some embodiments, the enhancer element is A1MB2. In some embodiments, the enhancer element comprises multiple copies of the AIMB2 enhancer sequence. In some embodiments, the A1MB2 enhancer is located 5' to a nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, the A1MB2 enhancer is located 5' to a promoter sequence, such as a mTTR promoter. In some embodiments, the enhancer element is an A1MB2 enhancer comprising the nucleic acid sequence of SEQ ID NO: 15.

[0141] In some embodiments, the nucleic acid molecules disclosed herein include 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 intron / rabbit beta-globin intron, modified to eliminate five existing ATG sequences to reduce false translation initiation. In certain embodiments, the intron sequence includes an SV40 small T intron. In some embodiments, the intron sequence is located 5' to a nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, the chimeric intron is located 5' to a promoter sequence, such as the mTTR promoter. In some embodiments, the chimeric intron includes the nucleic acid sequence of SEQ ID NO: 17.

[0142] In some embodiments, the nucleic acid molecules disclosed herein comprise a post-transcriptional regulatory element. In certain embodiments, the regulatory element comprises a mutant woodchuck hepatitis virus regulatory element (WPRE). The WPRE is believed to enhance expression of a transgene delivered by a viral vector. Examples of WPREs are described in Zufferey et al. (1999), J Virol., 73(4):2886-2892; Loeb et al. (1999), Hum Gene Ther., 10(14):2295-2305. In some embodiments, the WPRE is located 3' to the nucleic acid sequence encoding the FVIII polypeptide. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO:18.

[0143] In some embodiments, the nucleic acid molecules disclosed herein comprise a transcription terminator. In some embodiments, the transcription terminator is a polyadenylation (poly(A)) sequence. Non-limiting examples of transcription terminators include those derived from bovine growth hormone polyadenylation signal (BGHpA), simian virus 40 polyadenylation signal (SV40pA), or synthetic polyadenylation signals. In one embodiment, the 3'UTR poly(A) tail comprises an actin poly(A) site. In one embodiment, the 3'UTR poly(A) tail comprises a hemoglobin poly(A) site. In some embodiments, the transcription terminator is BGHpA. An example of a BGHpA transcription terminator is described in Woychik et al. (1984), PNAS, 81:3944-3948. In some embodiments, the transcription terminator is located at the 3' end of the gene cassette encoding the nucleic acid sequence encoding the FVIII polypeptide. In some embodiments, the transcription terminator is BGHpA, which comprises the nucleic acid sequence of SEQ ID NO:19.

[0144] In some embodiments, the nucleic acid molecule disclosed herein comprises one or more DNA nuclear targeting sequences (DTS). The DTS facilitates the translocation of DNA molecules containing such sequences into the nucleus. In certain embodiments, the DTS comprises an SV40 enhancer sequence. In certain embodiments, the DTS comprises a c-Myc enhancer sequence. In some embodiments, the nucleic acid molecule comprises a DTS located between the first ITR and the second ITR. In some embodiments, the nucleic acid molecule comprises a DTS located 3' to the first ITR and 5' to the transgene (e.g., FVIII protein). In some embodiments, the nucleic acid molecule comprises a DTS located 3' to the transgene and 5' to the second ITR on the nucleic acid molecule.

[0145] 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.

[0146] Inverted terminal repeat (ITR) sequences Certain embodiments of the present disclosure are directed to nucleic acid molecules that include a first ITR, e.g., a 5' ITR, and a second ITR, e.g., a 3' ITR. Typically, ITRs are involved in the replication and rescue or excision of parvovirus (e.g., AAV) DNA from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; GottliebandMuzyczka, 1988). In addition, ITRs are also considered to be the minimal sequences required for the integration of AAV provirus and packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are essential for efficient replication of parvovirus genomes. It is hypothesized that the minimal canonical elements essential for ITR function are Rep binding sites and terminal separation sites plus a variable palindrome that allows hairpin formation. Palindromic nucleotide regions usually function together in cis as origins of DNA replication and packaging signals for viruses. Complementary sequences in ITRs fold into hairpin structures during DNA replication. In some embodiments, ITRs fold into T-shaped hairpin structures. In other embodiments, ITRs fold into hairpin structures other than T-shaped, such as U-shaped hairpin structures. Data suggest that the T-shaped hairpin structure of AAV ITRs can inhibit the expression of transgenes flanked by ITRs. See, for example, Zhou et al. (2017), Scientific Reports, 7:5432. By utilizing ITRs that do not form T-shaped hairpin structures, this form of inhibition is avoided. Thus, in certain aspects, polynucleotides that include non-AAV ITRs have improved transgene expression compared to polynucleotides that include AAV ITRs that form T-shaped hairpins.

[0147] As used herein, "inverted terminal repeat" (or "ITR") refers to a nucleic acid subsequence located at the 5' or 3' end of a single stranded nucleic acid sequence that comprises a set of nucleotides (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, an ITR described herein forms a hairpin structure. In some embodiments, an ITR forms a T-shaped hairpin structure. In some embodiments, an ITR forms a hairpin structure other than a T-shaped, e.g., a U-shaped hairpin structure. In some embodiments, an ITR promotes the survival of a nucleic acid molecule in a cell nucleus over an extended period of time. In some embodiments, an ITR promotes the permanent survival (e.g., for the entire lifespan of a cell) of a nucleic acid molecule in a cell nucleus. In some embodiments, an ITR promotes the stability of a nucleic acid molecule in a cell nucleus. In some embodiments, the ITRs promote the retention of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs promote the persistence of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs inhibit or prevent the degradation of the nucleic acid molecule in the cell nucleus.

[0148] Thus, an "ITR" as used herein may fold back on itself to form a double-stranded segment. For example, the sequence GATCXXXXGATC includes an initial sequence of GATC and its complement (3'CTAG5') such that when folded, they form a double helix. In some embodiments, an ITR includes a continuous palindromic sequence (e.g., GATCGATC) between the initial sequence and the reverse complement. In some embodiments, an ITR includes an interrupted palindromic sequence (e.g., GATCXXXXGATC) between the initial sequence and the reverse complement. In some embodiments, the complementary portions of the continuous or interrupted palindromic sequences interact with each other to form a "hairpin loop" structure. A "hairpin loop" structure, as used herein, occurs when at least two complementary sequences on a single-stranded nucleotide molecule base pair to form a double-stranded portion. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop.

[0149] In the present disclosure, at least one ITR is an ITR other than adeno-associated virus (other than AAV).In certain embodiments, the ITR is an ITR of a member of the Parvoviridae family other than AAV.In some embodiments, the ITR is an ITR of a member of the Dependovirus or Erythrovirus genus other than AAV.

[0150] In some embodiments, the ITRs are from a non-AAV genome from the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Abeparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstildensovirus, and any combination thereof. In certain embodiments, the ITRs are from human bocavirus (HBoV1). In certain embodiments, the ITRs are from Erythrovirus B19 (a human virus), a parvovirus. In some embodiments, the ITRs are from the Dependoparvovirus. In one embodiment, the Dependoparvovirus is a Dependovirus goose parvovirus (GPV) strain. In a specific embodiment, the GPV strain is an attenuated GPV strain, for example, GPV 82-0321V strain. In another specific embodiment, the GPV strain is a pathogenic GPV strain, such as GPV strain B. In some embodiments, the ITRs are ITRs of goose parvovirus (GPV) or Muscovy duck parvovirus (MDPV).

[0151] In some embodiments, the ITRs are those of the parvovirus, erythrovirus B19 (also known as parvovirus B19 (also referred to herein as "B19", primate type 1 erythroparvovirus, B19 virus, and erythrovirus)). In some embodiments, the ITRs are those of human bocavirus (HBoV1).

[0152] In certain embodiments, one of the two ITRs is the ITR of AAV.In other embodiments, one of the two ITRs in the construct is the ITR of AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and any combination thereof.In a particular embodiment, the ITR is derived from AAV serotype 2, for example, the ITR of AAV serotype 2.

[0153] In certain aspects of the present disclosure, the nucleic acid molecule comprises two ITRs, a 5' ITR and a 3' ITR, the 5' ITR being located at the 5' end of the nucleic acid molecule and the 3' ITR being located at the 3' end of the nucleic acid molecule. The first ITR and the second ITR of the nucleic acid molecule may be derived from the same genome, e.g., the genome of the same virus, or may be derived from different genomes, e.g., the genomes of two or more different viral genomes (also known as "hybrid" ITRs). In some embodiments, the first ITR is derived from the B19 genome and the second ITR is derived from GPV. In some embodiments, the first ITR is derived from the GPV genome and the second ITR is derived from B19.

[0154] In certain embodiments, the first ITR and / or the second ITR comprises or consists of all or a part of an ITR from human bocavirus (HBoV1). In certain embodiments, the first ITR and / or the second ITR comprises or consists of all or a part of an ITR from HBoV1. In some embodiments, the second ITR is the reverse complement of the first ITR. In some embodiments, the first ITR is the reverse complement of the second ITR. In some embodiments, the first ITR and / or the second ITR from HBoV1 is capable of forming a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0155] In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NOs: 1, 2, 21-30, where the first ITR and / or the second ITR retains the functional properties of the wild-type ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR is derived from a wild-type HBoV1 ITR. In some embodiments, the first ITR and / or the second ITR is derived from a wild-type B19 ITR. In some embodiments, the first ITR and / or the second ITR is derived from a wild-type GPV ITR.

[0156] In some embodiments, the first and / or second ITR comprises or consists of a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NOs: 1, 2, 21-30, where the first and / or second ITR is capable of forming a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0157] Those skilled in the art will appreciate that any of the first ITR sequences described herein can be matched with any of the second ITR sequences described herein. In some embodiments, the first ITR sequence described herein is the 5' ITR sequence. In some embodiments, the second ITR sequence described herein is the 3' ITR sequence. In some embodiments, the second ITR sequence described herein is the 5' ITR sequence. In some embodiments, the first ITR sequence described herein is the 3' ITR sequence. Those skilled in the art will be able to determine the appropriate orientation of the first ITR and second ITR described herein for the construction of the gene cassette.

[0158] In another specific embodiment, the ITR is a synthetic sequence engineered to contain an ITR at its 5'-end and 3'-end that is not derived from the AAV genome. In another specific embodiment, the ITR is a synthetic sequence engineered to contain an ITR at its 5'-end and 3'-end that is derived from one or more non-AAV genomes. The two ITRs present in the nucleic acid molecule of the present invention can be from the same non-AAV genome or from different non-AAV genomes. In particular, the ITRs can be from the same non-AAV genome. In a specific embodiment, the two ITRs present in the nucleic acid molecule of the present invention can be the same, in particular, AAV2 ITR.

[0159] In some embodiments, the ITR sequence comprises one or more palindromic sequences. Palindromic ITR sequences disclosed herein include, but are not limited to, naturally occurring palindromic sequences (i.e., sequences found in nature), synthetic sequences such as pseudopalindromic sequences (i.e., sequences not found in nature), and combinations or modifications thereof.

[0160] In some embodiments, the ITR forms a hairpin loop structure. In one embodiment, the first ITR forms a hairpin structure. In another embodiment, the second ITR forms a hairpin structure. In yet another embodiment, both the first ITR and the second ITR form a hairpin structure. In some embodiments, the first ITR and / or the second ITR do not form a T-shaped hairpin structure. In certain embodiments, the first ITR and / or the second ITR form a hairpin structure other than a T-shaped structure. In some embodiments, the hairpin structure other than a T-shaped structure comprises a U-shaped hairpin structure.

[0161] In some embodiments, the ITRs in the nucleic acid molecules described herein may be transcriptionally activating ITRs. The transcriptionally activating ITRs may comprise all or part of the wild-type ITRs that have been transcriptionally activated by the incorporation of at least one transcriptionally active element. Various types of transcriptionally active elements are suitable for use in this context. In some embodiments, the transcriptionally active element is a constitutive transcriptionally active element. Constitutive transcriptionally active elements provide sustained levels of gene transcription and are preferred when it is desired that the transgene be expressed on a sustained basis. In other embodiments, the transcriptionally active element is an inducible transcriptionally active element. Inducible transcriptionally active elements generally exhibit low activity in the absence of an inducer (or an inducing condition) and are upregulated in the presence of an inducer (or a switch to an inducing condition). Inducible transcriptionally active elements may be preferred when expression is desired only at a certain time or at a certain location, or when it is desired to titrate the expression level using an inducer. Transcriptionally active elements can also be tissue specific; that is, active only in certain tissues or cell types.

[0162] The transcriptionally active element is incorporated into the ITR in various ways. In some embodiments, the transcriptionally active element is incorporated 5' to any part of the ITR or 3' to any part 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 separated, these elements alternate with parts of the ITR. In some embodiments, the hairpin structure of the ITR is deleted and replaced by an inverted repeat of the transcription element. This latter arrangement would create a hairpin that mimics the deleted part in the structure. There may be multiple tandem transcriptionally active elements in the transcriptionally activating ITR, which may be adjacent or separated. In addition, protein binding sites (e.g., Rep binding sites) are also introduced into the transcriptionally active element of the transcriptionally activating ITR. The transcriptionally active element may include any sequence that allows the control of transcription of DNA by RNA polymerase to form RNA, and may include, for example, the transcriptionally active elements defined below.

[0163] Transcriptionally activating ITRs provide both transcriptional activation and ITR functions to a nucleic acid molecule in a relatively limited nucleotide sequence length, which effectively maximizes the length of the transgene that is carried and expressed from the nucleic acid molecule. The incorporation of transcriptionally activating elements into ITRs can be accomplished in a variety of ways. Comparison of ITR sequences and sequence requirements of transcriptionally activating elements can provide insight into the manner of encoding elements within the ITR. For example, transcriptional activity is added to an ITR through the introduction of specific changes in the ITR sequence that duplicate the functional elements of the transcriptionally activating element. There are numerous techniques in the art that efficiently add, delete, and / or change specific nucleotide sequences at specific sites (see, for example, Deng and Nickoloff (1992), Anal. Biochem., 200:81-88). Another way of creating transcriptionally activating ITRs involves the introduction of restriction sites at desired positions within the ITR. In addition, multiple transcriptionally activating elements are incorporated into transcriptionally activating ITRs using methods known in the art.

[0164] By way of example, transcriptionally activating ITRs are created by the incorporation of one or more transcriptionally active elements, such as a TATAbox, a GCbox, a CCAATbox, an Sp1 site, an Inr region, a CRE (cAMP regulatory element) site, an ATF-1 / CRE site, an APBβbox, an APBαbox, a CArGbox, a CCACbox, or any other element involved in transcription known in the art.

[0165] Vector Some embodiments of the present disclosure are directed to vectors comprising one or more codon-optimized nucleic acid molecules encoding a polypeptide with FVIII activity as described herein, host cells comprising the vectors, and methods of treating bleeding disorders using the vectors. The present disclosure fulfills an important need in the art by providing vectors comprising optimized FVIII sequences that support increased expression in a subject, potentially resulting in increased therapeutic efficacy when used in gene therapy methods.

[0166] Vectors suitable for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In one embodiment, the vector is a viral vector.

[0167] As used herein, "expression vector" refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, for replication and translation, when introduced into a suitable host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0168] The expression vector of the present disclosure will include 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 functionality. 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 the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence was capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript was translated into the desired protein or polypeptide.

[0169] Viral vectors include, but are not limited to, 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 virus; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus; and nucleic acid sequences derived from RNA viruses such as retroviruses. Other vectors that are well known in the art can be easily incorporated. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes are replaced by genes of interest. In one embodiment, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated viruses are engineered to be replication-deficient and capable of infecting a wide range of cell types and species.

[0170] According to the present disclosure, one or more of different AAV vector sequences from almost any serotype may be used. The selection of a particular AAV vector sequence will be guided by known parameters, such as the desired tropism, the required vector yield, etc. In general, AAV serotypes have genome sequences with significant homology at the amino acid and nucleic acid levels, resulting in a set of related gene functions, producing related virions, and replicating and assembling in the same way. For the genomic sequences of the various AAV serotypes, and an overview of the genomic similarities, see, e.g., 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. AAV6, AAV7, AAV8, or AAV9, or newly developed AAV-like particles, for example, obtained by capsid shuffling methods and AAV capsid libraries, or derived from newly designed, developed, or evolved ITRs, are also suitable for application in certain disclosures. See Dalkara et al. (2013), Sci.Transl.Med., 5(189):189-76; Kotterman MA (2014), Nat.Rev.Genet., 15(7):455.

[0171] Other vectors include plasmid vectors. Plasmid vectors have been described extensively in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. In the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they are unable to replicate and integrate into the host genome. These plasmids, however, having a promoter compatible with the host cell, are capable of expressing the peptide gene operably encoded within the plasmid. Some commonly used plasmids available from commercial sources include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1, model number: V79020; pcDNA3.1 / hygro, model number: V87020; pcDNA4 / myc-His, model number: V86320; and pBudCE4.1, model number: V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids are also known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology methods to remove and / or add specific segments of DNA.

[0172] In certain embodiments, it may be useful to incorporate one or more miRNA target sequences into the vector, for example, operably linked to the optimized FVIII transgene. More than one copy of the miRNA target sequence incorporated into the vector may increase the efficacy of the system. For example, a vector expressing more than one transgene may have the transgene under the control of more than one miRNA target sequence, which may be the same or different. The miRNA target sequences may be in tandem, but other configurations may also be incorporated. The expression cassette of the transgene, containing the miRNA target sequence, may also be inserted into the vector in antisense orientation. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which are incorporated herein by reference in their entirety. However, in certain other embodiments, the vector will not incorporate any miRNA target sequence. The choice of whether or not to incorporate a miRNA target sequence (and how much to incorporate) will be guided by known parameters, such as the intended tissue target, the level of expression required, and the like.

[0173] host cell The present disclosure also provides a host cell comprising the nucleic acid molecule or vector of the present disclosure. As used herein, the term "transformation" is used broadly to refer to the introduction of DNA into a recipient host cell, resulting in a change in the genotype and, as a result, in the change of the recipient cell.

[0174] "Host cell" refers to a cell that is transformed with a vector constructed using recombinant DNA methods and encoding at least one heterologous gene. The host cell of the present disclosure is preferably of mammalian origin; most preferably of human or murine origin. Those skilled in the art are considered to be capable of preferentially determining the particular host cell line that is best suited for their purpose. Exemplary host cell lines include, but are not limited to, CHO, DG44, and DUXB11 (Chinese hamster ovary cell line, DHFR deleted), HELA (human cervical carcinoma), CVI (monkey kidney cell line), COS (a derivative of CVI cells with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell 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 cells are 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 published literature.

[0175] Introduction of the isolated nucleic acid molecule or vector of the present disclosure into a host cell can be accomplished by a variety of techniques well known to those skilled in the art. These 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, pages 470-472, "Vectors," edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Plasmids are introduced into the host via electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0176] The host cells containing the isolated nucleic acid molecule or vector of the present disclosure are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium containing nutrients required for cell growth. Nutrients required 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 growth medium will generally select for cells containing the DNA construct, for example, by drug selection or deficiency of essential nutrients, complemented by a selectable marker on the DNA construct or 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 one of ordinary skill in the art.

[0177] In some embodiments, the host cell suitable for use in the present invention is derived from insects. In some embodiments, suitable insect host cells include, for example, cell lines isolated from Fall Armyworm (Sf) or cell lines isolated from Nettle Looper (Tni). Those skilled in the art can easily determine the suitability of any Sf or Tni cell line. Exemplary insect host cells include, without limitation, Sf9 cells, Sf21 cells, and High Five™ cells. Exemplary insect host cells also include, without limitation, any Sf or Tni cell line that does not contain accidental viral contamination, such as Sf-rhabdovirus negative (Sf-RVN) cells and Tn-nodavirus negative (Tn-NVN) cells. Other suitable host insect cells are also known to those skilled in the art. In a particular embodiment, the insect host cell is Sf9 cell.

[0178] An embodiment of the present disclosure provides a method for cloning a nucleic acid molecule described herein, comprising inserting a nucleic acid molecule capable of complex secondary structures 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 in bacterial host strains. For example, nucleic acid molecules of the present disclosure that include a first ITR and a second ITR (e.g., parvovirus ITRs other than AAV, e.g., HBoV1 ITR) can be difficult to clone using conventional methods. Long DNA palindromic sequences inhibit DNA replication and are unstable in the genomes of E. coli, Bacillus, Streptococcus, Streptomyces, Saccharomyces 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 that contributes to blocking the replication of long palindromic sequences. The SbcCD complex is a core with ATP-dependent double-stranded DNA exonuclease activity and ATP-independent single-stranded DNA endonuclease activity. SbcCD can collapse replication forks by recognizing DNA palindromic sequences and attacking the resulting hairpin structures.

[0179] In certain embodiments, suitable bacterial host strains are unable to degrade cruciform DNA structures. In certain embodiments, suitable bacterial host strains comprise disruption in the SbcCD complex. In some embodiments, disruption in the SbcCD complex comprises gene disruption in the SbcC gene and / or in the SbcD gene. In certain embodiments, disruption in the SbcCD complex comprises gene disruption in the SbcC gene. In the art, various bacterial host strains are known that comprise gene disruption in the SbcC gene. For example, without limitation, 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 complex secondary structures into a suitable vector and introducing the resulting vector into the 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 complex secondary structures into a suitable vector and introducing the resulting vector into the host strain PMC103.

[0180] Suitable vectors are known in the art and are 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.

[0181] Thus, the present disclosure provides a method of cloning a nucleic acid molecule comprising inserting a nucleic acid molecule capable of complex secondary structures into a suitable vector and introducing the resulting vector into a bacterial host strain containing a disruption in the SbcCD complex, wherein the nucleic acid molecule comprises a first inverted terminal repeat (ITR) and a second ITR, and wherein the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NOs: 12-23, or a functional derivative thereof.

[0182] Production of Polypeptides The present disclosure also provides a polypeptide encoded by the nucleic acid molecule of the present disclosure. In other embodiments, the polypeptide of the present disclosure is encoded by a vector comprising the 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 the isolated nucleic acid molecule of the present disclosure.

[0183] In other embodiments, the present disclosure also provides a method for producing a polypeptide with FVIII activity, comprising culturing a host cell of the present disclosure under conditions in which a polypeptide with FVIII activity is produced, and recovering the polypeptide with FVIII activity. In some embodiments, the expression of the polypeptide with FVIII activity is increased compared to a host cell cultured under the same conditions but comprising a reference nucleotide sequence, comprising the parent FVIII nucleotide sequence, SEQ ID NO: 32.

[0184] In other embodiments, the present disclosure provides a method for increasing expression of a polypeptide with FVIII activity, comprising culturing a host cell of the present disclosure under conditions in which a polypeptide with FVIII activity is expressed by a nucleic acid molecule, wherein expression of the polypeptide with FVIII activity is increased compared to a host cell cultured under the same conditions but containing a reference nucleic acid molecule comprising SEQ ID NO:32.

[0185] In other embodiments, the disclosure provides a method for improving the yield of a polypeptide with FVIII activity, comprising culturing a host cell under conditions in which a polypeptide with FVIII activity is produced by a nucleic acid molecule, wherein the yield of the polypeptide with FVIII activity is increased compared to a host cell cultured under the same conditions but comprising a reference nucleic acid sequence comprising SEQ ID NO:32.

[0186] A variety of methods are available for recombinantly producing FVIII proteins from the optimized nucleic acid molecules of the present disclosure. A polynucleotide with a desired sequence may be produced by de novo solid-phase DNA synthesis or may be produced through PCR-based mutagenesis of an already produced polynucleotide. Oligonucleotide-mediated mutagenesis is one method for producing substitutions, insertions, deletions, or changes (e.g., codon changes) in a nucleotide sequence. For example, the starting DNA is modified by hybridizing an oligonucleotide encoding a desired mutation with a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, incorporating the oligonucleotide primer. In one embodiment, genetic engineering, such as primer-based PCR mutagenesis, is sufficient to incorporate the changes defined herein to produce a polynucleotide of the present disclosure.

[0187] For production of recombinant proteins, the optimized polynucleotide sequences of the present disclosure encoding FVIII proteins are inserted into an appropriate expression vehicle, i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence, or, in the case of RNA viral vectors, the necessary elements for replication and translation.

[0188] The polynucleotide sequence of the present disclosure is inserted into a vector in the correct reading frame. The expression vector is then transfected into an appropriate target cell to express the polypeptide. Transfection methods 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). A variety of host-expression vector systems are utilized 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 may also encode a signal sequence that allows secretion of the FVIII protein. One skilled in the art will understand 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 incorporated, the FVIII protein is recovered by lysing the cells.

[0189] The FVIII protein of the present disclosure is synthesized in transgenic animals such as rodents, goats, sheep, pigs or cows. The term "transgenic animals" refers to non-human animals that incorporate foreign genes into their genome. This gene is present in germ line tissues and is passed from parent to offspring. Exogenous genes are introduced into single-cell embryos (Brinster et al., 1985, Proc. Natl. Acad. Sci. USA, 82:4438). Methods for producing transgenic animals are known in the art, including transgenic animals that produce immunoglobulin molecules (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).

[0190] The expression vector may code for a tag that allows easy purification or identification of recombinantly produced proteins. Examples include, but are not limited to, pUR278 (Ruther et al., 1983, EMBO J., 2:1791), a vector in which the coding sequence of the FVIII protein described herein is ligated into the vector in frame with the lac Z coding region, so that hybrid proteins are produced; pGEX vectors are used to express proteins with glutathione S-transferase (GST) tags. These proteins are usually soluble and 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)) for easy removal of the tag after purification.

[0191] For the purposes of this disclosure, numerous expression vector systems are employed. These expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Expression vectors may 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 may also utilize DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), cytomegalovirus (CMV), or SV40 virus. Other expression vectors involve the use of polycistronic systems with internal ribosome binding sites.

[0192] Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362) that permits detection of cells transformed with the desired DNA sequences. Cells that have integrated the DNA into their chromosomes are selected by introducing one or more markers that permit selection of transfected host cells. The marker may confer prototrophy to an auxotrophic host, may confer resistance to biocides (e.g., antibiotics), or may confer resistance to heavy metals such as copper. The selection marker gene may be directly linked to the DNA sequence to be expressed, or may be introduced into the same cell by cotransformation.

[0193] An example of a vector useful for expressing an optimized FVIII sequence is NEOSPLA (US 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 found to result in extremely high levels of antibody expression upon integration of variable and constant region genes, followed by transfection of cells and selection in G418-containing medium and methotrexate amplification. Vector systems are also taught in US 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, for example, >30 pg per cell per day. Other exemplary vector systems are disclosed, for example, in US Pat. No. 6,413,777.

[0194] In other embodiments, the polypeptide of the present disclosure is expressed using a polycistronic construct. In these expression systems, multiple gene products of interest, such as multiple polypeptides of multimeric binding proteins, are produced from a single polycistronic construct. These systems are advantageous because they 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.

[0195] More generally, once a vector or DNA sequence encoding a polypeptide is prepared, the expression vector is introduced into a suitable host cell. That is, the host cell is transformed. The introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled in the art, as discussed above. The transformed cell is grown under conditions suitable for the production of FVIII polypeptide, and assayed for the synthesis of FVIII polypeptide. Exemplary assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0196] In describing processes for the isolation of polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to denote the source of the polypeptide, unless expressly specified otherwise. In other words, recovery of the polypeptide from "cells" can mean recovery from spun down whole cells, or it can mean the cell culture containing both the medium and the suspended cells.

[0197] The host cell line used for protein expression is preferably of mammalian origin, since the isolated nucleic acid of the present disclosure is optimized for expression in human cells; most preferably of human or mouse origin.Exemplary host cell lines are described above.In one embodiment of the method for producing a polypeptide with FVIII activity, the host cell is HEK293 cell.In another embodiment of the method for producing a polypeptide with FVIII activity, the host cell is CHO cell.

[0198] The gene encoding the polypeptide of the present disclosure can also be expressed in non-mammalian cells, such as bacteria or yeast or plant cells. In this regard, it will be appreciated that a variety of unicellular microorganisms other than mammals, i.e., microorganisms capable of growth in culture or fermentation, such as bacteria, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the polypeptide typically becomes part of inclusion bodies. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0199] Alternatively, the optimized nucleotide sequences of the present disclosure are incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., US 5,741,957; Rosen, US 5,304,489; and Meade et al., US 5,849,992). A suitable transgene comprises a coding sequence for a polypeptide operably linked to a promoter and enhancer derived from a mammary gland-specific gene, such as casein or beta-lactoglobulin.

[0200] In vitro production allows for scale-up to provide large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art, including homogenous suspension cultures, for example in airlift or continuous stirred reactors, or immobilized or encapsulated cell cultures, for example in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and / or desired, the solution of the polypeptide is purified by conventional chromatographic methods, for example, gel filtration chromatography, ion exchange chromatography, chromatography through DEAE-cellulose, or (immuno) affinity chromatography, for example after the preferential biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein. Optionally, an affinity tag sequence (e.g., His(6) tag) may be attached to or incorporated into the polypeptide sequence to facilitate downstream purification.

[0201] Once expressed, the FVIII protein is purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, HPLC purification, gel electrophoresis, and the like (see generally, Scopes, "Protein Purification," Springer-Verlag, NY (1982)). Substantially pure proteins having at least about 90-95% homogeneity are preferred for pharmaceutical uses, with 98-99% or more homogeneity being most preferred.

[0202] Pharmaceutical Compositions A composition containing 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 may contain a suitable pharma- ceutically acceptable carrier. For example, the composition may contain excipients and / or adjuvants that facilitate processing of the active compound into a product designed for delivery to a site of action.

[0203] The pharmaceutical composition is formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) by bolus injection. The formulation for injection is presented in unit dosage form, for example, in ampoules or multi-dose containers with added preservatives. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and may contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable medium, for example, pyrogen-free water.

[0204] Preparations suitable for parenteral administration also include aqueous solutions of the active compound in water-soluble form, for example, in water-soluble salt form. In addition, suspensions of the active compound as appropriate oily injection suspensions are also administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and dextran. Optionally, the suspension may also contain a stabilizer. Liposomes are also 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, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the composition includes an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride. In other embodiments, the composition includes a pharma- ceutically acceptable substance, such as a humectant, or minor amounts of auxiliary substances, such as humectants or emulsifiers, preservatives or buffers, which enhance the shelf life or efficacy of the active ingredient.

[0205] The compositions of the present disclosure may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solids, and solids. The preferred form depends on the mode of administration and therapeutic application.

[0206] The composition is formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of the above-listed ingredients as required, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active ingredient into a sterile medium containing a basic dispersion medium and other required ingredients from the above-listed ingredients. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained by using a coating such as lecithin, or by maintaining the required particle size in the case of dispersions, or by using surfactants. Prolonged absorption of injectable compositions can be achieved by incorporating an agent that delays absorption, such as monostearate salts and gelatin, into the composition.

[0207] The active ingredient is formulated with controlled release formulation or device.The examples of such formulation and device include implant, transdermal patch and microencapsulated delivery system.Biodegradable polymer, biocompatible polymer, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid are used.The method for producing such formulation and device is known in the art.See, for example, "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0208] Injectable depot preparations are made by forming microencapsulated matrices of drugs in biodegradable polymers such as polylactide-polyglycolide.Depending on the drug-to-polymer ratio and the nature of the polymer used, the drug release rate is controlled.Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides.Injectable depot preparations are also made by encapsulating drugs in liposomes or microemulsions.

[0209] The composition may also incorporate an auxiliary active compound. In one embodiment, the chimeric protein of the present disclosure is formulated with another coagulation factor, or a variant, fragment, analog, or derivative thereof. For example, the coagulation factor includes, 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 an activated form of any of the foregoing. The coagulation factor of the hemostatic agent may also include an antifibrinolytic agent, such as epsilon-aminocaproic acid, tranexamic acid.

[0210] Dosage regimen is adjusted to obtain the desired optimal response. For example, a single bolus may be administered, or a number of divided doses may be administered over time, and the dose may be reduced or increased accordingly 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 unit dosage form. For example, see "Remington's Pharmaceutical Sciences" (Mack Pub.Co., Easton, Pa., 1980).

[0211] 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, glycerol, tetrahydrofururyl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0212] Non-limiting examples of suitable pharmaceutical carriers are also 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, nonfat dry milk, glycerol, propylene glycol, water, ethanol, etc. The composition may also contain a pH buffering agent and a humectant or emulsifier.

[0213] For oral administration, the pharmaceutical composition may take the form of a tablet or capsule, which is prepared by conventional means. The composition may also be prepared as a liquid, for example, a syrup or suspension. The liquid may contain a suspending agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifying agent (lecithin or gum acacia), a non-aqueous medium (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and a preservative (e.g., methyl-p-hydroxybenzoate or propyl-p-hydroxybenzoate, or sorbic acid). The preparation may also contain flavorings, colorings, and sweetening agents. Alternatively, the composition may be provided as a dry product for constitution with water or another suitable vehicle.

[0214] For buccal administration, the composition may take the form of tablets or lozenges following conventional protocols.

[0215] For inhalation administration, 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, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide, or other suitable gas.In the case of a pressurized aerosol, the dosage unit is determined by providing a valve that delivers a metered amount.Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator are formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0216] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0217] 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.

[0218] Treatment In some aspects, the present disclosure is directed to a method of treating a disease or condition in a subject in need thereof, the method comprising administering a nucleic acid molecule, vector, polypeptide, or pharmaceutical composition disclosed herein.

[0219] In some embodiments, the present disclosure is directed to a method of treating a bleeding disorder. In some embodiments, the present disclosure is directed to a method of treating hemophilia A.

[0220] The isolated nucleic acid molecule, vector, or polypeptide is administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, for example, via oral, sublingual, buccal, sublingual, intranasal, rectal, vaginal, or pulmonary routes. The isolated nucleic acid molecule, vector, or polypeptide is also administered intraneurally, intraocularly, and intrathecally. The coagulation factor protein may be implanted in or linked to a biopolymeric solid support that allows for sustained release of the chimeric protein to the desired site.

[0221] In one embodiment, the administration route of the isolated nucleic acid molecule, vector, or polypeptide is parenteral.As used herein, the term "parenteral" includes intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intrarectal administration, or intravaginal administration.In some embodiments, the isolated nucleic acid molecule, vector, or polypeptide is administered intravenously.Although all of these administration forms are expressly contemplated to be within the scope of the present disclosure, the form for administration will be injectable solution, particularly for intravenous or intraarterial injection or instillation.

[0222] The effective dose of the composition of the present disclosure for treating a condition varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other medicines administered, and whether the treatment is a preventive or therapeutic treatment.Usually, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated.Treatment dosages are titrated using routine methods known to those skilled in the art that optimize safety and efficacy.

[0223] The nucleic acid molecules, vectors, or polypeptides of the disclosure are optionally administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic treatment).

[0224] As used herein, administration of the disclosed isolated nucleic acid molecule, vector, or polypeptide with or in combination with adjunctive therapy refers to sequential administration or application, simultaneous administration or application, co-administration or application, co-administration or application, or parallel administration or application of the therapy and the disclosed polypeptide. Those skilled in the art will recognize that the administration or application of various components of the combined therapy regimen is timed to enhance the efficacy of treatment. Those skilled in the art (e.g., physicians) will be able to easily identify an effective combined therapy regimen based on the selected adjunctive therapy and the teachings of this specification without undue experimentation.

[0225] It will be further appreciated that the isolated nucleic acid molecules, vectors, or polypeptides of the present disclosure may be used with or in combination with one or more drugs (e.g., to provide a combination therapeutic regimen). Exemplary drugs to be combined with the polypeptides or polynucleotides of the present disclosure include drugs that represent the current standard of care for the particular disorder being treated. Such drugs may be chemicals or biopharmaceuticals in nature. The term "biopharmaceutical" or "biopharmaceutical agent" refers to any pharmacologic active agent made from living organisms and / or their products that is intended for use as a therapeutic agent.

[0226] The amount of drugs used in combination with the polynucleotides or polypeptides of the present disclosure may vary from subject to subject and may be administered according to what is known in the art. See, for example, Bruce A Chabner et al., "Antineoplastic Agents," GOODMAN and GILMAN, "PHARMACOLOGICAL BASIS OF THERAPEUTICS," pp. 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 of care.

[0227] In one embodiment, also disclosed herein is a kit comprising the 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 producing the nucleic acid molecule provided herein. The nucleic acid molecule is produced in insect cells. In another embodiment, provided is a nanoparticle delivery system for an expression construct. The expression construct comprises the nucleic acid molecule disclosed herein.

[0228] Gene Therapy In some embodiments, the nucleic acid molecule disclosed herein is used in gene therapy. The optimized FVIII nucleic acid molecule disclosed herein is used in any context where expression of FVIII is required. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35.

[0229] For example, somatic cell gene therapy is being explored as a possible treatment for hemophilia A. Gene therapy is a particularly attractive treatment for hemophilia due to its potential to cure the disease through sustained endogenous production of FVIII after a single administration of a vector. Hemophilia A is well suited to a gene replacement approach because its clinical symptoms are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200 ng / ml) in plasma.

[0230] In one aspect, the nucleic acid molecules described herein are used in AAV gene therapy. AAV can infect many mammalian cells. See, for example, Tratschin et al. (1985), Mol. Cell Biol., 5:3251-3260; and Grimm et al. (1999), Hum. Gene Ther., 10:2445-2450. rAAV vectors carry the nucleic acid sequence that codes for a gene of interest, or a fragment thereof, under the control of a regulatory sequence that directs the expression of the gene product in cells. In some embodiments, rAAV is formulated with a carrier and additional components suitable for administration.

[0231] In another aspect, the nucleic acid molecules described herein are used in lentivirus gene therapy. Lentiviruses are RNA viruses whose viral genome is RNA. When lentivirus infects a host cell, the genomic RNA is reverse transcribed into a DNA intermediate and is highly efficiently integrated into the chromosomal DNA of the infected cell. In some embodiments, lentiviruses are formulated with carriers and additional components suitable for administration. In another aspect, the nucleic acid molecules described herein are used in adenovirus therapy. A review of the use of adenovirus for gene therapy can be found, for example, in Wold et al. (1985), Curr Gene Ther., 13(6):421-33. In another aspect, the nucleic acid molecules described herein are used in non-viral gene therapy.

[0232] The optimized FVIII protein of the present disclosure is produced in vivo in a mammal, e.g., a human patient, and a gene therapy approach may be therapeutically beneficial for the treatment of bleeding diseases or disorders selected from the group consisting of bleeding coagulation disorders, hemarthrosis, intramuscular bleeding, oral bleeding, bleeding into muscle, oral bleeding, trauma, traumatic head, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding into the retropharyngeal space, bleeding into the retroperitoneal space, and bleeding into the iliopsoas sheath. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves the administration of an optimized FVIII-encoding nucleic acid operably linked to an appropriate expression control sequence. In certain embodiments, these sequences are incorporated into a viral vector. The viral vector suitable for such gene therapy includes adenoviral vector, lentiviral vector, baculoviral vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, and adeno-associated virus (AAV) vector.The viral vector can be a replication-defective viral vector.In another embodiment, the adenoviral vector is deleted its E1 gene or E3 gene.In another embodiment, the sequence is also incorporated into a non-viral vector, which is known to those skilled in the art.

[0233] In another aspect, the methods disclosed herein provide techniques for targeted and specific modification of genetic information (e.g., genome) of an organism. As used herein, the term "modification" or "modification of genetic information" refers to any change in the genome of a cell. In the context of treating genetic disorders, modification can include, but is not limited to, insertion, deletion, and / or correction.

[0234] In some embodiments, the alteration may also include knocking in, knocking out, or knocking down of a gene. As used herein, the term "knock-in" refers to the addition of a DNA sequence or a fragment thereof to a genome. Such DNA sequence to be knocked in may include the entire gene, or the entire gene or gene may include the regulatory sequences associated with the gene or any part or fragment thereof. For example, a cDNA encoding a wild-type protein is inserted into the genome of a cell carrying a mutant gene. A knock-in strategy does not require replacing a defective gene in whole or in part. In some cases, a knock-in strategy may further involve replacing an existing sequence with a prepared sequence, for example, replacing a mutant allele with a wild-type copy. The term "knock-out" refers to the loss of a gene or gene expression. For example, a gene is knocked out by the deletion or addition of a nucleotide sequence that results in a disruption of the reading frame. As another example, a gene is knocked out by replacing a part of the gene with a non-involved sequence. As used herein, "knock-down" refers to the reduction of expression of a gene or its gene product(s). Gene knockdown may result in attenuation of protein activity or function, or in reduced or abolished protein levels.

[0235] In some embodiments, the nucleic acid sequences disclosed herein are used for genome editing. Genome editing generally refers to a process of modifying the nucleotide sequence of a genome, preferably in a precise or predetermined manner. Examples of genome editing methods described herein include methods using site-directed nucleases to cleave deoxyribonucleic acid (DNA) at precise target positions in the genome, thereby creating single-stranded or double-stranded DNA breaks at specific positions in the genome. Such breaks can be repaired and are regularly repaired by natural endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), as recently reviewed in Cox et al. (2015), Nature Medicine, 21(2):121-31. These two major DNA repair processes comprise a family of alternative pathways. NHEJ directly connects the DNA ends resulting from the double-stranded break, but in some cases involves the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR utilizes homologous or donor sequences as templates for inserting a defined DNA sequence into the breakpoint. Homologous sequences can be present in endogenous genomes, such as sister chromatids. Alternatively, donor can be exogenous nucleic acid, such as plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, duplex oligonucleotide, or virus, which has a large region of homology with the locus that is cut by nuclease, but also contains additional sequences or sequence changes, including deletions, that are integrated into the cut target locus. The third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as "alternative NHEJ", whose genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cut site. MMEJ may use a small number of base-pair homologous sequences flanking the DNA break site to drive a more favorable repair outcome by joining the DNA ends, but recent reports have further elucidated the molecular mechanisms of this process (see, e.g., Cho and Greenberg (2015), Nature, 518, 174-76).In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the DNA break site.

[0236] Each of these genome editing mechanisms is used to create the desired genome modification.The step in genome editing process can be to create one DNA break or two DNA breaks as double-strand breaks or two single-strand breaks in the target locus as the adjacent site of intended mutation.This is achieved through the use of site-directed polypeptides, such as CRISPR endonuclease system.

[0237] In another embodiment, the nucleic acid molecule described herein is used in lipid nanoparticle (LNP) mediated delivery of FVIII ceDNA.To prevent the degradation of nucleic acid in plasma and facilitate the uptake of oligonucleotide into cells, lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides are used.Such lipid nanoparticles are used to deliver the nucleic acid molecule described herein to subjects.

[0238] The present disclosure provides a method of increasing expression of a polypeptide with FVIII activity in a subject, comprising administering to a subject in need thereof an isolated nucleic acid molecule of the present disclosure, wherein expression of the polypeptide is increased relative to a reference nucleic acid molecule comprising SEQ ID NO: 32. The present disclosure also provides a method of increasing expression of a polypeptide with FVIII activity in a subject, comprising administering to a subject in need thereof a vector of the present disclosure, wherein expression of the polypeptide is increased relative to a vector comprising the reference nucleic acid molecule.

[0239] All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

[0240] All publications, patents, and patent applications mentioned in this specification are herein 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.

[0241] Having generally described the present disclosure, a further understanding can be obtained by reference to the examples provided herein, which are intended for purposes of illustration only and are not intended to be limiting. EXAMPLES

[0242] Modified FVIIIXTEN expression cassette We hypothesized that the expression level of the transgene would be increased by codon-optimizing the coding sequence for the target host. Previous studies described in US Publication No. 20190185543 have demonstrated high levels of FVIII expression using the V1.0 FVIIIco6XTEN expression cassette (SEQ ID NO: 32) (Figure 1). However, to further improve target specificity and reduce immunogenicity, the FVIIIXTEN expression cassette was codon-optimized by deleting CpG motifs to reduce the natural immune response elicited against the DNA vector encoding the FVIIIXTEN expression cassette together with the parvovirus ITR. In this study, the modified V2.0 FVIIIXTEN expression cassette is composed of a codon-optimized cDNA encoding B-domain deleted human factor VIII (BDDcoFVIII) fused to XTEN 144 peptide (FVIIIXTEN) under the control of the liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) with enhancer element (A1MB2), a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal (SEQ ID NO: 14) (Figure 1). The in vivo functionality of the modified V2.0 FVIIIXTEN expression cassette with different parvoviral ITRs in the form of single-stranded (ss) DNA or closed-end (ce) DNA was demonstrated by systemic delivery via hydrodynamic tail vein injection to express hFVIIIR593C. + / + This has been confirmed in / HemA mice. EXAMPLES

[0243] Single-stranded FVIIIXTEN (ssFVIIIXTEN) DNA Modified FVIIIXTEN showed significantly higher levels of activity in vivo It was hypothesized that hairpins formed within the ITR regions would drive long-term, sustained, high-level transgene expression. To validate the functionality of the modified FVIIIXTEN expression cassettes in vivo, single-stranded DNA (ssDNA) consisting of V1.0 human FVIIIXTEN or V2.0 human FVIIIXTEN with preformed erythrovirus B19 ITRs was transformed into hFVIIIR593C + / + hFVIIIR593C / HemA mice were examined. These mice contain a human FVIII-R593C transgene designed with a mouse albumin (Alb) promoter driving the expression of a modified human coagulation factor VIII (FVIII) cDNA carrying a mutation frequently observed in patients with mild hemophilia A. These mice also carry a knockout of the FVIII gene and are deficient for endogenous FVIII protein. These double mutant mice tolerate injections of human FVIII and have no FVIII activity. These double mutant mice produce only trace amounts of inhibitory antibodies after treatment with human FVIII and lack FVIII-responsive T or B cells. + / + The / HemA mice are further described in Bril et al. (2006), Thromb. Haemost., 95(2):341-7.

[0244] ssFVIIIXTEN with preformed B19 ITRs was generated by denaturing the double-stranded DNA fragment products (FVIII expression cassette and plasmid backbone) of MscI digestion at 95°C (denaturation) followed by cooling at 4°C (renaturation) to allow the palindromic ITR sequences to fold (Figure 2). hFVIIIR593C was then introduced into the plasmid via hydrodynamic tail vein injection. + / +ssFVIIIXTEN was injected systemically at 800 μg per kg of / HemA mice. Plasma samples were collected from injected mice at the indicated intervals over a 5.5 month period and FVIII activity was measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0245] Plasma FVIII activity normalized to percent normal for animals injected with V1.0 ssFVIIIXTEN and V2.0 ssFVIIIXTEN is shown in FIG. 3. The results showed a significant improvement in FVIII activity in the V2.0 ssFVIIIXTEN-injected cohort compared to V1.0 ssFVIIIXTEN. However, an initial drop in FVIII expression was observed by day 56, followed by stabilization of levels by day 168, suggesting sustained expression of parvoviral ITR-flanked V2.0 ssFVIIIXTEN from the liver of injected animals. Thus, these results validate the functionality of modified FVIIIXTEN in vivo with sustained expression activity of FVIII over a prolonged period of time compared to V1.0.

[0246] Human bocavirus (HBoV1) ITRs induced supraphysiological levels of FVIII expression in vivo To determine the effect of ITRs on the stability and long-term persistence of transgene expression, improved forms of FVIIIXTEN with ITRs from human bocavirus (HBoV1), human erythrovirus B19, goose parvovirus (GPV), or their mutants were examined in vivo. These ITRs were engineered based on ITR-specific elements required for thermostability and long-term persistence of the viral genome in their respective hosts. The ITR mutants tested and predicted secondary structures are described in previous US patent application Ser. No. 63 / 069,114. Individual mutant ITRs were cloned into synthetic FVIIIXTEN expression constructs using Golden Gate Assembly and verified by sequencing at the Genewiz sequencing facility. The sequence-verified constructs were then used to generate ssFVIIIXTEN (ssDNA) as described above, and then transformed into hFVIIIR593C. + / + In 10 / HemA mice, 200, 800, or 1600 μg / kg of FVIII was injected systemically via hydrodynamic tail vein injection. Plasma samples were collected from injected mice at the indicated intervals over a 5.5 month period, and FVIII activity was measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0247] Plasma FVIII activity normalized to percent of normal for animals injected with V2.0 ssFVIIIXTEN is shown in FIG. 4. Results showed variable levels of sustained FVIIIXTEN expression over time in all tested parvovirus ITRs. All tested GPV ITR mutants or hybrids showed sustained reductions in FVIIIXTEN expression levels compared to other parvovirus ITRs. In contrast, HBoV1 and B19 ITRs showed an initial reduction in FVIIIXTEN by day 56, then stabilized through day 168, suggesting ITR-dependent persistence of the FVIIIXTEN transgene in vivo. Unlike GPV ITRs, both B19 and HBoV1 ITRs showed significantly higher levels of FVIII expression, regardless of mutant tested, suggesting ITR-dependent stability of the FVIIIXTEN transgene in vivo.

[0248] Among the different parvovirus ITRs tested, the HBoV1 ITR is hFVIIIR593C + / + In / HemA mice, the modified FVIIIXTEN expression cassettes with different parvoviral ITRs showed significantly higher levels (>1000%) of normal FVIII activity (Figure 4). These results validate the functionality of the modified FVIIIXTEN expression cassettes with different parvoviral ITRs and support the ITR-dependent stability as well as sustained transgene expression in vivo. EXAMPLES

[0249] Closed-end FVIIIXTEN (ceFVIIIXTEN) DNA Although ssFVIIIXTEN (ssDNA) was effective in expressing modified FVIIIXTEN expression cassette in vivo, there are several limitations associated with ssDNA used as a non-viral gene therapy vector. One of them is the level of endotoxin contamination due to the prokaryotic host (E. coli) used to generate plasmid DNA, which also contains foreign sequences such as antibiotic resistance genes and prokaryotic origin of replication required for selection and amplification in E. coli. To address these challenges and limitations, a eukaryotic cell-based system was developed to generate DNA therapeutic drug substance in the form of closed-end DNA (ceDNA) composed of FVIIIXTEN expression cassette with parvovirus ITR. Gene organization by ceDNA is similar to recombinant AAV vector DNA, but the conformation is different.

[0250] To generate this DNA vector, we utilized the baculovirus insect cell line, which is the only platform for the production of recombinant influenza vaccines that is widely used and FDA approved for the production of biopharmaceuticals. As described in US Patent Application No. 63 / 069,073, three different approaches of ceDNA production were employed in the baculovirus system. Exemplary purified ceDNA encoding modified FVIIIXTEN with AAV2 ITR or HBoV1 ITR compared to the starting material (SM) is shown in Figure 5A.

[0251] To verify the functionality of the modified FVIIIXTEN expressed from ceDNA, purified ceFVIIIXTEN was administered to hFVIIIR593C via hydrodynamic tail vein injection. + / + In / HemA mice, 0.3 μg, 1.0 μg, or 2.0 μg per mouse were injected systemically, which is equivalent to 12 μg, 40 μg, and 80 μg / kg, respectively. Plasma samples from injected mice were collected at the indicated intervals, and FVIII activity was measured by the chromogenic assay described above.

[0252] Plasma FVIII activity normalized to percent normal for animals injected with ceFVIIIXTEN is shown in FIG. 5B. Results showed a dose-dependent response in HemA mice, with FVIII expression observed at supraphysiological levels (>500% of normal levels) up to 56 days after injection at the highest dose tested. Interestingly, similar expression levels were achieved when mice were injected with ssFVIIIXTEN at 1600 μg / kg, at least 20-fold higher dose than ceFVIIIXTEN (80 μg / kg) (FIG. 4). This data suggests that ceDNA results in higher levels of FVIII expression compared to the ssDNA form. Thus, these studies validate the functionality of modified FVIIIXTEN expressed from ssDNA or ceDNA, and confirm that codon optimization with the use of optimized ITRs can result in a functional transgene and improve its long-term persistence. EXAMPLES

[0253] Modified FVIIIXTEN expression cassette The V2.0 FVIIIXTEN expression cassette contains the mTTR promoter and enhancer elements (see FIG. 1). However, this promoter is mouse liver specific and has not been thoroughly studied or characterized to determine liver specificity in large animal models or human patients. Therefore, in this study, the V3.0 FVIIIXTEN expression cassette (SEQ ID NO: 35) was created by replacing the mTTR promoter and enhancer elements with the human liver specific alpha 1 antitrypsin (A1AT) promoter (SEQ ID NO: 36) in the V2.0 expression cassette (FIG. 1). EXAMPLES

[0254] In vivo efficacy of FVIIIXTEN HBoV1 mTTR ssDNA versus FVIIIXTEN HBoV1 A1AT ssDNA To verify the functionality of the mTTR promoter versus the A1AT promoter in vivo, single-stranded DNA (ssDNA) constructs containing codon-optimized human FVIIIXTEN with preformed HBoV1 ITRs (ssFVIIIXTEN) were generated as depicted in FIG. 6A. ssFVIIIXTEN with preformed HBoV1 ITRs was generated by denaturing the double-stranded DNA (dsDNA) fragment products (mTTR expression cassette or A1AT FVIII expression cassette and plasmid backbone) of PmlI digestion at 95° C., then cooling at 4° C. to allow the palindromic ITR sequences to fold. The resulting ssFVIIIXTEN was verified by 0.8-1.2% agarose gel electrophoresis. Gel analysis showed half the size of dsDNA for ssFVIIIXTEN, suggesting efficient hairpin formation (FIG. 6B).

[0255] ssFVIIIXTEN was injected systemically into hFVIIIR593C+ / + / HemA mice at 10 μg per mouse via fluid tail vein injection. Plasma samples were collected from injected mice at 7-day intervals over a period of 5.5 months. Plasma FVIII activity was measured by Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0256] Plasma FVIII activity normalized to percent of normal for animals injected with ssFVIIIXTEN is shown in Figure 6C. These results showed comparable levels of FVIII expression up to 21 days post-injection, suggesting that there is no significant difference in FVIIIXTEN levels expressed by the mTTR or A1AT promoters in the hFVIIIR593C+ / + / HemA mouse animal model. EXAMPLES

[0257] In vivo efficacy of FVIIIXTEN AAV2 full-length ceDNA versus truncated FVIIIXTEN AAV2 ceDNA It is known that adeno-associated virus (AAV) vectors can generate different replication forms (e.g., monomer, dimer, or multimer) of viral genome through ITR-ITR concatamerization.The present inventors have previously observed that a closed-end DNA (ceDNA) vector that contains V2.0 codon-optimized FVIIIXTEN (ceFVIIIXTEN) flanked by AAV2 WT ITRs generates truncated molecular species of ceFVIIIXTEN along with monomeric and multimeric forms of vector genome in baculovirus system.See, for example, International Application No. PCT / US21 / 47218.

[0258] In this study, to further explore the properties of the truncated species of ceFVIIIXTEN, we purified both full-length and truncated species of ceFVIIIXTEN by continuous elution electrophoresis as described in International Application No. PCT / US21 / 47218. The purity of both species of ceFVIIIXTEN was determined by agarose gel electrophoresis, and the results showed major bands corresponding to the sizes of the full-length (8.3 kb) and truncated (6.0 kb) species of ceFVIIIXTEN (Figure 7A).

[0259] To further verify the nucleotide sequence of both species of ceFVIIIXTEN, we performed next-generation sequencing (NGS) analysis on the purified ceFVIIIXTEN material using a MiSeq Illumina Sequence Analyzer. The NGS results shown in Figure 7B showed >80% coverage for full-length ceFVIIIXTEN sequence reads (upper panel) and >75% coverage for truncated ceFVIIIXTEN species (lower panel), with some impurities from host cell and / or baculovirus genomes. Further analysis of the NGS data revealed that the truncated ceFVIIIXTEN reads lost most of the chimeric intron region while retaining the ITR sequence at the 5' end of ceFVIIIXTEN (Figure 7B, lower panel).

[0260] To further verify the functionality of the truncated species of ceFVIIIXTEN, purified full-length or truncated species of ceFVIIIXTEN were administered to hFVIIIR593C cells via hydrodynamic tail vein injection. + / + ceFVIIIXTEN was injected systemically at 40 or 80 μg / kg in / HemA mice. Plasma samples were collected from the injected mice at 7-day intervals and plasma FVIII activity was measured by Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions. Plasma FVIII activity, normalized to percent of normal, for animals injected with ceFVIIIXTEN is shown in FIG. 7C.

[0261] Results showed supra-physiologic levels of FVIII expression in the full-length ceFVIIIXTEN-injected cohort. However, animals injected with truncated ceFVIIIXTEN showed half-fold lower FVIII expression at all doses tested up to 21 days post-injection (FIG. 7C). This data further supports the contribution of the chimeric intron to improved expression levels of V2.0 codon-optimized FVIIIXTEN in vivo (FIG. 7C). EXAMPLES

[0262] Efficacy of closed-end FVIIIXTEN (ceFVIIIXTEN) DNA in vivo In this study, we explored the in vivo efficacy of ceDNA encoding modified FVIIIXTEN and flanked by AAV2 ITR or HBoV1 ITR. ceFVIIIXTEN DNA was produced in baculovirus system using AAV2 ITR or HBoV1 ITR as previously described (see, for example, International Application No. PCT / US21 / 47218). The agarose gel used to analyze the purity of each ceDNA compared to the starting material (SM) is shown in Figure 8A.

[0263] Administer purified ceFVIIIXTEN and hFVIIIR593C via hydrodynamic tail vein injection. + / + In / HemA mice, 1.0 or 2.0 μg per mouse were injected systemically, which is equivalent to 40 or 80 μg / kg, respectively. Plasma samples from the injected mice were collected at intervals and FVIII activity was measured by the chromogenic assay described above.

[0264] Plasma FVIII activity, normalized to percent of normal, for animals injected with ceFVIIIXTEN is shown in FIG. 8B.

[0265] Results showed comparable FVIII expression levels for ceDNA vectors flanked by AAV2 ITRs or HBoV1 ITRs. As previously seen, FVIII expression levels gradually declined in treated animals up to day 256, suggesting vector loss in hepatocytes over time. These studies validate the functionality and long-term persistence of modified V2.0 FVIIIXTEN expressed from ceDNA vectors containing AAV2 ITRs or HBoV1 ITRs.

[0266] The foregoing description of specific embodiments will fully reveal the general nature of the present disclosure such that other practitioners, by applying knowledge within the art, may readily modify and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept of the present disclosure. It is therefore to be understood that such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. The phraseology or terminology in this specification is intended to be descriptive and not limiting, as the terminology or terminology in this specification will be interpreted by those skilled in the art in light of the teaching and guidance.

[0267] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

[0268] All patents and publications cited herein are hereby incorporated by reference in their entirety.

[0269] array

[0270] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO:9, wherein the nucleotide sequence encodes a polypeptide with factor VIII (FVIII) activity.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to nucleotides 58 to 4824 of SEQ ID NO:

9.

3. a. the nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide; b. the nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence of SEQ ID NO: 11; and / or c) The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is codon-optimized to contain fewer CpG motifs compared to SEQ ID NO:

32.

4. An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 85% identical to SEQ ID NO: 33, wherein the nucleotide sequence encodes a polypeptide with factor VIII (FVIII) activity.

5. a. enhancer elements; b. intron sequences; c. post-transcriptional regulatory elements; and / or d. The first and second inverted terminal repeats (ITRs) flanking the gene cassette The isolated nucleic acid molecule of any one of claims 1 to 4, further comprising:

6. a. the enhancer element is the A1MB2 enhancer element; b. the enhancer element comprises the nucleotide sequence of SEQ ID NO: 15; c. the intron sequence is a chimeric intron, a hybrid intron, or a synthetic intron; d. the intron sequence comprises the nucleotide sequence of SEQ ID NO: 17; and / or e. The isolated nucleic acid molecule of claim 5, wherein the post-transcriptional regulatory element comprises a woodchuck post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence of SEQ ID NO:

18.

7. 6. The isolated nucleic acid molecule of claim 5, wherein the first ITR and / or the second ITR are derived from a member of the Parvoviridae family, and optionally the first ITR and / or the second ITR are derived from human bocavirus (HBoV1), human erythrovirus (B19), goose parvovirus (GPV), or a variant thereof.

8. 6. The isolated nucleic acid molecule of claim 5, wherein the first ITR and / or the second ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO: 1, 2, or 21-30.

9. 6. The isolated nucleic acid molecule of claim 5, wherein the first ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:1, and the second ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:

2.

10. (a) the A1MB2 enhancer element comprises the nucleotide sequence of SEQ ID NO: 15; (b) a liver-specific modified mouse transthyretin (mTTR) promoter comprising the nucleotide sequence of SEQ ID NO: 16; (c) the chimeric intron comprises the nucleotide sequence of SEQ ID NO: 17; (d) the nucleotide sequence encoding the FVIII protein comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO:9 or SEQ ID NO:33; (e) the woodchuck post-transcriptional regulatory element (WPRE) comprises the nucleotide sequence of SEQ ID NO: 18; and 7. The isolated nucleic acid molecule of claim 6, wherein (f) the bovine growth hormone polyadenylation (bGHpA) signal comprises the nucleotide sequence of SEQ ID NO:

19.

11. A vector comprising the nucleic acid molecule of claim 1.

12. A host cell comprising the nucleic acid molecule of claim 1.

13. A polypeptide produced by the host cell of claim 12.

14. A baculovirus system for producing the nucleic acid molecule of claim 1, which is produced in insect cells.

15. A method for producing a polypeptide with FVIII activity, comprising culturing a host cell described in claim 12 under conditions in which a polypeptide with FVIII activity is produced, and recovering the polypeptide with FVIII activity.

16. A pharmaceutical composition comprising the nucleic acid molecule of claim 1.

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