Nucleic Acid Molecules and Their Use for Non-Viral Gene Therapy - Patent application

Abstract

The present disclosure provides a nucleic acid molecule comprising a first inverted terminal repeat (ITR), a second ITR, and a gene cassette encoding a target sequence. In some embodiments, the target sequence encodes an miRNA and / or a therapeutic protein. In certain embodiments, the therapeutic protein comprises a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, and a combination thereof. In some embodiments, the first ITR and / or the second ITR is an ITR of a non-adeno-associated virus (AAV). The present disclosure also provides a method for treating a liver metabolic disorder in a subject, comprising administering the nucleic acid molecule or a polypeptide encoded thereby.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 716,826, filed August 9, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the Sequence Listing submitted electronically as an ASCII text file (Name: SA9-465PC_SL_ST25.txt; Size: 460,648 bytes; and Creation Date: August 8, 2019) are incorporated by reference herein in their entirety. [Background technology]

[0003] Background of the disclosure Gene therapy offers the possibility of a long-lasting means of treating various diseases. In the past, many gene therapy treatments have typically relied on the use of viruses. There are many viral agents that can be selected for this purpose, each with individual properties that are more or less suitable for gene therapy. Non-Patent Document 1. However, the undesirable properties of some viral vectors, including their immunogenic profile or their tendency to cause cancer, have raised clinical safety concerns, and until recently, their clinical use has been limited to certain applications, such as vaccines and oncolytic strategies. Non-Patent Document 2.

[0004] Adeno-associated virus (AAV) is one of the most widely studied gene therapy vectors. AAV is a protein shell that surrounds and protects a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). Non-patent literature 3. AAV belongs to the parvovirus family and depends on co-infection with other viruses, primarily adenoviruses, to replicate. Ibid. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid), and aap (association). Ibid. These coding sequences are flanked by inverted terminal repeats (ITRs) that are necessary for genome replication and packaging. Ibid. The two cis-acting AAV ITRs are approximately 145 nucleotides long and are interrupted by a batch structure sequence that can fold into a T-shaped hairpin structure that functions as a primer during the initiation of DNA replication.

[0005] However, the use of conventional AAV as a gene delivery vector has certain drawbacks. One of the main drawbacks is associated with AAV's limited viral packaging capacity of about 4.5 kb of heterologous DNA. (Non-Patent Document 4). In addition, administration of AAV vectors can induce immune responses in humans. Although AAV has been shown to be less immunogenic than some other viruses (i.e., adenovirus), the capsid protein can elicit various components of the human immune system. See Naso et al., 2017. AAV is a common virus in the human population, and most people have been exposed to AAV, and therefore most people have already developed an immune response against the specific variants to which they have been previously exposed. Pre-existing adaptive responses may include neutralizing antibodies (NAbs) and T cells, which may reduce the clinical efficacy of subsequent reinfection of AAV and / or clearance of transduced cells, which may render patients with pre-existing anti-AAV immunity ineligible for AAV-based gene therapy treatment. Furthermore, evidence suggests that the T-shaped hairpin loop of the AAV ITR is susceptible to inhibition by host cell proteins / protein complexes that bind to the T-shaped hairpin structure of the AAV ITR. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Zhou et al., Adv Drug Deliv Rev.106(Pt A):3-26, 2016 [Non-Patent Document 2] Cotter et al., Front Biosci. 10:1098-105 (2005) [Non-Patent Document 3] Naso et al., BioDrugs, 31(4): 317-334, 2017 [Non-Patent Document 4] Dong et al., Hum Gene Ther. 7(17): 2101-12, 1996 [Non-Patent Document 5] Zhou et al., Scientific Reports 7:5432 (July 14, 2017) Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need in the art to efficiently and sustainably express target sequences, such as therapeutic proteins and / or miRNAs, in in vitro and in vivo settings while avoiding some of the unintended consequences and limitations of existing AAV vector technology. [Means for solving the problem]

[0008] Summary of disclosure In certain embodiments, a nucleic acid molecule is provided comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0009] In certain exemplary embodiments, the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 180 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 181. In certain exemplary embodiments, the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 183 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 184. In certain exemplary embodiments, the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 185 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 186. In certain exemplary embodiments, the first ITR comprises the nucleotide sequence set forth in SEQ ID NO: 187 and the second ITR comprises the nucleotide sequence set forth in SEQ ID NO: 188.

[0010] In certain exemplary embodiments, the first ITR and / or the second ITR consists of the nucleotide sequence set forth in SEQ ID NO: 180, 181, 183, 184, 185, 186, 187 or 188. In certain exemplary embodiments, the first ITR and the second ITR are the reverse complement of each other.

[0011] In certain exemplary embodiments, the nucleic acid molecule further comprises a promoter. In certain exemplary embodiments, the promoter is a tissue-specific promoter. In certain exemplary embodiments, the promoter drives expression of the heterologous polynucleotide sequence in an organ selected from muscle, central nervous system (CNS), eye, liver, heart, kidney, pancreas, lung, skin, bladder, urinary tract, or any combination thereof. In certain exemplary embodiments, the promoter drives expression of the heterologous polynucleotide sequence in hepatocytes, endothelial cells, cardiac myocytes, skeletal muscle cells, sinusoidal cells, afferent nerves, efferent nerves, interneurons, glial cells, astrocytes, oligodendrocytes, microglia, ependymal cells, lung epithelial cells, Schwann cells, satellite cells, photoreceptor cells, retinal ganglion cells, or any combination thereof. In certain exemplary embodiments, the promoter is located 5' to the heterologous polynucleotide sequence. In certain exemplary embodiments, the promoter is selected from the group consisting of mouse thyretin promoter (mTTR), endogenous human factor VIII promoter (F8), human alpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, mouse albumin promoter, tristetraprolin (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, alpha 1-antitrypsin (AAT), muscle creatine kinase (MCK), myosin heavy chain alpha (αMHC), myoglobin (MB), desmin (DES), SPc5-12, 2R5Sc5-12, dMCK, tMCK, and phosphoglycerate kinase (PGK) promoter.

[0012] In certain exemplary embodiments, the heterologous polynucleotide sequence further comprises an intron sequence. In certain exemplary embodiments, the intron sequence is located 5' to the heterologous polynucleotide sequence. In certain exemplary embodiments, the intron sequence is located 3' to the promoter. In certain exemplary embodiments, the intron sequence comprises a synthetic intron sequence. In certain exemplary embodiments, the intron sequence comprises SEQ ID NO: 115 or 192.

[0013] In certain exemplary embodiments, the gene cassette further comprises a post-transcriptional regulatory element. In certain exemplary embodiments, the post-transcriptional regulatory element is located 3' to the heterologous polynucleotide sequence. In certain exemplary embodiments, the post-transcriptional regulatory element comprises a mutant Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), a microRNA binding site, a DNA nuclear targeting sequence, or any combination thereof. In certain exemplary embodiments, the microRNA binding site comprises a binding site for miR142-3p.

[0014] In certain exemplary embodiments, the gene cassette further comprises a 3'UTR poly(A) tail sequence. In certain exemplary embodiments, the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In certain exemplary embodiments, the 3'UTR poly(A) tail sequence comprises bGH poly(A).

[0015] In certain exemplary embodiments, the gene cassette further comprises an enhancer sequence, hi certain exemplary embodiments, the enhancer sequence is located between the first and second ITRs.

[0016] In certain exemplary embodiments, the nucleic acid molecule comprises, from 5' to 3': a first ITR, a gene cassette, and a second ITR; wherein the gene cassette comprises a tissue-specific promoter sequence, an intron sequence, a heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3'UTR poly(A) tail sequence. In certain exemplary embodiments, the gene cassette comprises, from 5' to 3': a tissue-specific promoter sequence, an intron sequence, a heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3'UTR poly(A) tail sequence. In certain exemplary embodiments, the tissue-specific promoter sequence comprises a TTT promoter; the intron is a synthetic intron; the post-transcriptional regulatory element comprises a WPRE, and the 3'UTR poly(A) tail sequence comprises bGHpA.

[0017] In certain exemplary embodiments, the gene cassette comprises a single stranded nucleic acid. In certain exemplary embodiments, the gene cassette comprises a double stranded nucleic acid.

[0018] In certain exemplary embodiments, the heterologous polynucleotide sequence comprises a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or a combination thereof.

[0019] In certain exemplary embodiments, the heterologous polynucleotide sequence is an antibody that encodes a polypeptide that is capable of binding to ... Glomerulonephritis stimulating factor (GM-CSF)), epidermal growth factor (EGF), ephrins (e.g., ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3), erythropoietin (EPO), fibroblast growth factors (FGFs) (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FG F22, FGF23), fetal bovine somatotropin (FBS), GDNF family members (e.g., glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma cell-derived growth factor (HDGF), insulin, insulin-like growth factor (e.g., insulin-like growth factor-1 (IGF-1) or IGF-2), interleukin (IL) (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7), keratinocyte growth factor (K GF), migration stimulating factor (MSF), macrophage stimulating protein (MSP or hepatocyte growth factor-like protein (HGFLP)), myostatin (GDF-8), neuregulins (e.g., neuregulin 1 (NRG1), NRG2, NRG3, NRG4), neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4, placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T cell growth factor (TCGF), thrombopoietin (TPO),Transforming growth factors (e.g., growth factors selected from the group consisting of transforming growth factor alpha (TGF-α), TGF-β, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), and any combination thereof.

[0020] In certain exemplary embodiments, the heterologous polynucleotide sequence encodes a hormone.

[0021] In certain exemplary embodiments, the heterologous polynucleotide sequence encodes a cytokine.

[0022] In certain exemplary embodiments, the heterologous polynucleotide sequence encodes an antibody or a fragment thereof.

[0023] In certain exemplary embodiments, the heterologous polynucleotide sequence encodes a gene selected from X-linked dystrophin, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha glucosidase), and any combination thereof.

[0024] In certain exemplary embodiments, the heterologous polynucleotide sequence encodes a microRNA (miRNA). In certain exemplary embodiments, the miRNA downregulates expression of a target gene selected from SOD1, HTT, RHO, and any combination thereof.

[0025] In certain exemplary embodiments, the heterologous polynucleotide sequence is selected from the group consisting of factor I (FI), factor II (FII), factor III (FIII), factor IV (FVI), factor V (FV), factor VI (FVI), factor VII (FVII), factor VIII (FVIII), factor IX (FIX), factor X (FX), factor XI (FXI), factor XII (FXII), factor XIII (FVIII), von Willebrand factor (VWF), prekallikrein, high molecular weight kininocytes (HKGs), and the like. The coagulation factors encoded by the antibodies are selected from the group consisting of plasminogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), and any combination thereof.

[0026] In certain exemplary embodiments, the clotting factor is FVIII. In certain exemplary embodiments, the FVIII is full-length mature FVIII. In certain exemplary embodiments, the FVIII comprises an amino acid sequence that is at least about 70%, 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 an amino acid sequence having SEQ ID NO:106.

[0027] In certain exemplary embodiments, FVIII comprises the A1 domain, the A2 domain, the A3 domain, the C1 domain, the C2 domain, and a partial B domain or no B domain. In certain exemplary embodiments, FVIII comprises an amino acid sequence that is at least about 70%, 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 amino acid sequence of SEQ ID NO:109.

[0028] In certain exemplary embodiments, the coagulation factor comprises a heterologous moiety. In certain exemplary embodiments, the heterologous moiety is selected from the group consisting of albumin or a fragment thereof, an immunoglobulin Fc region, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, an albumin binding moiety, a derivative thereof, or any combination thereof. In certain exemplary embodiments, the heterologous moiety is linked to the N-terminus or C-terminus of FVIII or inserted between two amino acids in FVIII. In certain exemplary embodiments, the heterologous moiety is inserted between two amino acids at one or more insertion sites selected from the insertion sites listed in Table 4.

[0029] In certain exemplary embodiments, the FVIII further comprises the A1 domain, the A2 domain, the C1 domain, the C2 domain, an optional B domain, and a heterologous moiety, wherein the heterologous moiety is inserted immediately downstream of amino acid 745, which corresponds to mature FVIII (SEQ ID NO: 106).

[0030] In certain exemplary embodiments, FVIII further comprises an FcRn binding partner. In certain exemplary embodiments, the FcRn binding partner comprises the Fc region of an immunoglobulin constant domain.

[0031] In certain exemplary embodiments, the nucleic acid sequence encoding FVIII is codon-optimized. In certain exemplary embodiments, the nucleic acid sequence encoding FVIII is codon-optimized for expression in humans.

[0032] In certain exemplary embodiments, the nucleic acid sequence encoding FVIII comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, 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 about 100% identical to the nucleotide sequence of SEQ ID NO:107.

[0033] In certain exemplary embodiments, the nucleic acid sequence encoding FVIII comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, 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 about 100% identical to the nucleotide sequence of SEQ ID NO:71.

[0034] In certain exemplary embodiments, the heterologous polynucleotide sequence is codon-optimized. In certain exemplary embodiments, the heterologous polynucleotide sequence is codon-optimized for expression in humans.

[0035] In certain exemplary embodiments, the nucleic acid molecule is formulated with a delivery agent. In certain exemplary embodiments, the delivery agent comprises a lipid nanoparticle. In certain exemplary embodiments, the delivery agent is selected from the group consisting of a liposome, a non-lipid polymer molecule, and an endosome, and any combination thereof.

[0036] In certain exemplary embodiments, the nucleic acid molecule is formulated for intravenous, transdermal, intradermal, subcutaneous, pulmonary, or oral delivery, or any combination thereof, hi certain exemplary embodiments, the nucleic acid molecule is formulated for intravenous delivery.

[0037] In certain embodiments, a vector is provided that comprises a nucleic acid molecule as described herein.

[0038] In certain embodiments, a host cell is provided that comprises a nucleic acid molecule as described herein.

[0039] In certain aspects, there is provided a pharmaceutical composition comprising a nucleic acid molecule or vector as described herein and a pharma- ceutically acceptable excipient.

[0040] In certain aspects, a pharmaceutical composition is provided comprising a host cell as described herein and a pharma- ceutically acceptable excipient.

[0041] In certain embodiments, kits are provided that contain a nucleic acid molecule as described herein and instructions for administering the nucleic acid molecule to a subject in need thereof.

[0042] In certain embodiments, a baculovirus system for the production of the nucleic acid molecules as described herein is provided.

[0043] In certain exemplary embodiments, the nucleic acid molecules as described herein are produced in insect cells.

[0044] In certain embodiments, there is provided a nanoparticle delivery system comprising a nucleic acid molecule as described herein.

[0045] In certain aspects, there is provided a method for producing a polypeptide comprising culturing a host cell as described herein under suitable conditions and recovering the polypeptide.

[0046] In certain aspects, methods are provided for producing a polypeptide having clotting activity, comprising culturing a host cell as described herein under suitable conditions and recovering the polypeptide having clotting activity.

[0047] In certain aspects, there is provided a method for expressing a heterologous polynucleotide sequence in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule as described herein, a vector described herein, or a pharmaceutical composition described herein.

[0048] In certain aspects, a method of expressing a coagulation factor in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule as described herein, a vector described herein, a polypeptide described herein, or a pharmaceutical composition described herein.

[0049] In certain aspects, a method of treating a disease or disorder in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule as described herein, a vector described herein, or a pharmaceutical composition described herein.

[0050] In certain aspects, a method of treating a subject having a coagulation factor deficiency is provided, the method comprising administering to the subject a nucleic acid molecule as described herein, a vector described herein, a polypeptide described herein, or a pharmaceutical composition described herein.

[0051] In certain aspects, there is provided a method of treating a coagulation factor deficiency in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule as described herein, a vector described herein, a polypeptide described herein, or a pharmaceutical composition described herein.

[0052] In certain exemplary embodiments, the nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonary, or any combination thereof, hi certain exemplary embodiments, the nucleic acid molecule is administered intravenously.

[0053] In certain exemplary embodiments, the method further comprises administering a second agent to the subject.

[0054] In certain exemplary embodiments, the subject is a mammal, hi certain exemplary embodiments, the subject is a human.

[0055] In certain exemplary embodiments, administration of a nucleic acid molecule to a subject results in increased FVIII activity compared to FVIII activity in the subject prior to administration, where FVIII activity is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold.

[0056] In certain exemplary embodiments, the subject has a bleeding disorder. In certain exemplary embodiments, the bleeding disorder is hemophilia. In certain exemplary embodiments, the bleeding disorder is hemophilia A.

[0057] In certain embodiments, a method of treating a bleeding disorder in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding a coagulation factor, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0058] In certain embodiments, a method of treating hemophilia A in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding factor VIII (FVIII), 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0059] In certain embodiments, a method of treating a liver metabolic disorder in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding a liver-related metabolic enzyme that is deficient in the subject, wherein the first ITR and / or the second ITR are non-adeno-associated virus (non-AAV) ITRs.

[0060] In certain exemplary embodiments, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0061] In certain embodiments, a method of treating a liver metabolic disorder in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding a liver-related metabolic enzyme deficient in the subject, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0062] In certain exemplary embodiments, the gene cassette comprises a single stranded nucleic acid. In certain exemplary embodiments, the gene cassette comprises a double stranded nucleic acid.

[0063] In certain exemplary embodiments, the hepatic metabolic disorder is selected from the group consisting of phenylketonuria (PKU), urea cycle disorders, lysosomal storage disorders, and glycogen storage diseases. In certain exemplary embodiments, the hepatic metabolic disorder is phenylketonuria (PKU).

[0064] In certain exemplary embodiments, the nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonary, or any combination thereof, hi certain exemplary embodiments, the nucleic acid molecule is administered intravenously.

[0065] In certain exemplary embodiments, the method further comprises administering a second agent to the subject.

[0066] In certain exemplary embodiments, the subject is a mammal, hi certain exemplary embodiments, the subject is a human.

[0067] In certain embodiments, a method of treating phenylketonuria (PKU) in a subject in need thereof is provided, the method comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding phenylalanine hydroxylase, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0068] In certain exemplary embodiments, the gene cassette comprises a single stranded nucleic acid. In certain exemplary embodiments, the gene cassette comprises a double stranded nucleic acid.

[0069] In certain exemplary embodiments, the nucleic acid molecule is formulated with a delivery agent. In certain exemplary embodiments, the delivery agent comprises a lipid nanoparticle.

[0070] In certain embodiments, a method for cloning a nucleic acid molecule is provided, comprising inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into a bacterial host strain that contains a disruption in the SbcCD complex.

[0071] In certain exemplary embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene and / or the SbcD gene. In certain exemplary embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene. In certain exemplary embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcD gene.

[0072] In certain exemplary embodiments, the nucleic acid molecule comprises a first inverted terminal repeat (ITR) and a second ITR, where the first and / or second ITR are non-adeno-associated virus (non-AAV) ITRs.

[0073] In certain exemplary embodiments, 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 SEQ ID NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0074] In certain exemplary embodiments, the nucleic acid molecule further comprises a gene cassette, the gene cassette being flanked by a first ITR and a second ITR.

[0075] In certain exemplary embodiments, the gene cassette comprises a heterologous polynucleotide sequence.

[0076] In certain exemplary embodiments, a suitable vector is a low copy vector. In certain exemplary embodiments, a suitable vector is pBR322.

[0077] In certain exemplary embodiments, the bacterial host strain is unable to degrade cruciform DNA structures.

[0078] In certain exemplary embodiments, the bacterial host strain is PMC103 with the genotype sbcC, recD, mcrA, ΔmcrBCF. In certain exemplary embodiments, the bacterial host strain is PMC107 with the genotype recBC, recJ, sbcBC, mcrA, ΔmcrBCF. In certain exemplary embodiments, the bacterial host strain is SURE with the genotype recB, recJ, sbcC, mcrA, ΔmcrBCF, umuC, uvrC.

[0079] In certain embodiments, a method for cloning a nucleic acid molecule is provided, the method comprising inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into a bacterial host strain comprising a disruption in the SbcCD complex, wherein the nucleic acid molecule comprises a first inverted terminal repeat (ITR) and a second ITR, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof. [Brief description of the drawings]

[0080] [Figure 1-1] 1A-1B are schematic diagrams of single chain coagulation factor (e.g., FVIII) expression cassettes. The positions of the non-AAV derived 5'ITR (with hairpin loop at the end of the ssDNA structure), the non-AAV derived 3'ITR (with hairpin loop), the promoter sequence (e.g., TTPp or CAGp), and the transgene sequence, e.g., FVIIIco6XTEN sequence with XTEN144 inserted in the B domain, are shown. The exemplary expression cassette also shows possible additional elements, e.g., intron sequences, WPREmut sequences, and bGHpA sequences. [Figure 1-2]Figures 1C-1F are schematic diagrams of plasmids used to produce single-chain coagulation factor expression cassettes such as those shown in Figures 1A-1B, where the ITRs of the cassette are derived from AAV2 (Figure 1C), B19 (Figure 1D), GPV (Figure 1E), or the wild-type B19 ITR sequences (Figure 1F). Plasmid constructs containing ssFVIII expression cassettes as shown here were digested with PvuII (at the PvuII site) (Figure 1C) or LguI (at the LguI site) (Figures 1D-1F) to precisely release the ITRs and sequences comprising the expression cassette. The double-stranded DNA was heat denatured at 95°C to generate ssDNA, which was then incubated at 4°C to allow the ITR structures to form. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 2A] Figure 2A is a phylogenetic tree illustrating the relationships among the various parvovirus family members, with B19, AAV-2, and GPV indicated by boxes. [Figure 2B] FIG. 2B is a schematic diagram of various cassettes containing hairpin structures. [Figure 3A] Figures 3A and 3B are alignments of the ITRs of B19, GPV, and AAV2 (Figure 3A) and B19 and GPV (Figure 3B). Grey shading indicates homology. [Figure 3B] Figures 3A and 3B are alignments of the ITRs of B19, GPV, and AAV2 (Figure 3A) and B19 and GPV (Figure 3B). Grey shading indicates homology. [Figure 4]Figures 4A-4C show FVIII plasma activity following administration of single-stranded FVIII-AAV naked DNA (ssAAV-FVIII; Figure 1C), ssDNA-B19 FVIII (Figure 1D), or ssDNA-GPV FVIII (Figure 1E) by hydrodynamic injection (HDI) in Hem A mice. FVIII activity in mice treated with a single HDI of ssDNA at 50 μg / mouse (Figure 4C), 20 μg / mouse (Figures 4A and 4B), 10 μg / mouse (Figures 4A, 4B, and 4C), or 5 μg / mouse (Figure 4A) was measured in plasma samples at 24 hours, 3 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months (as a percentage of normal physiological levels in humans). HDI of 5 μg plasmid DNA / mouse was administered as a control (FIGS. 4A, 4B, and 4C). [Diagram 5] FIG. 5 shows FVIII activity in hemophilia A mouse plasma after a single hydrodynamic injection of equimolar amounts of single-stranded naked DNA (ssAAV-FVIII, FIG. 1A), double-stranded AAV-FVIII DNA containing ITR sequences (dsDNA), double-stranded FVIII DNA without ITRs (dsDNA No ITR), or circular double-stranded FVIII DNA without ITR or bacterial sequences (minicircle). dsDNA was generated by enzymatic cleavage of the AAV-FVIII plasmid (FIG. 2C) with PvuII but without heat denaturation. dsDNA No ITR was generated by enzymatic cleavage of the AAV-FVIII plasmid (FIG. 2C) with AflII and subsequently purified. Minicircle DNA was generated by ligation of the AflII site of dsDNA No ITR DNA. Mouse plasma was collected over a period of 3 or 4 months, and FVIII was determined by a chromogenic activity assay. [Figure 6]Figure 6 shows FVIII activity in hemophilia A mouse plasma after hydrodynamic injection of 30 μg of single-stranded naked FVIII-DNA (Figure 1A, Figures 1D-1F). Plasma was collected weekly for 7 weeks, and FVIII activity was determined by chromogenic assay. After 35 days (indicated as black arrows), mice that received FVIII-B19d135 and FVIII-GPVd162 ssDNA were rechallenged with 30 μg by hydrodynamic injection. [Figure 7-1] FIG. 7A is a schematic diagram of a single-stranded mouse phenylalanine hydroxylase (e.g., PAH) expression cassette. The locations of the non-AAV-derived 5'ITR (with hairpin loop at the end of the ssDNA structure), the non-AAV-derived 3'ITR (with hairpin loop), the promoter sequence (e.g., CAGp), and the transgene sequence, e.g., the 3xFLAG_mPAH sequence, are shown. The exemplary expression cassette also shows additional possible elements, e.g., the WPREmut sequence, and the bGHpA sequence. FIG. 7B-7D show plasma concentrations of phenylalanine (Phe) in phenylketonuric (PKU) mice before (day 0) and after a single dose of single-stranded DNA containing mouse PAH cDNA and the non-AAV ITR B19d135 or GPVd162 by hydrodynamic injection. Plasma was collected 3, 7, 14, 28, 42, and 56 days after ssDNA administration. Residual phenylalanine levels are shown as concentration in μg / ml (FIGS. 7B-7C) or as a percent of pre-dose (FIG. 7D). The horizontal line indicates baseline Phe levels before dosing. FIG. 7E shows Western immunoblots of liver lysates from PKU mice treated with a mouse PAH transgene and ssDNA containing either the B19d135 or GPVd165 ITRs. Livers were collected 81 days after treatment and protein lysates were extracted. Each well represents a single animal. FLAG-tagged mouse PAH protein was detected using M2 anti-FLAG antibody and a GAPDH loading control was included for comparison. [Figure 7-2] Continued from Figure 7-1. [Figure 8]Figures 8A-B show FVIII activity levels in Huh7 cell supernatants after transduction with lipid nanoparticles encapsulating FVIII-AAV DNA (Figures 1A-1C). Plasmid FVIII-AAV under the CAGp promoter (Figure 1B) was encapsulated at three amine-to-phosphate (NP) ratios and applied to Huh7 cells at various concentrations determined by picogreen assay (Figure 8A). Plasmids, double-stranded linear (ds) and single-stranded (ss) AAV-FVIII under the TTPp promoter (Figure 1A) were also encapsulated in lipid nanoparticles at two NP ratios and used at various DNA concentrations to transduce Huh7 cells (Figure 8B). FVIII was measured by a chromogenic activity assay compared to human FACT plasma standards. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Detailed Description of the Disclosure The present disclosure describes a plasmid-like nucleic acid molecule comprising a first inverted terminal repeat (ITR), a second ITR, and, for example, a gene cassette encoding a target sequence (also referred to herein as a heterologous polynucleotide sequence), for example, a therapeutic protein or miRNA, where the first ITR and / or the second ITR are non-adeno-associated virus ITRs (e.g., the first ITR and / or the second ITR are from a non-AAV). In some embodiments, the gene cassette encodes a therapeutic protein, for example, the target sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a protein selected from a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or a combination thereof. In some embodiments, the gene cassette encodes dystrophin X-linked, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha glucosidase), or any combination thereof.

[0082] In some embodiments, the therapeutic protein comprises a clotting factor, hi one particular embodiment, the therapeutic protein comprises a FVIII or FIX protein.

[0083] In some embodiments, the gene cassette encodes a miRNA. In certain embodiments, the miRNA downregulates expression of a target gene selected from SOD1, HTT, RHO, or any combination thereof.

[0084] In certain embodiments, the non-AAV is selected from the group consisting of members of the virus family Parvoviridae and any combination thereof. The present disclosure further relates to a method of expressing a therapeutic protein, e.g., a clotting factor, e.g., FVIII, in a subject in need thereof, comprising administering to the subject a nucleic acid molecule comprising a first inverted terminal repeat (ITR), a second ITR, and a gene cassette encoding, e.g., a therapeutic protein or miRNA, wherein the first ITR and / or the second ITR are ITRs of a non-adeno-associated virus (non-AAV). In certain embodiments, the present disclosure describes an isolated nucleic acid molecule comprising a nucleotide sequence having sequence homology to a nucleotide sequence selected from SEQ ID NOs: 113 and 120.

[0085] In certain embodiments, the present disclosure provides a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence, wherein the first and / or second ITR are derived from parvovirus B19 or goose parvovirus (GPV).

[0086] Exemplary constructs of the present disclosure are illustrated in the accompanying figures and sequence listing. In order to provide a clear understanding of the specification and claims, the following definitions are set forth below.

[0087] I. Definition It should be noted that the term "a" or "an" entity refers to one or more of that entity: for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Similarly, "a therapeutic protein" and "a miRNA" should be understood to represent one or more therapeutic proteins and one or more miRNAs, respectively. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0088] The term "about" is used herein to mean approximately, roughly, in the region of, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical range set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up and down (higher or lower).

[0089] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as no combinations when interpreted in the alternative ("or").

[0090] "Nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to ribonucleotides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), either in single-stranded form, or as a double-stranded helix, or the phosphate ester polymeric forms of any of the phosphate ester analogs thereof, such as phosphorithioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The terms nucleic acid molecule, and in particular DNA or RNA molecule, refer to the primary and secondary structure of the molecule. The term refers only to double-stranded DNA found in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes, among others. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of only providing the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having the sequence homologous to the 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.

[0091] As used herein, an "inverted terminal repeat sequence" (or "ITR") refers to a nucleic acid sequence located at either the 5' or 3' end of a single stranded nucleic acid sequence that includes a set of nucleotides (initial sequence) followed downstream by its reverse complement, i.e., a batch sequence. The intervening sequence of nucleotides 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 includes one or more "batch sequences." An ITR can have any number of functions. In some embodiments, the ITRs described herein form a hairpin structure. In some embodiments, the ITRs form a T-shaped hairpin structure. In some embodiments, the ITRs form a non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. In some embodiments, the ITRs promote long-term persistence of a nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITRs promote persistent persistence of a nucleic acid molecule in the nucleus of a cell (e.g., throughout the lifespan of a cell). In some embodiments, the ITRs promote safety of a nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITRs promote retention of a nucleic acid molecule in the nucleus of a cell. In some embodiments, the ITRs enhance the persistence of the nucleic acid molecule in the nucleus of the cell, hi some embodiments, the ITRs inhibit or prevent degradation of the nucleic acid molecule in the nucleus of the cell.

[0092] In one embodiment, the initial sequence and / or the reverse complement comprises about 2-600 nucleotides, about 2-550 nucleotides, about 2-500 nucleotides, about 2-450 nucleotides, about 2-400 nucleotides, about 2-350 nucleotides, about 2-300 nucleotides, or about 2-250 nucleotides. In some embodiments, the initial sequence and / or the reverse complement comprises about 5-600 nucleotides, about 10-600 nucleotides, about 15-600 nucleotides, about 20-600 nucleotides, about 25-600 nucleotides, about 30-600 nucleotides, about 35-600 nucleotides, about 40-600 nucleotides, about 45-600 nucleotides, about 50-600 nucleotides, about 60-600 nucleotides, about 70-600 nucleotides, about 80-600 nucleotides, about 90-600 nucleotides, about 100-600 nucleotides, about 150-600 nucleotides, about 200-600 nucleotides, about 300-600 nucleotides, about 350-600 nucleotides, about 400-600 nucleotides, about 450-600 nucleotides, about 500-600 nucleotides, or about 550-600 nucleotides. In some embodiments, the initial sequence and / or the reverse complement comprises about 5-550 nucleotides, about 5-500 nucleotides, about 5-450 nucleotides, about 5-400 nucleotides, about 5-350 nucleotides, about 5-300 nucleotides, or about 5-250 nucleotides. In some embodiments, the initial sequence and / or the reverse complement comprises about 10-550 nucleotides, about 15-500 nucleotides, about 20-450 nucleotides, about 25-400 nucleotides, about 30-350 nucleotides, about 35-300 nucleotides, or about 40-250 nucleotides. In certain embodiments, the initial sequence and / or the reverse complement comprises about 225 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, about 500 nucleotides, about 525 nucleotides, about 550 nucleotides, about 575 nucleotides, or about 600 nucleotides. In certain embodiments, the initial sequence and / or the reverse complement comprises about 400 nucleotides.

[0093] In other embodiments, the initial sequence and / or the reverse complement comprises about 2-200 nucleotides, about 5-200 nucleotides, about 10-200 nucleotides, about 20-200 nucleotides, about 30-200 nucleotides, about 40-200 nucleotides, about 50-200 nucleotides, about 60-200 nucleotides, about 70-200 nucleotides, about 80-200 nucleotides, about 90-200 nucleotides, about 100-200 nucleotides, about 125-200 nucleotides, about 150-200 nucleotides, or about 175-200 nucleotides. In other embodiments, the initial sequence and / or the reverse complement comprises about 2-150 nucleotides, about 5-150 nucleotides, about 10-150 nucleotides, about 20-150 nucleotides, about 30-150 nucleotides, about 40-150 nucleotides, about 50-150 nucleotides, about 75-150 nucleotides, about 100-150 nucleotides, or about 125-150 nucleotides. In other embodiments, the initial sequence and / or the reverse complement comprises about 2-100 nucleotides, about 5-100 nucleotides, about 10-100 nucleotides, about 20-100 nucleotides, about 30-100 nucleotides, about 40-100 nucleotides, about 50-100 nucleotides, or about 75-100 nucleotides. In other embodiments, the initial sequence and / or the reverse complement comprises about 2-50 nucleotides, about 10-50 nucleotides, about 20-50 nucleotides, about 30-50 nucleotides, about 40-50 nucleotides, about 3-30 nucleotides, about 4-20 nucleotides, or about 5-10 nucleotides. In another embodiment, the initial sequence and / or the reverse complement consists of 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides.In other embodiments, the intervening nucleotides between the initial sequence and the reverse complement are (e.g., consist of) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0094] Thus, as used herein, an "ITR" can fold back on itself to form a double-stranded segment. For example, the sequence GATCXXXXGATC, when folded back to form a double helix, contains an initial sequence of GATC and its complementary strand (3'CTAG5'). In some embodiments, an ITR contains a continuous batch sequence (e.g., GATCGATC) between the initial sequence and the reverse complement. In some embodiments, an ITR contains an intervening batch sequence (e.g., GATCXXXXGATC) between the initial sequence and the reverse complement. In some embodiments, the complementary sections of the continuous or interrupted batch sequences interact with each other to form a "hairpin loop" structure. As used herein, a "hairpin loop" structure occurs when at least two complementary sequences on a single-stranded nucleotide molecule base pair to form a double-stranded section. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop.

[0095] In the present disclosure, at least one ITR is a non-adenovirus-associated virus (non-AAV) ITR. In certain embodiments, the ITR is a non-AAV member of the virus family Parvoviridae. In some embodiments, the ITR is a non-AAV member of the Dependovirus or Erythrovirus genus. In certain embodiments, the ITR is a goose parvovirus (GPV), Muscovy duck parvovirus (MDPV), or erythrovirus parvovirus B19 (also known as parvovirus B19, primate erythrovirus parvovirus 1, B19 virus, and erythrovirus). In certain embodiments, one ITR of the two ITRs is an AAV ITR. In other embodiments, one of the two ITRs in the construct is an ITR of an AAV serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and any combination thereof. In one particular embodiment, the ITR is from AAV serotype 2, e.g., is an AAV serotype 2 ITR.

[0096] In certain embodiments of the present disclosure, the nucleic acid molecule comprises two ITRs, 5'ITR and 3'ITR, where the 5'ITR is located at the 5' end of the nucleic acid molecule and the 3'ITR is located at the 3' end of the nucleic acid molecule. The 5'ITR and 3'ITR may be derived from the same virus or different viruses. In certain embodiments, the 5'ITR is from AAV and the 3'ITR is not derived from an AAV virus (e.g., non-AAV). In some embodiments, the 3'ITR is from AAV and the 5'ITR is not derived from an AAV virus (e.g., non-AAV). In other embodiments, the 5'ITR is not derived from an AAV virus (e.g., non-AAV) and the 3'ITR is from the same or different non-AAV virus.

[0097] As used herein, the term "parvovirus" encompasses the family Parvoviridae, which includes, but is not limited to, autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include, for example, members of the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, and Penstyldensovirus.

[0098] Exemplary autonomous parvoviruses include, but are not limited to, porcine parvovirus, minute virus of mice, canine parvovirus, mink enteritis virus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those of skill in the art. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

[0099] As used herein, the term "non-AAV" encompasses nucleic acids, proteins and viruses from the Parvoviridae family except for the adeno-associated viruses (AAV) of the Parvoviridae family. "Non-AAV" includes, but is not limited to, autonomously replicating members of the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, and Penstyldensovirus.

[0100] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and phylogenetic groups disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), as well as other AAVs now known or later discovered. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0101] As used herein, the term "derived from" refers to a component that is isolated from or produced using a particular molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from a particular molecule or organism. For example, a nucleic acid sequence (e.g., ITR) derived from a second nucleic acid sequence (e.g., ITR) may contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of a nucleotide or polypeptide, the derived species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive a nucleotide or polypeptide can be either intentionally directed or intentionally random, or a mixture of each. Mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from the first one can be a random event (e.g., caused by polymerase infidelity), and identification of the derived nucleotide or polypeptide can be performed, for example, by suitable screening methods as discussed herein. Mutagenesis of a polypeptide typically entails the manipulation of a polynucleotide that encodes the polypeptide.In some embodiments, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence is at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, 4%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In other embodiments, the ITR derived from a non-AAV (or AAV) ITR is at least 90% identical to the non-AAV ITR (or AAV ITR, respectively), where the non-AAV (or AAV) ITR retains the functional properties of the non-AAV ITR (or AAV ITR, respectively). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 80% identical to the non-AAV ITR (respectively or AAV ITR), where the non-AAV (or AAV) ITR retains a functional property of the non-AAV ITR (respectively or AAV ITR). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 70% identical to the non-AAV ITR (respectively or AAV ITR), where the non-AAV (or AAV) ITR retains a functional property of the non-AAV ITR (respectively or AAV ITR).In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 60% identical to the non-AAV ITR (respectively or AAV ITR), where the non-AAV (or AAV) ITR retains a functional property of the non-AAV ITR (respectively or AAV ITR). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 50% identical to the non-AAV ITR (respectively or AAV ITR), where the non-AAV (or AAV) ITR retains a functional property of the non-AAV ITR (respectively or AAV ITR).

[0102] In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR. In some embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, where the fragment is at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, at least about 125 nucleotides, at least about 150 nucleotides, at least about 175 nucleotides, at least about 200 nucleotides, at least about 225 nucleotides, at least about 250 nucleotides, at least about 275 nucleotides, at least about 300 nucleotides, at least about 325 nucleotides, at least about 350 nucleotides, at least about 375 nucleotides, at least about 400 nucleotides, at least about 425 nucleotides, at least about 450 nucleotides, at least about 475 nucleotides, at least about 500 nucleotides, at least about 525 nucleotides, at least about 550 nucleotides, at least about 575 nucleotides, or at least about 600 nucleotides; wherein the ITRs derived from a non-AAV (or AAV) ITR retain the functional properties of the non-AAV ITR (respectively, or AAV ITR).In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, wherein the fragment comprises at least about 129 nucleotides and wherein the ITR derived from a non-AAV (or AAV) ITR retains the functional properties of the non-AAV ITR (respectively or AAV ITR). In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, wherein the fragment comprises at least about 102 nucleotides and wherein the ITR derived from a non-AAV (or AAV) ITR retains the functional properties of the non-AAV ITR (respectively or AAV ITR).

[0103] In some embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, wherein the fragment comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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%, or at least about 99% of the length of the non-AAV (or AAV) ITR.

[0104] In certain embodiments, a nucleotide or amino acid sequence derived from a second nucleotide or amino acid sequence, when properly aligned, has a sequence identity of at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 12 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In other embodiments, the ITR from a non-AAV (or AAV) ITR is at least 90% identical to the homologous portion of the non-AAV ITR (respectively or AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITR derived from a non-AAV (or AAV) ITR is at least 80% identical to the homologous portion of the non-AAV ITR (respectively, or AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.In some embodiments, the ITR derived from a non-AAV (or AAV) ITR is at least 70% identical to the homologous portion of the non-AAV ITR (respectively or AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITR derived from a non-AAV (or AAV) ITR is at least 60% identical to the homologous portion of the non-AAV ITR (respectively or AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITR derived from a non-AAV (or AAV) ITR is at least 50% identical to the homologous portion of the non-AAV ITR (respectively or AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

[0105] A "capsid-free" or "capsid-less" vector or nucleic acid molecule refers to a vector construct that does not contain a capsid. In some embodiments, a capsid-less vector or nucleic acid molecule does not contain sequences encoding, for example, AAV Rep proteins.

[0106] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered part of the coding region, although 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 encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end that encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows that a single vector can contain only a single coding region, or it can comprise two or more coding regions.

[0107] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide, which is cleaved from the mature protein upon initiation of export of the elongating protein chain across the rough endoplasmic reticulum. Those skilled in the art will appreciate that the signal peptide is generally fused to the N-terminus of the polypeptide and is cleaved from the complete or "full-length" polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of its sequence retains the ability to direct the secretion of a polypeptide to which it is operably linked. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, may be used.

[0108] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence refers to the sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the start site of transcription.

[0109] The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence relates to a coding region or a sequence located 5' of a transcription start site. For example, most promoters are located upstream of a transcription start site.

[0110] As used herein, the term "gene regulatory region" or "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence.

[0111] A polynucleotide encoding a product, e.g., a miRNA or a gene product (e.g., a polypeptide such as a therapeutic protein), may include a promoter and / or other expression (e.g., transcription or translation) control elements operably linked to one or more coding regions. In operably linked, a coding region for a gene product, e.g., a polypeptide, is linked to 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. For example, a coding region and a promoter are "operably linked" if induction of promoter function results in transcription of an mRNA that encodes the gene product encoded by the coding region, and if the nature of the binding between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product, nor does it interfere with the ability of the DNA template to be transcribed. In addition to promoters, other expression control elements, e.g., enhancers, operators, repressors, and transcription termination signals, may also be operably linked to a coding region to direct gene product expression.

[0112] "Transcriptional control sequence" refers to DNA regulatory sequences such as promoters, enhancers, terminators, etc. that effect expression of a coding sequence in a host cell. A variety of transcriptional control regions are known to those skilled in the art. These include, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, associated with intron-A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences that can control gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0113] Likewise, various translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0114] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. This includes, without limitation, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and translation of an mRNA into a polypeptide. Expression produces a "gene product." A gene product as used herein can be either a nucleic acid, such as a messenger RNA resulting from transcription of a gene, or a polypeptide translated from a transcript. A gene product as described herein further includes a nucleic acid having a post-transcriptional modification, such as polyadenylation or splicing, or a polypeptide having a post-translational modification, such as 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 produced by expression of a gene.

[0115] A "vector" refers to a vehicle for cloning and / or transport of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached to effect replication of the attached segment. A "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 vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into an appropriate vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector having complementary cohesive termini.

[0116] Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that have incorporated and expressed other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include: genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered as reporters.

[0117] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be used as or have been used as recipients of ssDNA or vectors. The term includes the progeny of the original cell that has been transduced. Thus, as used herein, "host cell" generally refers to a cell that has been transduced with a foreign DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely morphologically identical to the original parent or may not be complementary in genomics or total DNA due to natural, accidental, or deliberate mutations. In some embodiments, the host cell may be an in vitro host cell.

[0118] The term "selectable marker" refers to an identification factor, usually an antibiotic or chemical resistance gene, colorimetric marker, enzyme, fluorescent marker, etc., that can be selected based on the effect of the marker gene, i.e., resistance to antibiotics, resistance to herbicides, which 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, bialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0119] The term "reporter gene" refers to a nucleic acid that encodes a distinguishing factor that can be distinguished based on the effect of the reporter gene, where the effect is used to track the inheritance of the nucleic acid of interest, to identify cells or organisms that have inherited the nucleic acid of interest, and / or to measure gene expression induction or transcription. 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), etc. Selectable markers may also be considered reporter genes.

[0120] "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 can be derived entirely from a natural gene, be composed of different elements derived from different promoters found in nature, or can also include synthetic DNA segments. Those skilled in the art will appreciate that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types at most times is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in a 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 causes a gene to be expressed after exposure or treatment of the cell 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 since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0121] A promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (in the 5' direction) and includes the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0122] In some embodiments, the nucleic acid molecule comprises a tissue-specific promoter.In certain embodiments, the tissue-specific promoter promotes the expression of therapeutic protein, such as coagulation factor, in liver, for example in hepatocytes and / or endothelial cells.In certain embodiments, the promoter is selected from the group consisting of mouse thyretin promoter (mTTR), endogenous human factor VIII promoter (F8), human alpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, mouse albumin promoter, tristetraprolin (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, and any combination thereof. In some embodiments, the promoter is selected from liver-specific promoters (e.g., α1-antitrypsin (AAT)), muscle-specific promoters (e.g., muscle creatine kinase (MCK), myosin heavy chain alpha (αMHC), myoglobin (MB) and desmin (DES)), synthetic promoters (e.g., SPc5-12, 2R5Sc5-12, dMCK and tMCK) and any combination thereof. In one particular embodiment, the promoter comprises the TTP promoter.

[0123] The terms "restriction endonucleases" and "restriction enzymes" are used interchangeably and refer to enzymes that bind and cut within specific nucleotide sequences within double-stranded DNA.

[0124] 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 linear, circular or supercoiled, autonomously replicating sequences, genomic integration sequences, phages or nucleotide sequences of single- or double-stranded DNA or RNA from any source, in which several nucleotide sequences are linked or recombined into a specific construct that allows the introduction of promoter fragments and DNA sequences for selected gene products with appropriate 3' non-translated sequences into cells.

[0125] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses, such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0126] "Cloning vector" refers to a "replicon" that is a unit-length nucleic acid, such as a plasmid, phage, or cosmid, that replicates sequentially and contains an origin of replication, to which another nucleic acid segment can be attached so as to result in replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another cell type, e.g., eukaryotic cells. Cloning vectors typically contain one or more sequences that can be used for selection of cells that contain the vector, and / or one or more multiple cloning sites for insertion of a nucleic acid sequence of interest.

[0127] The term "expression vector" refers to a vehicle designed to enable expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operable relationship with the regulatory regions described above.

[0128] The vector is introduced into the host cell by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), using a gene gun, or DNA vector transporter.As used herein, "culture", "cultivating" and "cultivating" refer to incubating cells under in vitro conditions that allow cells to grow or divide, or to maintaining cells in a viable state.As used herein, "cultured cells" refer to cells that are grown in vitro.

[0129] As used herein, the term "polypeptide" encompasses the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to the product of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including chemical synthesis.

[0130] 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). Non-traditional 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 non-naturally occurring amino acid residues, the techniques of Noren et al., Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these techniques involve chemically activating a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of non-traditional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term "polar amino acid" includes amino acids that have zero total charge but non-zero partial charges at different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids that can have a nonzero total charge on their side chains (e.g., R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

[0131] Polypeptide fragments or variants and any combinations thereof are also included in the present disclosure. The term "fragment" or "variant" when referring to a polypeptide binding domain or binding molecule of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity to an FcRn binding domain or Fc variant, coagulation activity of a FVIII variant, or FVIII binding activity to a VWF fragment). A fragment of a polypeptide includes proteolytic fragments as well as deletion fragments in addition to specific antibody fragments discussed elsewhere herein, but does not include a naturally occurring full-length polypeptide (or a mature polypeptide). A variant of a polypeptide binding domain or binding molecule of the present disclosure also includes the above fragments, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. A variant may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. A variant polypeptide can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0132] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. The art defines a family of amino acid residues with similar side chains, 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).Therefore, when 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, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0133] As known in the art, the term "percent identity" refers to the 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" can be easily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity are codified in publicly available computer programs.Sequence alignment and percent identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the GCG suite of programs (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)) and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). Within the context of this application, when sequence analysis software is used for analysis, it is understood that the results of the analysis are based on the "default values" of the program referenced, unless otherwise stated. As used herein, "default values" refers to a set of values ​​or parameters that are initially loaded with the software when it is first initialized. For the purpose of determining the percent identity between a therapeutic protein, e.g., a clotting factor, sequence of the present disclosure and a reference sequence, only nucleotides of the reference sequence that correspond to nucleotides in the therapeutic protein, e.g., a clotting factor, sequence of the present disclosure are used to calculate the percent identity. For example, when comparing a full-length FVIII nucleotide sequence containing the B domain with an optimized B-domain deleted (BDD) FVIII nucleotide sequence of the present disclosure, the portion of the alignment including the A1, A2, A3, C1 and C2 domains is used to calculate the percent identity. Nucleotides in the portion of the full-length FVIII sequence that encodes the B domain (which results in a large "gap" in the alignment) are not counted as mismatches. Furthermore, in determining the percent identity between an optimized BDD FVIII sequence of the present disclosure or a designated portion thereof (e.g., nucleotides 58-2277 and 2320-4374 of SEQ ID NO:3) 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 listed herein.

[0134] As used herein, nucleotides corresponding to nucleotides of a particular sequence of the present disclosure are identified by alignment of the sequences of the present disclosure to maximize identity with the reference sequence. The number used to identify an equivalent amino acid in the reference sequence is based on the number used to identify the corresponding amino acid in the sequence of the present disclosure.

[0135] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences that normally reside in separate proteins can be brought together in a fusion polypeptide, or amino acid sequences that normally reside in the same protein can be placed in a new arrangement in a fusion polypeptide, such as a fusion of the Factor VIII domain and the Ig Fc domain of the present disclosure. Fusion proteins are produced, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Chimeric proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide or non-covalent bond.

[0136] As used herein, the term "insertion site" refers to a position in a polypeptide or a fragment, variant or derivative thereof that is immediately upstream of a position where a heterologous moiety can be inserted. An "insertion site" is defined by a number, which is the number of an amino acid in a reference sequence. For example, an "insertion site" in FVIII refers to the number of the amino acid sequence in mature native FVIII (SEQ ID NO: 15) to which the insertion site corresponds, which is immediately N-terminal to the insertion site. For example, the phrase "a3 contains a heterologous moiety at an insertion site corresponding to amino acid 1656 of SEQ ID NO: 15" indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 15.

[0137] As used herein, the phrase "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid.

[0138] As used herein, the terms "inserted," "inserted," "inserted into," or grammatically related terms refer to the location of a heterologous moiety in a polypeptide, such as a coagulation factor, relative to the analogous location in a parent polypeptide. For example, in certain embodiments, "inserted," or the like, refers to the location of a heterologous moiety in a recombinant FVIII polypeptide relative to the analogous location in native mature human FVIII. As used herein, these terms refer to characteristics of the polypeptide and do not indicate, imply, or mean any method or process by which the polypeptide was made.

[0139] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be expressed as the time required for half of the amount administered to a subject to be removed from the circulation and / or other tissues in that animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid alpha phase and a longer beta phase. The alpha phase generally represents the equilibration of the administered Fc polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The beta phase generally represents the catabolism of the polypeptide in the intravascular space. In some embodiments, therapeutic proteins, such as clotting factors, e.g., FVIII, and chimeric proteins comprising same, are monophasic, thus having no alpha phase, 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 beta phase.

[0140] The term "linked" as used herein refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence, or alternatively, the first sequence can be covalently joined to the second sequence with an intervening 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 the entire first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (respectively or the first amino acid sequence). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (in the case of a polypeptide chain) or a nucleotide or nucleotide chain (in the case of a nucleotide chain) or any chemical moiety (both in the case of a polypeptide and a polynucleotide chain). The term "linked" may also be indicated by a hyphen (-).

[0141] 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.

[0142] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to an impaired or deficient ability to form fibrin clots. Examples of such disorders include hemophilia. The three main 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, deficient or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X or factor XIII, defects or deficiencies of GPIb. These include Bernard-Soulier syndrome, which is a condition in which the vWF receptor GPIb is defective, leading to loss of primary clot formation (primary hemostasis) and increased bleeding tendency, as well as Glanzman and Naegeli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of clotting factors by the liver, which may increase the risk of bleeding.

[0143] The isolated nucleic acid molecule, isolated polypeptide, or vector comprising the isolated nucleic acid molecule of the present disclosure can be used prophylactically. As used herein, the term "prophylactic treatment" refers to administration of the molecule before a bleeding episode. In one embodiment, the subject in need of a systemic hemostatic agent is undergoing or about to undergo surgery. The polynucleotide, polypeptide, or vector of the present disclosure can be administered before or after surgery as a prophylactic agent. The polynucleotide, polypeptide, or vector of the present disclosure can be administered during or after surgery to control acute bleeding episodes. Surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0144] The isolated nucleic acid molecule, isolated polypeptide, or vector of the present disclosure can also be used for custom treatment. The term "custom 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 custom treatment can be given when bleeding begins, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the custom treatment can be given before an activity that increases the risk of bleeding, such as contact sports.

[0145] As used herein, the term "acute bleeding" refers to bleeding episodes regardless of 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.

[0146] As used herein, treat, treatment, and managing refer to, for example, reducing the severity of a disease or condition; reducing the duration of a disease; ameliorating one or more symptoms associated with a disease or condition; and providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition or preventing one or more symptoms associated with the disease or condition. In one embodiment, the term "treating" or "treatment" refers to the administration of an isolated nucleic acid molecule, isolated polypeptide, or vector of the present disclosure to a subject, for example, to 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, 20 IU / dL, 25 IU / dL, 30 IU / dL, 35 IU / dL, 36 IU / dL, 37 IU / dL, 38 IU / dL, 39 IU / dL, 40 IU / dL, 41 IU / dL, 42 IU / dL, 43 IU / dL, 44 IU / dL, 45 IU / dL, 46 IU / dL, 47 IU / dL, 48 IU / dL, 49 IU / dL, 50 IU / dL, 51 IU / dL, 52 IU / dL, 53 IU / dL, 54 IU / dL, 55 IU / dL, 56 IU / dL, 57 IU / dL, 58 IU / dL, 59 IU / dL, 60 IU / dL, 61 IU / dL, 62 IU / dL, 63 IU / dL, 64 IU / dL, 65 IU / d "FVIII trough levels" means maintaining a FVIII trough level of 100 IU / dL, 30 IU / dL, 35 IU / dL, 40 IU / dL, 45 IU / dL, 50 IU / dL, 55 IU / dL, 60 IU / dL, 65 IU / dL, 70 IU / dL, 75 IU / dL, 80 IU / dL, 85 IU / dL, 90 IU / dL, 95 IU / dL, 100 IU / dL, 105 IU / dL, 110 IU / dL, 115 IU / dL, 120 IU / dL, 125 IU / dL, 130 IU / dL, 135 IU / dL, 140 IU / dL, 145 IU / dL, or 150 IU / dL.In another embodiment, the treating or treatment comprises increasing FVIII trough levels to between about 1 and about 150 IU / dL, between about 1 and about 125 IU / dL, between about 1 and about 100 IU / dL, between about 1 and about 90 IU / dL, between about 1 and about 85 IU / dL, between about 1 and about 80 IU / dL, between about 1 and about 75 IU / dL, between about 1 and about 70 IU / dL, between about 1 and about 65 IU / dL, between about 1 and about 60 IU / dL, between about 1 and about 55 IU / dL, between about 1 and about 50 IU / dL, between about 1 and about 45 IU / dL, between about 1 and about 40 IU / dL, between about 1 and about 35 IU / dL, By this is meant maintaining the blood concentration at between about 1 and about 30 IU / dL, between about 1 and about 25 IU / dL, between about 25 and about 125 IU / dL, between about 50 and about 100 IU / dL, between about 50 and about 75 IU / dL, between about 75 and about 100 IU / dL, between about 1 and about 20 IU / dL, between about 2 and about 20 IU / dL, between about 3 and about 20 IU / dL, between about 4 and about 20 IU / dL, between about 5 and about 20 IU / dL, between about 6 and about 20 IU / dL, between about 7 and about 20 IU / dL, between about 8 and about 20 IU / dL, between about 9 and about 20 IU / dL, or between about 10 and about 20 IU / dL. Treating or treating a disease or condition can 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%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% of the FVIII activity in a non-hemophilic subject. The minimum trough level required for treatment can be determined by one or more known methods and can be adjusted (increased or decreased) for each individual.

[0147] As used herein, "administering" means giving a pharma- ceutically acceptable nucleic acid molecule, a polypeptide expressed therefrom, or a vector comprising a nucleic acid molecule of the present disclosure to a subject through a pharma- ceutically acceptable route. The administration route can be intravenous, for example, intravenous injection and intravenous infusion. Additional administration routes include, for example, subcutaneous, intramuscular, oral, nasal and pulmonary administration. The nucleic acid molecule, polypeptide and vector can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0148] The term "pharmacologically acceptable" as used herein refers to molecular entities and compositions that, when administered to humans, are physiologically tolerable and generally do not produce toxicity, allergic or similar untoward reactions, such as stomach upset, dizziness, etc. In some cases, as used herein, the term "pharmacologically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals, and more particularly in humans.

[0149] As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure, for example, to improve hemostasis. In one embodiment, the subject includes, but is not limited to, an individual with hemophilia. In another embodiment, the subject includes, but is not limited to, an individual who develops an inhibitor to a therapeutic protein, such as a clotting factor, such as FVIII, and therefore requires bypass therapy. The subject may be an adult or a minor (e.g., under the age of 12).

[0150] As used herein, the term "therapeutic protein" refers to any polypeptide known in the art that can be administered to a subject. In some embodiments, the therapeutic protein comprises a protein selected from a clotting factor, a growth factor, an antibody, a functional fragment thereof, or a combination thereof. As used herein, the term "clotting factor" refers to a naturally occurring or recombinantly produced molecule or analog thereof that prevents or shortens the duration of bleeding episodes in a subject. In other words, it means a molecule that has procoagulant activity, i.e., is responsible for the conversion of fibrinogen into a mesh of insoluble fibrin that causes blood clotting or coagulation. As used herein, "clotting factor" includes activated clotting factors, their zymogens, or activatable clotting factors. An "activatable clotting factor" is a clotting factor in an inactive form (e.g., in the form of its zymogen) that is capable of being converted to an activated form. The term "clotting factor" includes, but is not limited to, Factor I (FI), Factor II (FII), Factor V (FV), FVII, FVIII, FIX, Factor X (FX), Factor XI (FXI), Factor XII (FXII), Factor XIII (FXIII), von Willebrand factor (VWF), prekallikrein, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), a zymogen thereof, an activated form thereof, or any combination thereof.

[0151] As used herein, coagulation activity means the ability to complete the formation of a fibrin clot and / or participate in a cascade of biochemical reactions that reduce the severity, duration or frequency of hemorrhage or bleeding episodes.

[0152] As used herein, "growth factor" includes any growth factor known in the art, including cytokines and hormones. In some embodiments, the growth factor is adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP) (e.g., BMP2, BMP4, BMP5, BMP7), ciliary neurotrophic factor family members (e.g., ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6)), colony stimulating factors (e.g., macrophage colony stimulating factor (m-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage ... -stimulating factor (GM-CSF)), epidermal growth factor (EGF), ephrins (e.g., ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3), erythropoietin (EPO), fibroblast growth factors (FGFs) (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF 20, FGF21, FGF22, FGF23), fetal bovine somatotropin (FBS), GDNF family members (e.g., glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factors (e.g., insulin-like growth factor-1 (IGF-1) or IGF-2), interleukins (IL) (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39 ...9, IL-39, IL-31, IL-32, IL-33, IL-34, IL-35, IL-35, IL-36, IL-37, IL-38, IL-39, IL-39, IL-39, IL , IL-6, IL-7), keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP or hepatocyte growth factor-like protein (HGFLP)), myostatin (GDF-8), neuregulins (e.g., neuregulin 1 (NRG1), NRG2, NRG3, NRG4), neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4, placental growth factor (PGF), platelet-derived growth factor (PDGF),renalase (RNLS), T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor (e.g., selected from transforming growth factor alpha (TGF-α), TGF-β, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

[0153] In some embodiments, the therapeutic protein is encoded by a gene selected from X-linked dystrophin, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha-glucosidase), or any combination thereof.

[0154] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., a cell or polypeptide. An exogenous or heterologous molecule can be introduced into a cell and is present only following manipulation of the cell, e.g., by transfection or other forms of genetic engineering, or a heterologous amino acid sequence can be present in a protein in which it is not found in nature.

[0155] 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 a therapeutic protein, e.g., a clotting factor, e.g., FVIII. In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of a therapeutic protein, e.g., a clotting factor, e.g., FVIII. In other embodiments, the heterologous nucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of a therapeutic protein without significantly affecting its biological activity or function (e.g., procoagulant activity). In some embodiments, the therapeutic protein is linked or connected to the polypeptide encoded by the heterologous nucleotide sequence by a linker. Non-limiting examples of polypeptide moieties encoded by a heterologous nucleotide sequence include immunoglobulin constant regions or portions thereof, albumin or fragments thereof, albumin binding moieties, transferrin, the PAS polypeptides of U.S. Patent Application Publication No. 20100292130, HAP sequences, transferrin or fragments thereof, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, albumin binding small molecules, XTEN sequences, FcRn binding moieties (e.g., a complete Fc region or portion thereof that binds to FcRn), monoclonal antibodies, and the like. These include chain Fc regions (ScFc regions, e.g., those described in US2008 / 0260738, WO2008 / 012543 or WO2008 / 1439545), polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), especially with varying degrees of secondary structure from less than 50% to more than 50%, or combinations of two or more thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety.Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid, hydroxyethyl starch (HES), derivatives thereof, or any combination thereof.

[0156] As used herein, the term "Fc region" is defined as that portion of a polypeptide that corresponds to the Fc region of a native Ig, i.e., formed by the dimeric association of the Fc domains of each of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, as used herein, the term "genetically fused Fc region" or "single-chain Fc region" (scFc region) refers to a synthetic dimeric Fc region composed of Fc domains that are genetically linked (i.e., encoded in a single contiguous gene sequence) within a single polypeptide chain.

[0157] In one embodiment, "Fc region" refers to that portion of a single Ig heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 for IgG, where the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2 and CH3 domains.

[0158] The Fc region of the Ig constant region can include CH2, CH3 and CH4 domains, as well as a hinge region, depending on the Ig isotype. Chimeric proteins that include the Fc region of an Ig provide the chimeric protein with several desirable properties, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors, such as the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1), which are incorporated herein by reference in their entirety.

[0159] As used herein in comparison to a nucleotide sequence of the present disclosure, a "reference nucleotide sequence" is a polynucleotide sequence that is substantially identical to a nucleotide sequence of the present disclosure, except that the sequence is not optimized. For example, a reference nucleotide sequence for a nucleic acid molecule consisting of a codon-optimized BDD FVIII of SEQ ID NO:1 and a heterologous nucleotide sequence encoding a single-chain Fc region linked at its 3' end to SEQ ID NO:1 is a nucleic acid molecule consisting of the original (or "parent") BDD FVIII of SEQ ID NO:16 and the same heterologous nucleotide sequence encoding a single-chain Fc region linked at its 3' end to SEQ ID NO:16.

[0160] As used herein, the term "optimized" with respect to a nucleotide sequence refers to a polynucleotide sequence encoding a polypeptide that has been mutated to enhance the properties of the polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of a polypeptide produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize it include codon optimization, G / C content optimization, removal of repetitive sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis-acting elements that repress transcription or translation, adding or removing poly-T or poly-A sequences, adding sequences that enhance transcription such as Kozak consensus sequences around the transcription start site, removal of sequences that may form stem-loop structures, removal of destabilizing sequences, and combinations of two or more thereof.

[0161] II. Nucleic acid molecules The present disclosure relates to a plasmid-like capsid-free nucleic acid molecule that encodes a gene, e.g., miRNA, that can modulate the expression of a therapeutic or target protein. Capsid, the protein shell of a virus, encloses the genetic material of the virus. It is known that capsid helps the function of virion by protecting the viral genome, delivering the genome to the host, and interacting with the host. Nevertheless, viral capsid may be a factor in limiting the packaging capacity of vectors and / or in inducing immune responses, especially when used in gene therapy.

[0162] AAV vector has emerged as one of the more common types of gene therapy vectors.However, the existence of capsid limits the usefulness of AAV vector in gene therapy.In particular, capsid itself can limit the size of the transgene contained in vector to as low as less than 4.5kb.Various therapeutic proteins that can be useful in gene therapy can easily exceed this size even before regulatory elements are added.

[0163] Furthermore, the proteins that make up the capsid can act as antigens that can be targeted by the subject's immune system. AAV is very common in the general population, and most people have been exposed to AAV during their lifetime. As a result, most potential gene therapy recipients may already have mounted an immune response to AAV and are therefore more likely to reject the therapy.

[0164] Certain aspects of the present disclosure aim to overcome these deficiencies of AAV vectors. In particular, certain aspects of the present disclosure relate to a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding, for example, a therapeutic protein and / or a miRNA. In some embodiments, the first ITR and the second ITR are adjacent to a gene cassette comprising a heterologous polynucleotide sequence. In some embodiments, the nucleic acid molecule does not comprise a gene encoding a capsid protein, a replication protein, and / or an assembly protein. 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 certain embodiments, the gene cassette is located between the first ITR and the second ITR. In some embodiments, the nucleic acid molecule further comprises one or more non-coding regions. In certain embodiments, the one or more non-coding regions comprise a promoter sequence, an intron, a post-transcriptional regulatory element, a 3'UTR poly(A) sequence, or any combination thereof.

[0165] In one embodiment, the gene cassette is a single-stranded nucleic acid. In another embodiment, the gene cassette is a double-stranded nucleic acid.

[0166] In one embodiment, the nucleic acid molecule is: (a) a first ITR that is an ITR of a non-AAV family member of the Parvoviridae (e.g., a B19 or GPV ITR); (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding miRNA or therapeutic proteins, e.g., clotting factors; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) 3'UTR poly(A) tail sequence, e.g., bGHpA; (g) a second ITR that is an ITR of a non-AAV family member of the Parvoviridae family (e.g., a B19 or GPV ITR). Includes.

[0167] In one embodiment, the nucleic acid molecule is: (a) The first ITR, which is an ITR of a non-AAV family member of the Parvoviridae (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding a miRNA, [wherein the miRNA downregulates expression of a target gene selected from SOD1, HTT, RHO, and any combination thereof]; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) 3'UTR poly(A) tail sequence, e.g., bGHpA; (g) A second ITR that is an ITR of a non-AAV family member of the Parvoviridae family. Includes.

[0168] In one embodiment, the nucleic acid molecule is: (a) The first ITR, which is an ITR of a non-AAV family member of the Parvoviridae; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding X-linked dystrophin, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha glucosidase), or any combination thereof; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) 3'UTR poly(A) tail sequence, e.g., bGHpA; (g) A second ITR that is an ITR of a non-AAV family member of the Parvoviridae family. Includes.

[0169] In one embodiment, the nucleic acid molecule is: (a) the first ITR, which is an ITR of an AAV, e.g., AAV serotype 2 genome; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a nucleotide sequence encoding FVIII; wherein the nucleotide sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence selected from SEQ ID NOs:1-14 or SEQ ID NO:71, and wherein the FVIII encoded by the nucleotide sequence retains FVIII activity; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) a second ITR that is an ITR of an AAV, e.g., an AAV serotype 2 genome; Includes.

[0170] In one embodiment, the nucleic acid molecule is: (a) the first ITR, which is an ITR of an AAV, e.g., AAV serotype 2 genome; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding a miRNA, [wherein the miRNA downregulates expression of a target gene, e.g., SOD1, HTT, RHO, and any combination thereof]; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) a second ITR that is an ITR of an AAV, e.g., an AAV serotype 2 genome; Includes.

[0171] In one embodiment, the nucleic acid molecule is: (a) the first ITR, which is an ITR of an AAV, e.g., AAV serotype 2 genome; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding X-linked dystrophin, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPS I), IDS (MPS II), PAH, GAA (acid alpha glucosidase), or any combination thereof; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) a second ITR that is an ITR of an AAV, e.g., an AAV serotype 2 genome; Includes.

[0172] In another embodiment, the nucleic acid molecule is: (a) First ITR; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding miRNA or therapeutic proteins, e.g., clotting factors; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) Second ITR; wherein one of the first or second ITR is an ITR of a non-AAV family member of the Parvoviridae, and the other ITR is an ITR of an AAV, e.g., an AAV serotype 2 genome.

[0173] In another embodiment, the nucleic acid molecule is: (a) a 5' ITR having the AAV2 5' ITR sequence set forth in SEQ ID NO:111; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the AAV2 3'ITR sequence set forth in SEQ ID NO: 124. Includes.

[0174] In another embodiment, the nucleic acid molecule is: (a) a 5' ITR having the AAV2 5' ITR sequence set forth in SEQ ID NO:111; (b) a tissue-specific promoter sequence, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the AAV2 3'ITR sequence set forth in SEQ ID NO: 193. Includes.

[0175] In another embodiment, the nucleic acid molecule is: (a) First ITR; (b) tissue-specific promoter sequence, the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) nucleotides encoding miRNA or therapeutic proteins, e.g., clotting factors; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) Second ITR; wherein the first ITR is a synthetic ITR, the second ITR is a synthetic ITR, or both the first ITR and the second ITR are synthetic ITRs.

[0176] In another embodiment, the nucleic acid molecule is: (a) First B19 ITR; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a therapeutic protein selected from the group consisting of clotting factors, growth factors, hormones, cytokines, antibodies, fragments thereof, and combinations thereof; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second B19 ITR Includes.

[0177] In another embodiment, the nucleic acid molecule is: (a) First GPV ITR; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a therapeutic protein selected from the group consisting of clotting factors, growth factors, hormones, cytokines, antibodies, fragments thereof, and combinations thereof; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second GPV ITR Includes.

[0178] In another embodiment, the nucleic acid molecule is: (a) First B19 ITR; (b) ubiquitous promoter sequences, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a therapeutic protein selected from the group consisting of clotting factors, growth factors, hormones, cytokines, antibodies, fragments thereof, and combinations thereof; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second B19 ITR Includes.

[0179] In another embodiment, the nucleic acid molecule is: (a) First GPV ITR; (b) ubiquitous promoter sequences, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a therapeutic protein selected from the group consisting of clotting factors, growth factors, hormones, cytokines, antibodies, fragments thereof, and combinations thereof; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second GPV ITR Includes.

[0180] In another embodiment, the nucleic acid molecule is: (a) First B19 ITR; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a phenylalanine hydroxylase (PAH); (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second B19 ITR Includes.

[0181] In another embodiment, the nucleic acid molecule is: (a) First GPV ITR; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a phenylalanine hydroxylase (PAH); (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) Second GPV ITR Includes.

[0182] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the B19d135 5'ITR sequence set forth in SEQ ID NO: 180; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the B19d135 3'ITR sequence shown in SEQ ID NO: 181 Includes.

[0183] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the GPVd162 5'ITR sequence set forth in SEQ ID NO: 183; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the GPVd162 3'ITR sequence shown in SEQ ID NO: 184 Includes.

[0184] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the full length B19 5'ITR sequence as set forth in SEQ ID NO:185; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the full length B19 3'ITR sequence shown in SEQ ID NO: 186 Includes.

[0185] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the full-length GPV 5'ITR sequence set forth in SEQ ID NO:187; (b) a tissue-specific promoter sequence, e.g., the TTP promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding FVIII, e.g., FVIIIco6XTEN; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the full-length GPV 3'ITR sequence shown in SEQ ID NO: 188 Includes.

[0186] In another embodiment, the nucleic acid molecule is: (a) a 5' ITR having the B19d135 5' ITR sequence set forth in SEQ ID NO: 180; (b) a tissue-specific promoter sequence, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a PAH; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the B19d135 3'ITR sequence shown in SEQ ID NO: 181 Includes.

[0187] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the GPVd162 5'ITR sequence set forth in SEQ ID NO: 183; (b) a tissue-specific promoter sequence, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a PAH; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the GPVd162 3'ITR sequence shown in SEQ ID NO: 184 Includes.

[0188] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the full length B19 5'ITR sequence as set forth in SEQ ID NO:185; (b) a tissue-specific promoter sequence, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a PAH; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the full length B19 3'ITR sequence shown in SEQ ID NO: 186 Includes.

[0189] In another embodiment, the nucleic acid molecule is: (a) a 5'ITR having the full-length GPV 5'ITR sequence set forth in SEQ ID NO:187; (b) a tissue-specific promoter sequence, e.g., the CAG promoter; (c) an intron, e.g., a synthetic intron; (d) a heterologous polynucleotide sequence encoding a PAH; (e) post-transcriptional regulatory elements, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and / or (g) a 3'ITR having the full-length GPV 3'ITR sequence shown in SEQ ID NO: 188 Includes.

[0190] A. Inverted terminal repeats Certain embodiments of the present disclosure relate to nucleic acid molecules that include a first ITR, such as a 5'ITR, and a second ITR, such as a 3'ITR. In general, ITRs are involved in the replication and release, or excision, of parvovirus (e.g., AAV) DNA from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; Gottlieb and Muzyczka, 1988). In addition, ITRs appear to be the minimal sequences required for AAV proviral incorporation and packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are essential for efficient propagation of parvovirus genomes. The minimum defined elements essential for ITR function are hypothesized to be a Rep binding site (e.g., RBS; GCGCGCTCGCTCGCTC for AAV2 (SEQ ID NO: 104)) and a terminal resolution site (e.g., TRS; AGTTGG for AAV2 (SEQ ID NO: 105)), plus a variable palindromic sequence that allows hairpin formation. The palindromic nucleotide regions usually function together in cis as origins of DNA replication and as packaging signals for the virus. Complementary sequences within the ITRs fold into a hairpin structure during DNA replication. In some embodiments, the ITRs fold into a hairpin T-shaped structure. In other embodiments, the ITRs fold into a non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. Data suggest that the T-shaped hairpin structure of the AAV ITRs can suppress expression of transgenes adjacent to the ITRs. See, e.g., Zhou et al., Scientific Reports 7:5432 (July 14, 2017). This form of inhibition can be avoided by utilizing ITRs that do not form T-hairpin structures. Thus, in certain embodiments, polynucleotides that include non-AAV ITRs have improved transgene expression compared to polynucleotides that include AAV ITRs that form T-hairpins.

[0191] In some embodiments, the ITR comprises a naturally occurring ITR, e.g., the ITR comprises all or a portion of a parvovirus ITR. In some embodiments, the ITR comprises a synthetic sequence. In one embodiment, the first ITR or the second ITR comprises a synthetic sequence. In another embodiment, each of the first ITR and the second ITR comprises a synthetic sequence. In some embodiments, the first ITR or the second ITR comprises a naturally occurring sequence. In another embodiment, each of the first ITR and the second ITR comprises a naturally occurring sequence.

[0192] In some embodiments, the ITR comprises or consists of a portion of a naturally occurring ITR, such as a truncated ITR. In some embodiments, the ITR comprises or consists of a fragment of a naturally occurring ITR, where the fragment is at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, at least about 125 nucleotides, at least about 150 nucleotides, at least about 175 nucleotides, at least about 200 nucleotides, at least about 225 nucleotides, at least about 250 nucleotides, at least about 275 nucleotides, at least about 300 nucleotides, at least about 325 nucleotides, at least about 350 nucleotides, at least about 375 nucleotides, at least about 400 nucleotides, at least about 425 nucleotides, at least about 450 nucleotides, at least about 475 nucleotides, at least about 500 nucleotides, at least about 525 nucleotides, at least about 550 nucleotides, at least about 575 nucleotides, or at least about 600 nucleotides; wherein the ITR retains the functional characteristics of a naturally occurring ITR. In certain embodiments, the ITR comprises or consists of a fragment of a naturally occurring ITR, wherein the fragment comprises at least about 129 nucleotides, and wherein the ITR retains the functional characteristics of a naturally occurring ITR. In certain embodiments, the ITR comprises or consists of a fragment of a naturally occurring ITR, wherein the fragment comprises at least about 102 nucleotides, and wherein the ITR retains the functional properties of the naturally occurring ITR.

[0193] In some embodiments, the ITR comprises or consists of a portion of a naturally occurring ITR, where the fragment comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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%, or at least about 99% of the length of a naturally occurring ITR; where the fragment retains a functional characteristic of the naturally occurring ITR.

[0194] In certain embodiments, the ITR, when properly aligned, has a sequence similar to that of a naturally occurring ITR, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least In another embodiment, the ITR comprises or consists of a sequence having at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the naturally occurring ITR; wherein the ITR retains the functional properties of the naturally occurring ITR. In another embodiment, the ITR comprises or consists of a sequence having at least 90% sequence identity with the homologous portion of the naturally occurring ITR when properly aligned; wherein the ITR retains the functional properties of the naturally occurring ITR. In some embodiments, the ITR comprises or consists of a sequence that, when properly aligned, has at least 80% sequence identity with the homologous portion of the naturally occurring ITR; wherein the ITR retains the functional properties of the naturally occurring ITR. In some embodiments, the ITR comprises or consists of a sequence that, when properly aligned, has at least 70% sequence identity with the homologous portion of the naturally occurring ITR; wherein the ITR retains the functional properties of the naturally occurring ITR. In some embodiments, the ITR comprises or consists of a sequence that, when properly aligned, has at least 60% sequence identity with the homologous portion of the naturally occurring ITR; wherein the ITR retains the functional properties of the naturally occurring ITR.In some embodiments, the ITR comprises or consists of a sequence that, when properly aligned, has at least 50% sequence identity with a homologous portion of a naturally occurring ITR; wherein the ITR retains the functional properties of the naturally occurring ITR.

[0195] In some embodiments, ITR comprises ITR from AAV genome.In some embodiments, ITR is the ITR of AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and any combination thereof.In certain embodiments, ITR is the ITR of AAV2 genome.In another embodiment, ITR is a synthetic sequence that is engineered to comprise ITR from one or more AAV genomes at its 5' and 3' ends.

[0196] In some embodiments, the ITRs are not derived from an AAV genome. In some embodiments, the ITRs are non-AAV ITRs. In some embodiments, the ITRs are ITRs of a non-AAV genome from the Parvoviridae family of viruses, including, but not limited to, the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and any combination thereof. In certain embodiments, the ITRs are derived from Erythrovirus parvovirus B19 (a human virus). In another embodiment, the ITRs are derived from a Muscovy Duck Parvovirus (MDPV) strain. In certain embodiments, the MDPV strain is attenuated, e.g., MDPV strain FZ91-30. In other embodiments, the MDPV strain is pathogenic, e.g., MDPV strain YY. In some embodiments, the ITRs are derived from a porcine parvovirus, e.g., porcine parvovirus U44978. In some embodiments, the ITRs are derived from minute virus of mice, e.g., minute virus of mice U34256. In some embodiments, the ITRs are derived from a canine parvovirus, e.g., canine parvovirus M19296. In some embodiments, the ITRs are derived from a mink enteritis virus, e.g., mink enteritis virus D00765. In some embodiments, the ITRs are derived from a Dependoparvovirus.In one embodiment, the Dependoparvovirus is a Dependovirus goose parvovirus (GPV) strain. In a specific embodiment, the GPV strain is attenuated, for example, GPV strain 82-0321V. In another specific embodiment, the GPV strain is pathogenic, for example, GPV strain B.

[0197] The first ITR and the second ITR of the nucleic acid molecule can be derived from the same genome, for example the genome of the same virus, or from different genomes, for example the genomes of two or more different virus genomes. In certain embodiments, the first ITR and the second ITR are derived from the same AAV genome. In a specific embodiment, the two ITRs present in the nucleic acid molecule of the present invention are the same, and may in particular be AAV2 ITRs. In other embodiments, the first ITR is derived from an AAV genome, and the second ITR is not derived from an AAV genome (e.g., a non-AAV genome). In other embodiments, the first ITR is not derived from an AAV genome (e.g., a non-AAV genome), and the second ITR is derived from an AAV genome. In yet other embodiments, the first ITR and the second ITR are both not derived from an AAV genome (e.g., a non-AAV genome). In a particular embodiment, the first ITR and the second ITR are identical.

[0198] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from a genome selected from the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and any combination thereof. In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from a genome selected from the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and any combination thereof.In other embodiments, the first ITR and the second ITR are derived from a genome selected from the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and any combination thereof, wherein the first ITR and the second ITR are derived from the same genome. In other embodiments, the first ITR and the second ITR are derived from a genome selected from the group consisting of Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Aveparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstyldensovirus, and any combination thereof, wherein the first ITR and the second ITR are derived from different genomes.

[0199] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from an erythrovirus parvovirus B19 (a human virus). In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from an erythrovirus parvovirus B19 (a human virus).

[0200] In certain embodiments, the first and / or second ITR comprises or consists of all or a portion of an ITR from B19. In some embodiments, the first and / or second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 167, 168, 169, 170, and 171, wherein the first and / or second ITR retains the functional properties of the B19 ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 167, 168, 169, 170 and 171, wherein the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0201] In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 167, 168, 169, 170 and 171. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 167. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 168. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 169. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 170. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 171.

[0202] [Table 1] [Table 2] [Table 3]

[0203] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 167. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:167. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 168. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:168. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO:169, wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO:167 and retains the functional properties of the B19 ITR from which it is derived.In some embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO: 169, and wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 167, and wherein the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0204] In certain embodiments, the first ITR and / or the second ITR comprises or consists of all or a portion of an ITR from B19. 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 comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 180, 181, 185, and 186, or a functional derivative thereof. In some embodiments, the functional derivative retains the functional properties of the B19 ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 180, 181, 185, and 186, or a functional derivative thereof. In some embodiments, the functional derivative is capable of forming a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0205] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 180. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:180. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 181. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:181. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 185. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:185.In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 186. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:186.

[0206] In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 180, 181, 185, and 186. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 180. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 181. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 185. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 186. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 180. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 181. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 185. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 186.

[0207] In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 180 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 181. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 181 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 180. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 185 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 186. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 186 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 185.

[0208] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from a GPV, hi other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from a GPV.

[0209] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 172. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:172. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 173. In certain embodiments, the first ITR and / or the second ITR comprises or consists of all or a portion of an ITR from a GPV. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 172, 173, 174, 175, and 176, wherein the first ITR and / or the second ITR retains the functional properties of the GPV ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises or consists of all or a portion of an ITR from a GPV.In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 172, 173, 174, 175, and 176, where the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not include a T-shaped hairpin. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 172, 173, 174, 175, and 176. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 172. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 173. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 174. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 175. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 176.

[0210] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO:174, and wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO:172, and wherein the first ITR and / or the second ITR retains the functional properties of the GPV ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO: 174, and wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 172, and wherein the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0211] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO:176, and wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO:172, and wherein the first ITR and / or the second ITR retains the functional properties of the GPV ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence comprising the minimal nucleotide sequence set forth in SEQ ID NO: 176, and wherein the nucleotide sequence is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 172, and wherein the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0212] In certain embodiments, the first ITR and / or the second ITR comprises or consists of all or a portion of an ITR derived from GPV. 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 comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 183, 184, 187, and 188, or a functional derivative thereof. In some embodiments, the functional derivative retains the functional properties of the GPV ITR from which it is derived. In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 a nucleotide sequence selected from SEQ ID NOs: 183, 184, 187, and 188, or a functional derivative thereof. In some embodiments, the functional derivative is capable of forming a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0213] In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 183. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:183. In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 184. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:184. In certain embodiments, the first ITR and / or the second ITR comprises nucleotides that are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 187. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:187.In certain embodiments, the first ITR and / or the second ITR comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 188. In certain embodiments, the first ITR and / or the second ITR consists of SEQ ID NO:188.

[0214] In some embodiments, the first ITR and / or the second ITR comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 183, 184, 187 and 188. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 183. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 184. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 187. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 188. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 183. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 184. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 187. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 188.

[0215] In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 183 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 184. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 184 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 183. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 187 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 188. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 188 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 187.

[0216] In certain embodiments, one of the first ITR or the second ITR comprises or consists of all or a portion of an ITR from AAV2. In some embodiments, the first ITR or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 177 or 178, wherein the first ITR and / or the second ITR retains the functional properties of the AAV2 ITR from which it is derived. In some embodiments, the first ITR or the second ITR comprises or consists of a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 65%, at least about 70%, 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 NO: 177 or 178, where the first ITR and / or the second ITR can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 177 or 178. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 177. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO:178.

[0217] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from a Muscovy Duck Parvovirus (MDPV) strain. In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from a Muscovy Duck Parvovirus (MDPV) strain. In certain embodiments, the MDPV strain is attenuated, e.g., MDPV strain FZ91-30. In other embodiments, the MDPV strain is pathogenic, e.g., MDPV strain YY.

[0218] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from Dependoparvovirus. In some embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from Dependoparvovirus. In other embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from a Dependovirus goose parvovirus (GPV) strain. In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from a Dependovirus GPV strain. In certain embodiments, the GPV strain is attenuated, e.g., GPV strain 82-0321V. In other embodiments, the GPV strain is pathogenic, e.g., GPV strain B.

[0219] In certain embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from a genome selected from the group consisting of porcine parvovirus, e.g., porcine parvovirus strain U44978; minute virus of mice, e.g., minute virus of mice strain U34256; canine parvovirus, e.g., canine parvovirus strain M19296; mink enteritis virus, e.g., mink enteritis virus strain D00765; and any combination thereof. In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from a genome selected from the group consisting of porcine parvovirus, e.g., porcine parvovirus strain U44978; minute virus of mice, e.g., minute virus of mice strain U34256; canine parvovirus, e.g., canine parvovirus strain M19296; mink enteritis virus, e.g., mink enteritis virus strain D00765; and any combination thereof.

[0220] In another specific embodiment, the ITR is a synthetic sequence engineered to contain an ITR at its 5' and 3' ends 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' and 3' ends that is derived from one or more non-AAV genomes. The two ITRs present in the nucleic acid molecule of the present invention may be the same non-AAV or different non-AAV. In particular, the ITRs may be derived from the same non-AAV genome. In a specific embodiment, the two ITRs present in the nucleic acid molecule of the present invention may be the same, and in particular, the AAV2 ITR.

[0221] In some embodiments, the ITR sequence comprises one or more palindromic sequences. The palindromic sequences of the ITRs disclosed herein include, but are not limited to, naturally occurring palindromic sequences (i.e., sequences found in nature), synthetic sequences (i.e., sequences not found in nature), such as pseudopalindromic sequences, and combinations or modifications thereof. A "pseudopalindromic sequence" is a palindromic DNA sequence that includes an imperfect palindromic sequence that shares less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%, or less than 80%, including no identity, with a sequence in a naturally occurring AAV or non-AAV palindromic sequence that forms a secondary structure. A naturally occurring palindromic sequence can be obtained or derived from any genome disclosed herein. A synthetic palindromic sequence can be based on any genome disclosed herein.

[0222] The palindromic sequence may be continuous or interrupted. In some embodiments, the palindromic sequence is interrupted, where the palindromic sequence comprises the insertion of a second sequence. In some embodiments, the second sequence comprises a promoter, an enhancer, an integration site for an integrase (e.g., a site for Cre or Flp recombinase), an open reading frame for a gene product, or a combination thereof.

[0223] 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 non-T-shaped hairpin structure. In some embodiments, the non-T-shaped hairpin structure comprises a U-shaped hairpin structure.

[0224] In some embodiments, the ITRs in the nucleic acid molecules as described herein may be transcriptionally activating ITRs. The transcriptionally activating ITRs may comprise all or part of a wild-type ITR that has been transcriptionally activated by the inclusion of at least one transcriptionally active element. A variety of transcriptionally active elements are suitable for use in this context. In some embodiments, the transcriptionally active elements are constitutive transcriptionally active elements. Constitutive transcriptionally active elements provide a sustained level of gene transcription and are preferred when it is desired that the transgene be continuously expressed. In other embodiments, the transcriptionally active elements are inducible transcriptionally active elements. Inducible transcriptionally active elements generally exhibit low activity in the absence of an inducer (or 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 certain times or in certain locations, or when it is desired to titrate expression levels using an inducing agent. Transcriptionally active elements may be tissue specific; i.e., they are only active in certain tissues or cell types.

[0225] Transcriptionally active elements can be incorporated into the ITRs in a variety of ways. In some embodiments, the transcriptionally active elements are incorporated 5' of any portion of the ITR or 3' of any portion of the ITR. In other embodiments, the transcriptionally active elements of the transcriptionally activating ITR are between two ITR sequences. If the transcriptionally active elements contain two or more elements that must be spaced apart, the elements can alternate portions of the ITR. In some embodiments, the hairpin structure of the ITR is deleted and replaced with an inverted repeat of the transcription element. This latter arrangement would form a hairpin that mimics the deleted portion of the structure. There can be multiple tandem transcriptionally active elements in the transcriptionally activating ITR, which can be adjacent or spaced apart. Additionally, protein binding sites (e.g., Rep binding sites) can be introduced into the transcriptionally active elements of the transcriptionally activating ITR. The transcriptionally active elements can include any sequence that allows for controlled transcription of DNA by RNA polymerase to form RNA, and can include, for example, the transcriptionally active elements defined below.

[0226] Transcriptionally activating ITRs provide both transcriptional activation and ITR functions to a nucleic acid molecule of relatively limited nucleotide sequence length, which effectively maximizes the length of the transgene that can be carried and expressed from the nucleic acid molecule. Incorporation of transcriptionally active elements into ITRs can be accomplished in a variety of ways. Comparison of ITR sequences and sequence requirements of transcriptionally active elements can provide insight into how to encode elements within the ITR. For example, transcriptional activity can be added to an ITR through the introduction of specific alterations in the ITR sequence that duplicate functional elements of the transcriptionally active element. Several techniques exist in the art for efficiently adding, deleting, and / or modifying specific nucleotide sequences at specific sites (see, for example, Deng and Nickoloff (1992) Anal. Biochem. 200:81-88). Another method of creating transcriptionally activating ITRs includes the introduction of restriction sites at desired positions in the ITR. Additionally, multiple transcriptionally active elements can be incorporated into a transcriptionally activating ITR using methods known in the art.

[0227] By way of illustration, transcriptionally activating ITRs can be generated by the inclusion of one or more transcriptionally active elements, such as a TATA box, a GC box, a CCAAT box, an Sp1 site, an Inr region, a CRE (cAMP regulatory element) site, an ATF-1 / CRE site, an APBβ box, an APBα box, a CArG box, a CCAC box, or any other element involved in transcription as known in the art.

[0228] Aspects of the present disclosure provide a method for cloning a nucleic acid molecule as described herein, comprising inserting a nucleic acid molecule capable of adopting a complex secondary structure into a suitable vector and introducing the resulting vector into a suitable bacterial host strain. As is known in the art, complex secondary structures of nucleic acids (e.g., long palindromic regions) may be unstable and difficult to clone into bacterial host strains. For example, a nucleic acid molecule comprising a first ITR and a second ITR (e.g., a non-AAV parvovirus ITR, e.g., B19 or GPV ITR) of the present disclosure may be difficult to clone using conventional methodologies. Long DNA palindromes inhibit DNA replication and are unstable in the genomes of E. coli, Bacillus, Steptococcus, Streptomyces, S. cerevisiae, mice, and humans. These effects result from the formation of hairpin or cruciform structures by intrastrand base pairing. In E. coli, inhibition of DNA replication can be overcome in SbcC or SbcD mutants. SbcD is the nuclease subunit and SbcC is the ATPase subunit of the SbcCD complex. The E. coli SbcCD complex is an exonuclease complex responsible for preventing the replication of long palindromes. The SbcCD complex is the core of ATP-dependent double-stranded DNA exonuclease activity and ATP-dependent single-stranded DNA endonuclease activity. SbcCD can recognize DNA palindromes and collapse the replication fork by attacking the resulting hairpin structure.

[0229] In certain embodiments, a suitable bacterial host strain cannot degrade cruciform DNA structures. In certain embodiments, a suitable bacterial host strain comprises a disruption in the SbcCD complex. In some embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene and / or the SbcD gene. In certain embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene. Various bacterial host strains comprising a gene disruption in the SbcC gene are known in the art. For example, and 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; and bacterial host strain SURE comprises the genotypes recB, recJ, sbcC, mcrA, ΔmcrBCF, umuC, uvrC. Thus, in some embodiments, a method for cloning a nucleic acid molecule as described herein comprises inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into host strain PMC103, PMC107, or SURE. In certain embodiments, a method for cloning a nucleic acid molecule as described herein comprises inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into host strain PMC103.

[0230] 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.

[0231] Thus, the present disclosure provides a method for cloning a nucleic acid molecule, the method comprising inserting a nucleic acid molecule capable of adopting complex secondary structures into a suitable vector and introducing the resulting vector into a bacterial host strain comprising a disruption in the SbcCD complex, wherein the nucleic acid molecule comprises a first inverted terminal repeat (ITR) and a second ITR, 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 NO: 180, 181, 183, 184, 185, 186, 187 or 188, or a functional derivative thereof.

[0232] B. Therapeutic Proteins Certain aspects of the present disclosure relate to a nucleic acid molecule comprising a gene cassette encoding a first ITR, a second ITR, and a target sequence, where the target sequence encodes a therapeutic protein. In some embodiments, the gene cassette encodes one therapeutic protein. In some embodiments, the gene cassette encodes more than one therapeutic protein. In some embodiments, the gene cassette encodes two or more copies of the same therapeutic protein. In some embodiments, the gene cassette encodes two or more variants of the same therapeutic protein. In some embodiments, the gene cassette encodes two or more different therapeutic proteins.

[0233] Certain embodiments of the present disclosure relate to a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a therapeutic protein, wherein the therapeutic protein comprises a coagulation factor. In some embodiments, the coagulation factor is selected from the group consisting of FI, FII, FIII, FIV, FV, FVI, FVII, FVIII, FIX, FX, FXI, FXII, FXIII), VWF, prekallikrein, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), any zymogen thereof, any active form thereof, and any combination thereof. In one embodiment, the coagulation factor comprises FVIII or a variant or fragment thereof. In another embodiment, the clotting factor comprises FIX or a variant or fragment thereof. In another embodiment, the clotting factor comprises FVII or a variant or fragment thereof. In another embodiment, the clotting factor comprises VWF or a variant or fragment thereof.

[0234] 1. Clotting factors In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, where the target sequence encodes a therapeutic protein, where the therapeutic protein comprises a factor VIII polypeptide. "Factor VIII," abbreviated as "FVIII" throughout this application, as used herein, unless otherwise specified, refers to a FVIII polypeptide that is functional in its normal role in coagulation. Thus, the term FVIII includes variant polypeptides that are functional. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, act as a cofactor for factor IX, or inhibit Ca. 2+The FVIII protein may be a human, porcine, canine, rat, or murine FVIII protein. Furthermore, comparisons between human-derived FVIII and FVIII from other species have identified conserved residues likely required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632). Full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified versions. Various FVIII amino acid and nucleotide sequences are disclosed, for example, in U.S. Patent Publication Nos. 2015 / 0158929, 2014 / 0308280, and 2014 / 0370035, and International Publication WO 2015 / 106052. FVIII polypeptides include, for example, full-length FVIII, full-length FVIII minus the N-terminal Met, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and / or FVIII with a full or partial deletion of the B domain. FVIII variants include deletions of the B domain, whether partial or full deletions.

[0235] a. Polynucleotide sequences encoding FVIII and FVIII proteins In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, where the target sequence encodes a therapeutic protein, where the therapeutic protein comprises a factor VIII polypeptide. "Factor VIII," abbreviated as "FVIII" throughout this application, as used herein, unless otherwise specified, refers to a FVIII polypeptide that is functional in its normal role in coagulation. Thus, the term FVIII includes variant polypeptides that are functional. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, act as a cofactor for factor IX, or to inhibit Ca. 2+ The FVIII protein may be a human, porcine, canine, rat, or murine FVIII protein. Furthermore, comparisons between human-derived FVIII and FVIII from other species have identified conserved residues likely required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632). Full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified versions. Various FVIII amino acid and nucleotide sequences are disclosed, for example, in U.S. Patent Publication Nos. 2015 / 0158929, 2014 / 0308280, and 2014 / 0370035, and International Publication WO 2015 / 106052. FVIII polypeptides include, for example, full-length FVIII, full-length FVIII minus the N-terminal Met, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and / or FVIII with a full or partial deletion of the B domain. FVIII variants include deletions of the B domain, whether partial or full deletions.

[0236] As used herein, the FVIII portion of the chimeric protein has FVIII activity. FVIII activity can be measured by any method known in the art. Numerous tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM assay, prothrombin time (PT) test (also used to determine the INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing tests (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimers, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0237] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" coagulation pathway (also called the contact activation pathway) and the conventional coagulation pathway. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, e.g., FVIII. This test is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0238] ROTEM analysis provides information on the entire dynamics of hemostasis: clotting time, clot formation, clot stability and lysis. The various parameters of thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or the many factors that affect their interactions. This assay can provide a complete view of secondary hemostasis.

[0239] The mechanism of the chromogenic assay is based on the principle of the blood coagulation cascade, where activated FVIII accelerates the conversion of factor X to factor Xa in the presence of activated factor IX, phospholipids and calcium ions. Factor Xa activity is assessed by hydrolysis of the factor Xa-specific p-nitroanilide (pNA) substrate. The initial rate of p-nitroaniline release, measured at 405 nM, is directly proportional to the factor Xa activity, and hence to the FVIII activity, in the sample.

[0240] Chromogenic assays are recommended by the FVIII and Factor IX Subcommittees of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostatsis (ISTH). Since 1994, chromogenic assays are also the reference method of the European Pharmacopoeia for the assignment of potency of FVIII concentrates. Thus, in one embodiment, the chimeric polypeptide comprising FVIII has a FVIII activity comparable to that of a chimeric polypeptide comprising mature FVIII or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0241] In another embodiment, the FVIII-containing chimeric proteins of this disclosure have a factor Xa generation rate comparable to that of chimeric proteins containing mature FVIII or BDD FVIII (e.g., ADVATE®, REACTO®, or ELOCTATE®).

[0242] To activate factor X to factor Xa, activated factor IX (factor IXa) hydrolyzes one arginine-isoleucine bond of factor X to form factor Xa in the presence of Ca2+, membrane phospholipids, and FVIII cofactor. Therefore, the interaction between FVIII and factor IX is crucial in the aggregation pathway. In certain embodiments, chimeric polypeptides comprising FVIII can interact with factor IXa at a rate comparable to chimeric polypeptides comprising mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REACTO®, or ELOCTATE®).

[0243] Furthermore, FVIII binds to von Willebrand factor but is inactive in the circulating blood. If FVIII is not bound to VWF, it is rapidly degraded and released from VWF by the action of thrombin. In some embodiments, the chimeric polypeptide comprising FVIII binds to von Willebrand factor at a level comparable to that of the chimeric polypeptide comprising mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REACTO®, or ELOCTATE®).

[0244] FVIII can be inactivated by activated protein C in the presence of calcium and phospholipids. Activated protein C cleaves the FVIII heavy chain after arginine 336 in the A1 domain, destroying the substrate interaction site for factor X, and after arginine 562 in the A2 domain, promoting dissociation of the A2 domain and destroying the interaction site with factor IXa. This cleavage also bisects the A2 domain (43 kDa) and generates the A2-N domain (18 kDa) and the A2-C domain (25 kDa). Thus, activated protein C can catalyze multiple cleavage sites in the heavy chain. In one embodiment, a chimeric polypeptide containing FVIII is inactivated by activated protein C at a level comparable to that of a chimeric polypeptide containing mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0245] In other embodiments, the chimeric protein comprising FVIII has in vivo FVIII activity comparable to chimeric polypeptides comprising mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®). In certain embodiments, the chimeric polypeptide comprising FVIII is capable of protecting HemA mice in a tail vein transection model at a level comparable to chimeric polypeptides comprising mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0246] The "B domain" of FVIII, as used herein, is identical to the B domains known in the art, defined by internal amino acid sequence identity and site of proteolytic cleavage by thrombin, e.g., residues Ser741 to Arg1648 of mature human FVIII. Other human FVIII domains are defined by the following amino acid residues relative to mature human FVIII: A1, residues Ala1 to Arg372 of mature FVIII; A2, residues Ser373 to Arg740 of mature FVIII; A3, residues Ser1690 to Ile2032 of mature FVIII; C1, residues Arg2033 to Asn2172 of mature FVIII; C2, residues Ser2173 to Tyr2332 of mature FVIII. Unless otherwise specified, sequence residue numbers used herein without reference to any SEQ ID NO correspond to the FVIII sequence without the sequence of the signal peptide (19 amino acids). The A3-C1-C2 sequence, also known as the FVIII heavy chain, comprises residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is usually referred to as the FVIII light chain activation peptide. The locations of the boundaries for all of the domains, including the B domain, for porcine, murine and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B domain deleted FVIII" or "BDD FVIII"). One example of a BDD FVIII is REFACTO® (recombinant BDD FVIII). In one particular embodiment, the B domain deleted FVIII variant comprises a deletion of amino acid residues 746 to 1648 of mature FVIII.

[0247] "B domain deleted FVIII" is described in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5, Nos. 543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, and International Publication WO2015106052A1 (PCT / US2015 / 010738). In some embodiments, the B domain deleted FVIII sequence used in the disclosed method comprises any one of the deletions disclosed in U.S. Patent No. 6,316,226 (also US6,346,513) at column 4, line 4 to column 5, line 28 and in Examples 1-5. In another embodiment, the B domain deleted FVIII is S743 / Q1638 B domain deleted FVIII (SQ BDD FVIII) (e.g., a factor VIII having a deletion of amino acids 744 to 1637, e.g., a factor VIII having amino acids 1-743 and amino acids 1638-2332 of mature FVIII). In some embodiments, the B domain deleted FVIII used in the methods of the disclosure has a deletion as disclosed in column 2, lines 26-51 and Examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US 6,228,620).In some embodiments, the B domain deleted factor VIII is described in U.S. Pat. No. 5,972,885, column 1, line 25 to column 2, line 40; U.S. Pat. No. 6,048,720, column 6, lines 1-22 and Example 1; U.S. Pat. No. 5,543,502, column 2, lines 17-46; U.S. Pat. No. 5,171,844, column 4, line 22 to column 5, line 36; U.S. Pat. No. 5,112,950, column 2, lines 55-68. , FIG. 2, and Example 1; column 2, line 2 to column 19, line 21 and Table 2 of U.S. Patent No. 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. Patent No. 7,041,635; or column 4, lines 25-53 of U.S. Patent No. 6,458,563. In some embodiments, as disclosed in WO91 / 09122, the B domain deleted FVIII has most of the B domain deleted, but still contains the amino terminal sequence of the B domain that is essential for in vivo protein processing of the primary translation product into two polypeptide chains. In some embodiments, the B domain deleted FVIII is constructed by deleting amino acids 747-1638, i.e., substantially complete deletion of the B domain. Hoeben RC et al. J. Biol. Chem. 265(13):7318-7323 (1990). B-domain deleted factor VIII may contain deletions of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P. et al. Protein Eng. 2(4):301-6 (1988).Additional B domain deletions that are part of the present disclosure include deletions of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), 797 to 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741 to 1646 (Kaufman, PCT Publication No. 87 / 04187), 747 to 1560 (Sarver et al., DNA 6:553-564 (1987)), 741 to 1648 (Pasek, PCT Application No. 88 / 00831), or 816 to 1598 or 741 to 1648 (Lagner, Behring Inst. Mitt. (1988) No. 82:16-25; EP 295597). In one particular embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 746 to 1648 of mature FVIII. In another embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 745 to 1648 of mature FVIII. In some embodiments, the BDD FVIII comprises a single chain FVIII containing a deletion of amino acids 765 to 1652, which corresponds to mature full length FVIII (also known as rVIII-SingleChain and AFSTYLA®). See U.S. Patent No. 7,041,635.

[0248] In other embodiments, the BDD FVIII comprises a FVIII polypeptide containing a fragment of the B domain that retains one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, corresponding to the amino acid sequence of the full-length FVIII sequence. Examples of B domain fragments include 226 or 163 amino acids of the B domain disclosed in Miao, HZ et al., Blood 103(a):3412-3419 (2004), Kasuda, A et al., J. Thromb. Haemost. 6:1352-1359 (2008) and Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011) (i.e., the first 226 or 163 amino acids of the B domain are retained). In yet other embodiments, the BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of the BDD FVIII protein. See Miao, HZ et al., Blood 103(a):3412-3419 (2004). In yet other embodiments, the BDD FVIII comprises a FVIII polypeptide that contains a portion of the B domain but does not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011). In some embodiments, the BDD FVIII comprises a single chain FVIII that contains a deletion from amino acids 765 to 1652, which corresponds to mature full-length FVIII (also known as rVIII-SingleChain and AFSTYLA®). See U.S. Patent No. 7,041,635. Each of the above deletions can be made in any FVIII sequence.

[0249] Numerous functional FVIII variants are known, as discussed above and below.In addition, hundreds of non-functional mutations in FVIII have been identified in hemophilia patients, and the effect of these mutations on FVIII function has been determined to be due to their location in the three-dimensional structure of FVIII rather than the nature of the substitution (Cutler et al., Hum.Mutat.19:274-8 (2002)) (incorporated herein by reference in its entirety).In addition, comparison of human-derived FVIII with FVIII from other species has identified conserved residues that may be required for function (Cameron et al., Thromb.Haemost.79:317-22 (1998); US6,251,632) (incorporated herein by reference in its entirety).

[0250] In some embodiments, the FVIII polypeptide comprises a FVIII variant or a fragment thereof, and the FVIII variant or a fragment thereof has FVIII activity. In some embodiments, the gene cassette encodes a full-length FVIII polypeptide. In other embodiments, the gene cassette 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 gene cassette 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: 106, 107, 109, 110, 111, or 112. In some embodiments, the gene cassette encodes a polypeptide having an amino acid sequence of SEQ ID NO: 17, or a fragment thereof. In some embodiments, the gene cassette encodes a polypeptide having an amino acid sequence of SEQ ID NO: 106, or a fragment thereof. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 107. In some embodiments, the gene cassette encodes a polypeptide having the amino acid sequence of SEQ ID NO: 109, or a fragment thereof. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 109.

[0251] In some embodiments, the gene cassette of the present disclosure encodes a FVIII polypeptide comprising a signal peptide or a fragment thereof. In other embodiments, the gene cassette encodes a FVIII polypeptide lacking a signal peptide. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO:17.

[0252] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is codon-optimized. In certain 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 comprises a nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is codon-optimized. In certain embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 1-14. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 71. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19.

[0253] i. Codon-optimized nucleotide sequences encoding FVIII polypeptides In some embodiments, the nucleic acid molecule of the present disclosure comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, wherein the target sequence encodes a therapeutic protein, wherein the first ITR and the second ITR are derived from an AAV genome, and the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide. In some embodiments, the codon-optimized nucleotide sequence encodes a full-length FVIII polypeptide. In other embodiments, the codon-optimized nucleotide sequence 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 codon-optimized nucleotide sequence encodes a polypeptide or a fragment thereof 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: 17. In one embodiment, the codon optimized nucleotide sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO: 17, or a fragment thereof.

[0254] In some embodiments, the codon-optimized nucleotide sequence encodes a FVIII polypeptide that includes a signal peptide or a fragment thereof. In other embodiments, the codon-optimized sequence encodes a FVIII polypeptide that lacks a signal peptide. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO:17.

[0255] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO:3 or (ii) nucleotides 58-1791 of SEQ ID NO:4; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity. In a particular embodiment, the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO:3. In another embodiment, the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 1791 of SEQ ID NO: 4. In other embodiments, the first nucleotide sequence comprises nucleotides 58 to 1791 of SEQ ID NO: 3 or nucleotides 58 to 1791 of SEQ ID NO: 4.

[0256] In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1-1791 of SEQ ID NO:3 or (ii) nucleotides 1-1791 of SEQ ID NO:4; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity. In one embodiment, the first nucleotide sequence comprises nucleotides 1-1791 of SEQ ID NO:3 or nucleotides 1-1791 of SEQ ID NO:4. In another embodiment, the second nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792 to 4374 of SEQ ID NO: 3 or 1792 to 4374 of SEQ ID NO: 4. In a particular embodiment, the second nucleotide sequence comprises nucleotides 1792 to 4374 of SEQ ID NO: 3 or 1792 to 4374 of SEQ ID NO: 4. In yet another embodiment, the second nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:3 or 1792-4374 of SEQ ID NO:4 (i.e., nucleotides 1792-4374 of SEQ ID NO:3 or 1792-4374 of SEQ ID NO:4 that do not include nucleotides encoding the B domain or a fragment of the B domain).In a particular embodiment, the second nucleotide sequence comprises nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:3 or 1792 to 2277 and 2320 to 4374 of SEQ ID NO:4 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:3 or 1792 to 4374 of SEQ ID NO:4 that do not include the nucleotides encoding the B domain or a fragment of the B domain).

[0257] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO:5 or (ii) nucleotides 1792-4374 of SEQ ID NO:6; and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity. In certain embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO:5. In other embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 6. In a particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-4374 of SEQ ID NO: 5 or 1792-4374 of SEQ ID NO: 6. In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO: 5 or nucleotides 58-1791 of SEQ ID NO: 6. In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence recited above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1-1791 of SEQ ID NO:5 or nucleotides 1-1791 of SEQ ID NO:6.

[0258] In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:5 (i.e., nucleotides 1792-4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or a fragment of the B domain) or (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 1792-4374 of SEQ ID NO:6 without the nucleotides encoding the B domain or a fragment of the B domain); and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity. In certain embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:5 (i.e., nucleotides 1792-4374 of SEQ ID NO:5 without the nucleotides encoding the B domain or a fragment of the B domain). In other embodiments, the second nucleic acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 1792-4374 of SEQ ID NO:6 without the nucleotides encoding the B domain or a fragment of the B domain).In a particular embodiment, the second nucleic acid sequence comprises nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:5 or 1792-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 1792-4374 of SEQ ID NO:5 and 1792-4374 of SEQ ID NO:6 that do not include the nucleotides encoding the B domain or a fragment of the B domain). In some embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence listed above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-1791 of SEQ ID NO:5 or nucleotides 58-1791 of SEQ ID NO:6. In other embodiments, the first nucleic acid sequence linked to the second nucleic acid sequence recited above has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1-1791 of SEQ ID NO:5 or nucleotides 1-1791 of SEQ ID NO:6.

[0259] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 58-1791 of SEQ ID NO:1, (ii) nucleotides 58-1791 of SEQ ID NO:2, (iii) nucleotides 58-1791 of SEQ ID NO:70, or (iv) nucleotides 58-1791 of SEQ ID NO:71; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity. In other embodiments, the first nucleotide sequence comprises nucleotides 58-1791 of SEQ ID NO:1, nucleotides 58-1791 of SEQ ID NO:2, (iii) nucleotides 58-1791 of SEQ ID NO:70, or (iv) nucleotides 58-1791 of SEQ ID NO:71.

[0260] In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1-1791 of SEQ ID NO:1, (ii) nucleotides 1-1791 of SEQ ID NO:2, (iii) nucleotides 1-1791 of SEQ ID NO:70, or (iv) nucleotides 1-1791 of SEQ ID NO:71; and wherein the N-terminal portion and the C-terminal portion together have a FVIII polypeptide activity. In one embodiment, the first nucleotide sequence comprises nucleotides 1-1791 of SEQ ID NO:1, nucleotides 1-1791 of SEQ ID NO:2, (iii) nucleotides 1-1791 of SEQ ID NO:70, or (iv) nucleotides 1-1791 of SEQ ID NO:71. In another embodiment, the second nucleotide sequence linked to the first nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO:1, nucleotides 1792-4374 of SEQ ID NO:2, (iii) nucleotides 1792-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-4374 of SEQ ID NO:71. In a particular embodiment, the second nucleotide sequence linked to the first nucleotide sequence comprises (i) nucleotides 1792-4374 of SEQ ID NO:1, (ii) nucleotides 1792-4374 of SEQ ID NO:2, (iii) nucleotides 1792-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-4374 of SEQ ID NO:71.In other embodiments, the second nucleotide sequence linked to the first nucleotide sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:1, (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:71. In one embodiment, the second nucleotide sequence comprises (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:1, (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:71.

[0261] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) nucleotides 1792-4374 of SEQ ID NO:1, (ii) nucleotides 1792-4374 of SEQ ID NO:2, (iii) nucleotides 1792-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-4374 of SEQ ID NO:71; and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity. In a particular embodiment, the second nucleic acid sequence comprises (i) nucleotides 1792 to 4374 of SEQ ID NO:1, (ii) nucleotides 1792 to 4374 of SEQ ID NO:2, (iii) nucleotides 1792 to 4374 of SEQ ID NO:70, or (iv) nucleotides 1792 to 4374 of SEQ ID NO:71.In some embodiments, the codon-optimized sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:1, (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:71. 792-2277 and 2320-4374 (i.e., nucleotides 1792-4374 of SEQ ID NO:1, nucleotides 1792-4374 of SEQ ID NO:2, nucleotides 1792-4374 of SEQ ID NO:70, or nucleotides 1792-4374 of SEQ ID NO:71, not including any nucleotides encoding the B domain or a fragment of the B domain); and wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity. In one embodiment, the second nucleic acid sequence comprises (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:1, (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:2, (iii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:70, or (iv) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:71 (i.e., nucleotides 1792-4374 of SEQ ID NO:1, nucleotides 1792-4374 of SEQ ID NO:2, nucleotides 1792-4374 of SEQ ID NO:70, or nucleotides 1792-4374 of SEQ ID NO:71 that do not include the nucleotides encoding the B domain or a fragment of the B domain).

[0262] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 1. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO: 1 (i.e., nucleotides 58 to 4374 of SEQ ID NO: 1 that do not include nucleotides encoding the B domain or a B domain fragment). In other embodiments, the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 58-4374 of SEQ ID NO: 1 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO: 1. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 1 (i.e., nucleotides 1-4374 of SEQ ID NO: 1 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO: 1.

[0263] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 2. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2. In other embodiments, the nucleic acid sequence has at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:2 (i.e., nucleotides 58-4374 of SEQ ID NO:2 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO:2. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO:2 (i.e., nucleotides 1-4374 of SEQ ID NO:2 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 1 to 4374 of SEQ ID NO:2.

[0264] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 70. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 58-4374 of SEQ ID NO: 70 that do not include nucleotides encoding the B domain or B domain fragment). In other embodiments, the nucleic acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 58-4374 of SEQ ID NO: 70 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO: 70. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 70 (i.e., nucleotides 1-4374 of SEQ ID NO: 70 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO: 70.

[0265] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 71. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 71 that do not include nucleotides encoding the B domain or a B domain fragment). In other embodiments, the nucleic acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 71. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 58-4374 of SEQ ID NO: 71 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO: 71. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO: 71 (i.e., nucleotides 1-4374 of SEQ ID NO: 71 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO: 71.

[0266] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 3. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 3 (i.e., nucleotides 58-4374 of SEQ ID NO: 3 that do not include nucleotides encoding the B domain or B domain fragment). In certain embodiments, the nucleic acid sequence has at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:3 (i.e., nucleotides 58-4374 of SEQ ID NO:3 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO:3. In yet other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:3 (i.e., nucleotides 1-4374 of SEQ ID NO:3 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 1 to 4374 of SEQ ID NO:3.

[0267] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 4. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4 (i.e., nucleotides 58-4374 of SEQ ID NO: 4 that do not include nucleotides encoding the B domain or a B domain fragment). In other embodiments, the nucleic acid sequence has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4. In other embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:4 (i.e., nucleotides 58-4374 of SEQ ID NO:4 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO:4. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO:4 (i.e., nucleotides 1-4374 of SEQ ID NO:4 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO:4.

[0268] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 5. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 5 (i.e., nucleotides 58-4374 of SEQ ID NO: 5 that do not include nucleotides encoding the B domain or a B domain fragment). In certain embodiments, the nucleic acid sequence has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5. In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:5 (i.e., nucleotides 58-4374 of SEQ ID NO:5 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 58-4374 of SEQ ID NO:5. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO:5 (i.e., nucleotides 1-4374 of SEQ ID NO:5 that do not include nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO:5.

[0269] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-4374 of SEQ ID NO: 6. In other embodiments, the nucleotide sequence comprises a nucleic acid sequence having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 6 (i.e., nucleotides 58-4374 of SEQ ID NO: 6 that do not include nucleotides encoding the B domain or B domain fragment). In certain embodiments, the nucleic acid sequence has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6. In some embodiments, the nucleotide sequence comprises nucleotides 58-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 58-4374 of SEQ ID NO:6 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 58 to 4374 of SEQ ID NO:6. In yet other embodiments, the nucleotide sequence comprises nucleotides 1-2277 and 2320-4374 of SEQ ID NO:6 (i.e., nucleotides 1-4374 of SEQ ID NO:6 without the nucleotides encoding the B domain or a B domain fragment), or nucleotides 1-4374 of SEQ ID NO:6.

[0270] In some embodiments, the codon-optimized nucleotide sequence encoding the FVIII polypeptide comprises a nucleic acid sequence encoding a signal peptide. In certain embodiments, the signal peptide is a FVIII signal peptide. In some embodiments, the nucleic acid sequence encoding the signal peptide is codon-optimized. In a particular embodiment, the nucleic acid sequence encoding the signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (i) nucleotides 1 to 57 of SEQ ID NO:1; (ii) nucleotides 1 to 57 of SEQ ID NO:2; (iii) nucleotides 1 to 57 of SEQ ID NO:3; (iv) nucleotides 1 to 57 of SEQ ID NO:4; (v) nucleotides 1 to 57 of SEQ ID NO:5; (vi) nucleotides 1 to 57 of SEQ ID NO:6; (vii) nucleotides 1 to 57 of SEQ ID NO:70; (viii) nucleotides 1 to 57 of SEQ ID NO:71; or (ix) nucleotides 1 to 57 of SEQ ID NO:68.

[0271] SEQ ID NOs: 1-6, 70, and 71 are optimized versions of SEQ ID NO: 16, the starting or "parent" or "wild-type" FVIII nucleotide sequence. SEQ ID NO: 16 encodes a B-domain deleted human FVIII. Although SEQ ID NOs: 1-6, 70, and 71 are derived from a specific B-domain deleted form of FVIII (SEQ ID NO: 16), it should be understood that the present disclosure also includes optimized versions of nucleic acids encoding other versions of FVIII. For example, other versions of FVIII can include full length FVIII, other B-domain deletions of FVIII (described herein), or other fragments of FVIII that retain FVIII activity.

[0272] In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Tables 2A-2F. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence shown in Table 2A. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Table 2B. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Table 2C. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Table 2D. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Table 2E. In one embodiment, the gene cassette comprises a FVIII construct comprising a polynucleotide sequence listed in Table 2F.

[0273] In certain embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 179, 182, 189, or 194. In some embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 179. In some embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 182. In some embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 189. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO:194.In some embodiments, the isolated nucleic acid molecule retains the ability to express a functional FVIII protein.

[0274] [Table 4] [Table 5] [Table 6] [Table 7]

[0275] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0276] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17]

[0277]

Table 18

Table 19

Table 20

Table 21

Table 22

[0278]

Table 23

Table 24

Table 25

Table 26

Table 27

[0279]

Table 28

Table 29

Table 30

Table 31

Table 32

[0280] In one embodiment, the gene cassette comprises a phenylalanine hydroxylase (PAH) construct comprising a polynucleotide sequence listed in Tables 10A and 10B. In one embodiment, the gene cassette comprises a PAH construct comprising a polynucleotide sequence shown in Table 10A. In one embodiment, the gene cassette comprises a PAH construct comprising a polynucleotide sequence shown in Table 10B.

[0281] In certain embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 197 or 198. In some embodiments, an isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 197. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, 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 about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 198. In some embodiments, the isolated nucleic acid molecule retains the ability to express a functional phenylalanine hydroxylase.

[0282] A. Codon Adaptation Index In one embodiment, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the human codon adaptation index of the codon-optimized nucleotide sequence is increased relative to SEQ ID NO: 16. For example, the codon-optimized nucleotide sequence has a human codon adaptation index of at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about The codon-optimized nucleotide sequence may have a Human Codon Adaptation Index that is at least about 0.87 (87%), at least about 0.88 (88%), at least about 0.89 (89%), at least about 0.90 (90%), at least about 0.91 (91%), at least about 0.92 (92%), at least about 0.93 (93%), at least about 0.94 (94%), at least about 0.95 (95%), at least about 0.96 (96%), at least about 0.97 (97%), at least about 0.98 (98%), or at least about 0.99 (99%). In some embodiments, the codon-optimized nucleotide sequence has a Human Codon Adaptation Index that is at least about 0.88 (88%). In other embodiments, the codon-optimized nucleotide sequence has a Human Codon Adaptation Index that is at least about 0.91 (91%). In other embodiments, the codon-optimized nucleotide sequence has a Human Codon Adaptation Index that is at least about 0.97 (97%).

[0283] In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; the first nucleic acid sequence is identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4 and at least having 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the human codon adaptation index of the nucleotide sequence is increased compared to SEQ ID NO:16. In some embodiments, the nucleotide sequence has a Human Codon Adaptation Index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), at least about 0.88 (88%), at least about 0.89 (89%), at least about 0.90 (90%), or at least about .91 (91%). In a particular embodiment, the nucleotide sequence has a Human Codon Adaptation Index that is at least about .88 (88%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .91 (91%).

[0284] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence comprising a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to (i) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:5, or (ii) nucleotides 1792-2277 and 2320-4374 of SEQ ID NO:6; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the human codon adaptation index of the nucleotide sequence is increased compared to SEQ ID NO:16. In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), or at least about 0.88 (88%). In a particular embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.83 (83%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about 0.88 (88%).

[0285] In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a nucleotide sequence encoding a polypeptide having FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 58-2277 and 2320-4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58-4374 of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, or 71 that do not contain nucleotides encoding a B domain or a B domain fragment); and the human codon adaptation index of the nucleotide sequence is increased compared to SEQ ID NO:16. In some embodiments, the nucleotide sequence has a human codon adaptation index that is at least about 0.75 (75%), at least about 0.76 (76%), at least about 0.77 (77%), at least about 0.78 (78%), at least about 0.79 (79%), at least about 0.80 (80%), at least about 0.81 (81%), at least about 0.82 (82%), at least about 0.83 (83%), at least about 0.84 (84%), at least about 0.85 (85%), at least about 0.86 (86%), at least about 0.87 (87%), or at least about 0.88 (88%). In a particular embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .75 (75%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .83 (83%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .88 (88%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .91 (91%). In another embodiment, the nucleotide sequence has a human codon adaptation index that is at least about .97 (97%).

[0286] In some embodiments, a codon-optimized nucleotide sequence encoding a FVIII polypeptide of the present disclosure has an increased frequency of optimal codon (FOP) compared to SEQ ID NO: 16. In certain embodiments, the FOP of a codon-optimized nucleotide sequence encoding a FVIII polypeptide is at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 64, at least about 65, at least about 70, at least about 75, at least about 79, at least about 80, at least about 85, or at least about 90.

[0287] In other embodiments, the codon-optimized nucleotide sequence encoding the FVIII polypeptide of the present disclosure has an increased relative synonymous codon usage (RCSU) compared to SEQ ID NO: 16. In some embodiments, the RCSU of the isolated nucleic acid molecule is greater than 1.5. In other embodiments, the RCSU of the isolated nucleic acid molecule is greater than 2.0. In certain embodiments, the RCSU of the isolated nucleic acid molecule is at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, or at least about 2.7.

[0288] In still other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide of the present disclosure has an effective number of codons that is reduced compared to SEQ ID NO: 16. In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has an effective number of codons that is less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, or less than about 25. In a particular embodiment, the isolated nucleic acid molecule has an effective number of codons of about 40, about 35, about 30, about 25, or about 20.

[0289] Optimization of BG / C content In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, or at least about 60%.

[0290] In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence is at least about 80%, at least about 85%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 40 ... 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16. In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, or at least about 58%. In one particular embodiment, the nucleotide sequence encoding a polypeptide having FVIII activity has a G / C content that is at least about 58%.

[0291] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is selected from the group consisting of (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment), or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment). the N-terminal portion has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the N-terminal portion of SEQ ID NO:6 (nucleotides 1792-4374 of SEQ ID NO:6, not including nucleotides encoding the FVIII fragment); where the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides of SEQ ID NO:16. In other embodiments, the codon-optimized nucleotide sequence encoding the FVIII polypeptide has a G / C content of at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, or at least about 57%. In a particular embodiment, the codon-optimized nucleotide sequence encoding the FVIII polypeptide has a G / C content of at least about 52%. In another embodiment, the codon-optimized nucleotide sequence encoding the FVIII polypeptide has a G / C content of at least about 55%.In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 57%.

[0292] In other embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is selected from (i) nucleotides 58 to 4374 or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, or 71 that do not include nucleotides encoding a B domain or a B domain fragment). 58-4374); and wherein the nucleotide sequence contains a higher percentage of G / C nucleotides compared to the percentage of G / C nucleotides in SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 45%. In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 52%. In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 55%. In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 57%. In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 58%. In yet another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide has a G / C content that is at least about 60%.

[0293] "G / C content" (or guanine-cytosine content), or "percentage of G / C nucleotides" refers to the percentage of nitrogenous bases in a DNA molecule that are either guanine or cytosine. G / C content is calculated according to the following formula:

number

[0294] The G / C content of human genes is highly heterogeneous, with some genes having a G / C content as low as 20% and others as high as 95%. In general, G / C-rich genes are more highly expressed. In fact, it has been demonstrated that an increase in the G / C content of a gene can lead to increased gene expression, which is mostly due to increased transcription and much more stable mRNA levels. See Kudla et al., PLoS Biol., 4(6):e180 (2006).

[0295] C. Matrix-binding domain-like sequence In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 1 MARS / ARS sequence. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain a MARS / ARS sequence.

[0296] In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence is at least about 80%, ... 5%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 1 MARS / ARS sequence. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain a MARS / ARS sequence.

[0297] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is selected from the group consisting of (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (all of which are incorporated herein by reference). i.e., 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO: 6, not including nucleotides encoding a B domain or a B domain fragment; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 1 MARS / ARS sequence. In yet other embodiments, the codon-optimized nucleotide sequence encoding the FVIII polypeptide does not contain a MARS / ARS sequence.

[0298] In other embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is (i) nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71 (i.e., SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71, which does not include nucleotides encoding a B domain or a B domain fragment). 16); and wherein the codon-optimized nucleotide sequence contains fewer MARS / ARS sequences compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 6, up to 5, up to 4, up to 3, or up to 2 MARS / ARS sequences. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 1 MARS / ARS sequence. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains no MARS / ARS sequences.

[0299] An AT-rich element in the human FVIII nucleotide sequence has been identified that shares sequence similarity with the Saccharomyces cerevisiae autonomously replicating sequence (ARS) and nuclear matrix attachment region (MAR). (Fallux et al., Mol. Cell. Biol. 16:4264-4272 (1996). One of these elements has been demonstrated to bind nuclear factors in vitro and repress expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Id. It was hypothesized that these sequences may contribute to transcriptional repression of the human FVIII gene. Thus, in one embodiment, all MAR / ARS sequences were eliminated in the codon-optimized nucleotide sequences encoding the FVIII polypeptides of the present disclosure. There are four MAR / ARS ATATTT sequences (SEQ ID NO:21) and three MAR / ARS AAATAT sequences (SEQ ID NO:22) in the parent FVIII sequence (SEQ ID NO:16). All of these sites were mutated to disrupt the MAR / ARS sequences in the optimized FVIII sequence (SEQ ID NOs:1-6). The location of each of these elements, and the corresponding nucleotide sequence in the optimized sequence, are shown in Table 3 below.

[0300] [Table 33] [Table 34]

[0301] D. Destabilizing Sequences In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain a destabilizing element.

[0302] In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence is at least about 80%, at least about 80%, or at least about 80% identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. 5%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; wherein the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain a destabilizing element.

[0303] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is selected from the group consisting of (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment); the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the nucleotide sequence encoding a polypeptide having FVIII activity contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the codon-optimized nucleotide sequence encoding the FVIII polypeptide does not contain a destabilizing element.

[0304] In other embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., a sequence that does not include nucleotides encoding a B domain or a B domain fragment). and a nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 70, or 71; and the codon-optimized nucleotide sequence contains fewer destabilizing elements compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 destabilizing elements. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 destabilizing element. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain a destabilizing element.

[0305] There are ten destabilizing elements in the parent FVIII sequence (SEQ ID NO: 16): six ATTTA sequences (SEQ ID NO: 23) and four TAAAT sequences (SEQ ID NO: 24). In one embodiment, the sequences at these sites were mutated to destroy the destabilizing elements in optimized FVIII SEQ ID NOs: 1-6, 70, and 71. The location of each of these elements, and the corresponding nucleotide sequence in the optimized sequence, are shown in Table 3.

[0306] E. Potential promoter binding sites In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 potential promoter binding sites. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 potential promoter binding site. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain any potential promoter binding sites.

[0307] In one particular embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence is at least about 80%, ... 5%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 potential promoter binding sites. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 potential promoter binding site. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain any potential promoter binding sites.

[0308] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is selected from the group consisting of (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding the B domain or B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., has 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 1792-4374 of SEQ ID NO:6, not including nucleotides encoding the B domain or B domain fragment; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO:16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 potential promoter binding sites. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 potential promoter binding site. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide does not contain any potential promoter binding sites.

[0309] In other embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, which do not include nucleotides encoding a B domain or a B domain fragment). and the codon-optimized nucleotide sequence contains fewer potential promoter binding sites compared to SEQ ID NO: 16. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 9, up to 8, up to 7, up to 6, or up to 5 potential promoter binding sites. In other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains up to 4, up to 3, up to 2, or up to 1 potential promoter binding sites. In yet other embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide contains no potential promoter binding sites.

[0310] TATA boxes are regulatory sequences that are often found in eukaryotic promoter regions. They serve as binding sites for the general transcription factor TATA binding protein (TBP). TATA boxes usually contain the sequence TATAA (SEQ ID NO: 28) or a close variant. However, TATA boxes in coding sequences can inhibit translation of full-length proteins. There are 10 potential promoter binding sequences in the wild-type BDD FVIII sequence (SEQ ID NO: 16): 5 TATAA sequences (SEQ ID NO: 28) and 5 TTATA sequences (SEQ ID NO: 29). In some embodiments, at least 1, at least 2, at least 3, or at least 4 promoter binding sites are eliminated in the FVIII gene of the present disclosure. In some embodiments, at least 5 promoter binding sites are eliminated in the FVIII gene of the present disclosure. In other embodiments, at least 6, at least 7, or at least 8 promoter binding sites are eliminated in the FVIII gene of the present disclosure. In one embodiment, at least 9 promoter binding sites are eliminated in the FVIII gene of the present disclosure. In a particular embodiment, all promoter binding sites are eliminated in the FVIII gene of the present disclosure. The location of each potential promoter binding site and the corresponding nucleotide sequence in the optimized sequence are shown in Table 3.

[0311] F. Other Cis-Acting Negative Regulatory Elements In addition to the above-mentioned MAR / ARS sequences, destabilizing elements, and potential promoter sites, several additional potential inhibitory sequences can be identified in the wild-type BDD FVIII sequence (SEQ ID NO: 16). Two AU-rich sequence elements (AREs) can be identified (ATTTTATT (SEQ ID NO: 30); and ATTTTTAA (SEQ ID NO: 31)), together with a poly A site (AAAAAAA; SEQ ID NO: 26), a poly T site (TTTTTT; SEQ ID NO: 25), and a splice site (GGTGAT; SEQ ID NO: 27) in the non-optimized BDD FVIII sequence. One or more of these elements can be removed from the optimized FVIII sequence. The location of each of these sites and the corresponding nucleotide sequence in the optimized sequence are shown in Table 3.

[0312] In certain embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence is at least about 80%, at least about 85%, at least about 86%, at least about 87%, or at least about 89% identical to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. , at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain one or more cis-acting negative regulatory elements, such as a splice site, a polyT sequence, a polyA sequence, an ARE sequence, or any combination thereof.

[0313] In another embodiment, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the second nucleic acid sequence is selected from the group consisting of (i) nucleotides 1792 to 4374 of SEQ ID NO:5; (ii) nucleotides 1792 to 4374 of SEQ ID NO:6; (iii) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:5 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment); or (iv) nucleotides 1792 to 2277 and 2320 to 4374 of SEQ ID NO:6 (i.e., nucleotides 1792 to 4374 of SEQ ID NO:5 that do not include nucleotides encoding a B domain or a B domain fragment). the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain one or more cis-acting negative regulatory elements, such as a splice site, a polyT sequence, a polyA sequence, an ARE sequence, or any combination thereof.

[0314] In other embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, or 71 that do not include nucleotides encoding a B domain or a B domain fragment). and the codon-optimized nucleotide sequence does not contain one or more cis-acting negative regulatory elements, such as a splice site, a polyT sequence, a polyA sequence, an ARE sequence, or any combination thereof.

[0315] In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has a similar sequence to at least about 80%, at least about 80%, of (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO:27). In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80% similarity to (i) nucleotides 58-1791 of SEQ ID NO:3; (ii) nucleotides 1-1791 of SEQ ID NO:3; (iii) nucleotides 58-1791 of SEQ ID NO:4; or (iv) nucleotides 1-1791 of SEQ ID NO:4. have 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain a poly-T sequence (SEQ ID NO:25).In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has at least about 80% similarity to (i) nucleotides 58-1791 of SEQ ID NO:3; (ii) nucleotides 1-1791 of SEQ ID NO:3; (iii) nucleotides 58-1791 of SEQ ID NO:4; or (iv) nucleotides 1-1791 of SEQ ID NO:4. have 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain a polyA sequence (SEQ ID NO:26). In some embodiments, the codon-optimized nucleotide sequence encoding a FVIII polypeptide comprises a first nucleic acid sequence encoding an N-terminal portion of a FVIII polypeptide and a second nucleic acid sequence encoding a C-terminal portion of a FVIII polypeptide; wherein the first nucleic acid sequence has a codon-optimized sequence that is at least about 80%, at least about 80%, relative to (i) nucleotides 58 to 1791 of SEQ ID NO:3; (ii) nucleotides 1 to 1791 of SEQ ID NO:3; (iii) nucleotides 58 to 1791 of SEQ ID NO:4; or (iv) nucleotides 1 to 1791 of SEQ ID NO:4. having 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; the N-terminal portion and the C-terminal portion together have FVIII polypeptide activity; and the codon-optimized nucleotide sequence does not contain an ARE element (SEQ ID NO:30 or SEQ ID NO:31).

[0316] In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., does not include nucleotides encoding a B domain or a B domain fragment). the nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO:1, 2, 3, 4, 5, 6, 70, or 71; and the codon-optimized nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO:27). In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides encoding a B domain or a B domain fragment). nucleotides 58-4374 of SEQ ID NO:1, 2, 3, 4, 5, 6, 70, or 71, not including SEQ ID NO:1; and the codon-optimized nucleotide sequence does not contain a poly-T sequence (SEQ ID NO:25).In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, wherein the codon-optimized nucleotide sequence is selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., nucleotides encoding a B domain or a B domain fragment). nucleotides 58-4374 of SEQ ID NO:1, 2, 3, 4, 5, 6, 70, or 71, not including SEQ ID NO:1; and the codon-optimized nucleotide sequence does not contain a polyA sequence (SEQ ID NO:26). In some embodiments, the gene cassette comprises a codon-optimized nucleotide sequence encoding a FVIII polypeptide, the codon-optimized nucleotide sequence being selected from (i) nucleotides 58 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71, or (ii) nucleotides 58 to 2277 and 2320 to 4374 of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 70, and 71 (i.e., sequence numbers that do not include nucleotides encoding a B domain or a B domain fragment). the nucleic acid sequence having at least about 80%, at least about 85%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to nucleotides 58 to 4374 of SEQ ID NO:1, 2, 3, 4, 5, 6, 70, or 71; and the codon-optimized nucleotide sequence does not contain an ARE element (SEQ ID NO:30 or SEQ ID NO:31).

[0317] In other embodiments, the optimized FVIII sequences of the present disclosure do not include one or more of the antiviral motifs, stem loop structures, and repeat sequences.

[0318] In yet another embodiment, the nucleotides surrounding the transcription start site are replaced with the Kozak consensus sequence (GCCGCCACC ATG C (SEQ ID NO:32), where the underlined nucleotides are the start codon.) In other embodiments, restriction sites may be added or removed to facilitate the cloning process.

[0319] b. FIX and polynucleotide sequences encoding the FIX protein In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, wherein the target sequence encodes a therapeutic protein, and the therapeutic protein comprises a FIX polypeptide. In some embodiments, the FIX polypeptide comprises FIX or a variant or fragment thereof, and the FIX or variant or fragment thereof has FIX activity.

[0320] Human FIX is a serine protease that is an important component of the intrinsic pathway of the blood coagulation cascade. "Factor IX" or "FIX" as used herein refers to coagulation factor proteins and their species and sequence variants, including but not limited to the 461 single amino acid sequence of human FIX precursor polypeptide ("prepro"), the 415 single amino acid sequence of mature human FIX (SEQ ID NO: 125), and the R338L FIX (Padua) variant (SEQ ID NO: 126). FIX includes any form of FIX molecule that has the typical characteristics of blood coagulation FIX. As used herein, "Factor IX" and "FIX" are intended to encompass polypeptides that contain the domains Gla (the region containing gamma-carboxyglutamic acid residues), EGF1 and EGF2 (the region containing sequences homologous to human epidermal growth factor), the activation peptide ("AP" formed by residues R136-R180 of mature FIX), and the C-terminal protease domain ("Pro"), or synonyms of these domains known in the art, or may be truncated fragments or sequence variants that retain at least a portion of the biological activity of the native protein. FIX or sequence variants have been cloned as described in U.S. Patent Nos. 4,770,999 and 7,700,734, and cDNA encoding human FIX has been isolated, characterized, and cloned into an expression vector (see, e.g., Choo et al., Nature 299:178-180 (1982); Fair et al., Blood 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad. Sci., USA 79:6461-6464 (1982)). One particular variant of FIX characterized by Simioni et al., 2009, the R338L FIX (Padua) variant (SEQ ID NO: 2), contains a gain-of-function mutation that correlates with an almost 8-fold increase in activity of the Padua variant over native FIX (Table 4). A FIX variant can also include any FIX polypeptide with one or more conservative amino acid substitutions that do not affect the FIX activity of the FIX polypeptide.In some embodiments, the FIX variant comprises a rFIX albumin fused by a cleavable linker, e.g., IDELVION®. See US 7,939,632, which is incorporated by reference in its entirety.

[0321] [Table 35] [Table 36] [Table 37]

[0322] The FIX polypeptide is 55 kDa and is synthesized as a prepropolypeptide chain (SEQ ID NO: 125) composed of three regions: a 28 amino acid signal peptide (amino acids 1 to 28 of SEQ ID NO: 127), an 18 amino acid propeptide (amino acids 29 to 46) required for gamma-carboxylation of glutamic acid residues, and the 415 amino acid mature factor IX (SEQ ID NO: 125 or 126). The propeptide is a sequence of 18 amino acid residues N-terminal to the gamma-carboxyglutamate domain. The propeptide binds to vitamin K-dependent gamma-carboxylase and is then cleaved from the precursor polypeptide of FIX by an endogenous protease, most likely PACE (paired basic amino acid cleaving enzyme), also known as furin or PCSK3. Without gamma-carboxylation, the Gla domain cannot bind calcium to adopt the correct conformation required to anchor the protein to negatively charged phospholipid surfaces, rendering factor IX non-functional. Even when carboxylated, the Gla domain also depends on cleavage of the propeptide for proper function, because the retained propeptide interferes with the conformational changes of the Gla domain necessary for optimal binding to calcium and phospholipids. In humans, the resulting mature factor IX is secreted into the bloodstream by hepatocytes as an inactive zymogen, a single-chain protein of 415 amino acid residues that contains approximately 17% carbohydrate by weight (Schmidt, AE et al. (2003) Trends Cardiovasc Med, 13:39).

[0323] Mature FIX is composed of several domains, in an N-to-C-terminal configuration: the GLA domain, the EGF1 domain, the EGF2 domain, the activation peptide (AP) domain, and the protease (or catalytic) domain. A short linker connects the EGF2 domain, which contains the AP domain. FIX contains two activation peptides, formed by R145-A146 and R180-V181, respectively. After activation, single-chain FIX becomes a two-chain molecule in which the two chains are linked by a disulfide bond. Coagulation factors can be engineered by replacing their activation peptides, resulting in altered activation specificity. In mammals, mature FIX must be activated by activated factor XI to obtain factor IXa. Upon activation of FIX to FIXa, the protease domain provides the catalytic activity of FIX. Activated factor VIII (FVIIIa) is the specific cofactor for the full expression of FIXa activity.

[0324] In certain embodiments, the FIX polypeptide comprises the Thr148 allele of plasma-derived FIX and has structural and functional characteristics similar to endogenous FIX.

[0325] Many functional FIX variants are known in the art. International Patent Application Publication No. WO02 / 040544A3 discloses on page 4, lines 9-30 and on page 15, lines 6-31 variants exhibiting increased resistance to inhibition by heparin. International Patent Application Publication No. WO03 / 020764A2 discloses in Tables 2 and 3 (pages 14-24) and on page 12, lines 1-27 variants of FIX with reduced T-cell immunogenicity. International Patent Application Publication No. WO2007 / 149406A2 discloses on page 4, line 1 to page 19, line 11 variants of FIX molecules exhibiting increased protein stability, increased in vivo and in vitro half-life, and increased resistance to proteases. WO2007 / 149406A2 also discloses chimeric and other variant FIX molecules on page 19, line 12 to page 20, line 9. International Patent Application Publication No. WO08 / 118507A2 discloses FIX variants exhibiting increased coagulation activity on page 5, line 14 to page 6, line 5. International Patent Application Publication No. WO09 / 051717A2 discloses FIX variants with an increased number of N-linked and / or O-linked glycosylation sites resulting in increased and / or restored half-life on page 9, line 11 to page 20, line 2. International Patent Application Publication No. WO09 / 137254A2 discloses factor IX variants with an increased number of glycosylation sites on page 2, paragraphs

[0006] to 5,

[0011] and on pages 16,

[0044] to 24,

[0057] . International Patent Application Publication No. WO09 / 130198A2 discloses, on page 4, line 26 to page 12, line 6, functional mutant FIX molecules with an increased number of glycosylation sites resulting in increased half-life. International Patent Application Publication No. WO09 / 140015A2 discloses, on page 11, paragraph

[0043] to page 13, paragraph

[0053] , functional FIX mutants with an increased number of Cys residues that can be used for conjugation of polymers (e.g., PEG). The FIX polypeptides described in International Patent Application No. PCT / US2011 / 043569, filed July 11, 2011 and published January 12, 2012 as WO2012 / 006624, are also incorporated herein by reference in their entirety.In some embodiments, the FIX polypeptide comprises a FIX polypeptide fused to albumin, e.g., FIX-albumin. In certain embodiments, the FIX polypeptide is IDELVION® or rIX-FP.

[0326] Additionally, hundreds of non-functional mutations in FIX have been identified in hemophilia subjects, many of which are disclosed in Table 6 of International Patent Application Publication No. WO09 / 137254A2, pages 11-14. Such non-functional mutations are not included in the present invention, but provide additional guidance as to which mutations are more or less likely to result in a functional FIX polypeptide.

[0327] In one embodiment, the FIX polypeptide (or the Factor IX portion of the fusion polypeptide) comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1 or 2 (amino acids 1 to 415 of SEQ ID NO: 125 or 126), or alternatively, the propeptide sequence, or the propeptide and signal sequence (full length FIX). In another embodiment, the FIX polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2.

[0328] FIX clotting activity is expressed as International Units (IU). One IU of FIX activity roughly corresponds to the amount of FIX in one milliliter of normal human plasma. Several assays are available for measuring FIX activity, including one-stage clotting assay (activated partial thromboplastin time; aPTT), thrombin generation time (TGA) and rotational thromboelastometry (ROTEM®). The present invention contemplates FIX sequences, sequences with homology to sequence fragments and non-natural sequence variants derived from natural, e.g., human, non-human primates, mammals (including domestic animals), that retain at least a portion of the bioactivity or biological function of FIX and / or are useful for preventing, treating, mediating or ameliorating diseases, deficiencies, disorders or conditions associated with clotting factors (e.g., bleeding episodes associated with trauma, surgery, deficiencies of clotting factors). Sequences with homology to human FIX can be found by standard homology search techniques, e.g., NCBI BLAST.

[0329] In certain embodiments, the FIX sequence is codon-optimized. Examples of codon-optimized FIX sequences include, but are not limited to, SEQ ID NOs: 1 and 54-58 of International Publication No. WO2016 / 004113 A1, which is incorporated herein by reference in its entirety.

[0330] C. FVII and polynucleotide sequences encoding the FVII protein In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, where the target sequence encodes a therapeutic protein, and the therapeutic protein comprises a Factor VII polypeptide. In some embodiments, the FVII polypeptide comprises FVII or a variant or fragment thereof having FVII activity.

[0331] "Factor VII" ("FVII", or "F7"; also called factor 7, clotting factor VII, serum factor VII, serum prothrombin converting accelerator, SPCA, proconvertin, and eptacog alfa) is a serine protease that is part of the coagulation cascade. In one embodiment, the coagulation factor in the nucleic acids described herein is FVII. Recombinant activated factor VII ("FVII") has become widely used to treat major bleeding, such as that occurring in patients with hemophilia A or B, clotting factor XI, FVII deficiency, defective platelet function, thrombocytopenia, or von Willebrand's disease.

[0332] Recombinant activated FVII (rFVIIa; NOVOSEVEN®) is used to treat bleeding episodes in (i) hemophilia patients with neutralizing antibodies (inhibitors) against FVIII or FIX, (ii) patients with FVII deficiency, or (iii) patients with hemophilia A or B, via surgical procedures with inhibitors. However, NOVOSEVEN® shows poor efficacy. Due to its low affinity for activated platelets in the absence of tissue factor, its short half-life, and its insufficient enzymatic activity, repeated administration of FVIIa at high concentrations is often required to control bleeding. Therefore, there is an unmet medical need for better treatment and prevention options for hemophilia patients with FVIII and FIX inhibitors and / or with FVII deficiency.

[0333] In one embodiment, the gene cassette encodes the mature form of FVII or its variants. FVII contains a Gla domain, two EGF domains (EGF-1 and EGF-2), and a serine protease domain (or peptidase S1 domain) that is highly conserved among all members of the peptidase S1 family of serine proteases, such as for example chymotrypsin. FVII occurs as a single-chain zymogen (i.e., activatable FVII) and a fully activated two-chain form.

[0334] C. Growth factors In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, wherein the target sequence encodes a therapeutic protein, and the therapeutic protein comprises a growth factor. The growth factor can be selected from any growth factor known in the art. In some embodiments, the growth factor is a hormone. In other embodiments, the growth factor is a cytokine. In some embodiments, the growth factor is a chemokine.

[0335] In some embodiments, the growth factor is adrenomedullin (AM). In some embodiments, the growth factor is an angiopoietin (Ang). In some embodiments, the growth factor is an autocrine cell motility stimulating factor. In some embodiments, the growth factor is a bone morphogenetic protein (BMP). In some embodiments, the BMP is selected from BMP2, BMP4, BMP5, and BMP7. In some embodiments, the growth factor is a member of the ciliary neurotrophic factor family. In some embodiments, the member of the ciliary neurotrophic factor family is selected from ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and interleukin-6 (IL-6). In some embodiments, the growth factor is a colony stimulating factor. In some embodiments, the colony stimulating factor is selected from macrophage colony stimulating factor (m-CSF), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the growth factor is an epidermal growth factor (EGF). In some embodiments, the growth factor is an ephrin. In some embodiments, the ephrin is selected from ephrinA1, ephrinA2, ephrinA3, ephrinA4, ephrinA5, ephrinB1, ephrinB2, and ephrinB3. In some embodiments, the growth factor is erythropoietin (EPO). In some embodiments, the growth factor is fibroblast growth factor (FGF). In some embodiments, the FGF is selected from FGF1, FGF2, F...

Claims

1. A nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR adjacent to a gene cassette comprising a heterologous polynucleotide sequence, wherein the first ITR consists of the nucleotide sequence set forth in SEQ ID NO:180 and the second ITR consists of the nucleotide sequence set forth in SEQ ID NO:181; or the first ITR consists of the nucleotide sequence set forth in SEQ ID NO:183 and the second ITR consists of the nucleotide sequence set forth in SEQ ID NO:

184.

2. The gene cassette further comprises a promoter, the promoter is positioned 5' to the heterologous polynucleotide sequence; the promoter is a tissue-specific promoter; the promoter drives expression of the heterologous polynucleotide sequence in an organ selected from muscle, the central nervous system (CNS), eye, liver, heart, kidney, pancreas, lung, skin, bladder, urinary tract, or any combination thereof; the promoter drives expression of the heterologous polynucleotide sequence in a hepatocyte, an endothelial cell, a cardiac muscle cell, a skeletal muscle cell, a sinusoidal cell, an afferent nerve, an efferent nerve, an interneuron, a glial cell, an astrocyte, an oligodendrocyte, a microglia, an ependymal cell, a pulmonary epithelial cell, a Schwann cell, a satellite cell, a photoreceptor cell, a retinal ganglion cell, or any combination thereof; and / or 2. The nucleic acid molecule of claim 1, wherein the promoter is selected from the group consisting of mouse thyretin promoter (mTTR), endogenous human factor VIII promoter (F8), human alpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, mouse albumin promoter, tristetraprolin (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, alpha 1-antitrypsin (AAT), muscle creatine kinase (MCK), myosin heavy chain alpha (αMHC), myoglobin (MB), desmin (DES), SPc5-12, 2R5Sc5-12, dMCK, tMCK, and phosphoglycerate kinase (PGK) promoter.

3. The nucleic acid molecule of claim 1 , wherein the heterologous polynucleotide sequence further comprises an intron sequence.

4. the intron sequence is located 5' to the heterologous polynucleotide sequence; the intron sequence is located 3' to the promoter; and / or The intron sequence comprises a synthetic intron sequence; The heterologous polynucleotide sequence encodes a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or any combination thereof. A nucleic acid molecule according to claim 3.

5. The heterologous polynucleotide sequence may be any of the following: adrenomedullin (AM), angiopoietin (Ang), autocrine motility stimulating factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor family members, colony stimulating factors, epidermal growth factor (EGF), ephrin, erythropoietin (EPO), fibroblast growth factor (FGF), fetal bovine somatotropin (FBS), GDNF family members, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma cell-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), ... , encoding a growth factor selected from the group consisting of interleukin (IL), keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein, myostatin (GDF-8), neuregulin, neurotrophin, placental growth factor (PGF), platelet derived growth factor (PDGF), renalase (RNLS), T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), and any combination thereof; the heterologous polynucleotide sequence encodes a hormone; The heterologous polynucleotide sequence encodes a cytokine; or the heterologous polynucleotide sequence encodes an antibody or fragment thereof; or The heterologous polynucleotide sequence encodes a gene selected from X-linked dystrophin, MTM1 (myotubularin), tyrosine hydroxylase, AADC, cyclohydrolase, SMN1, FXN (frataxin), GUCY2D, RS1, CFH, HTRA, ARMS, CFB / CC2, CNGA / CNGB, Prf65, ARSA, PSAP, IDUA (MPSI), IDS (MPSII), PAH, GAA (acid alpha glucosidase), and any combination thereof. A nucleic acid molecule according to any one of claims 1 to 4.

6. The heterologous polynucleotide sequence encodes a microRNA (miRNA); and / or The heterologous polynucleotide sequence is codon optimized. A nucleic acid molecule according to any one of claims 1 to 5.

7. The nucleic acid molecule of claim 6 , wherein the miRNA downregulates expression of a target gene selected from SOD1, HTT, RHO, and any combination thereof.

8. The nucleic acid molecule of claim 6 or 7, wherein the heterologous polynucleotide sequence is codon-optimized for expression in humans.

9. The nucleic acid molecule of claim 1 , wherein the gene cassette further comprises a post-transcriptional regulatory element.

10. the post-transcriptional regulatory element is located 3' to the heterologous polynucleotide sequence; and / or The post-transcriptional regulatory element is a mutant woodchuck hepatitis virus post-transcriptional regulatory element ( WPRE), a microRNA binding site, a DNA nuclear targeting sequence, or any combination thereof, or the microRNA binding site comprises a binding site for miR142-3p; The gene cassette further comprises a 3'UTR poly(A) tail sequence; The gene cassette further comprises an enhancer sequence, the enhancer sequence being located between the first ITR and the second ITR; the gene cassette comprises a single stranded nucleic acid; and / or The gene cassette comprises a double-stranded nucleic acid, The nucleic acid molecule of claim 9.

11. The nucleic acid molecule of claim 10, wherein the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof, or the 3'UTR poly(A) tail sequence comprises bGH poly(A).

12. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises, from 5' to 3': the first ITR, the gene cassette, and the second ITR; the gene cassette comprises a tissue-specific promoter sequence, an intron sequence, the heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3'UTR poly(A) tail sequence.

13. The nucleic acid molecule of claim 12, wherein the gene cassette comprises, from 5' to 3': a tissue-specific promoter sequence, an intron sequence, the heterologous polynucleotide sequence, a post-transcriptional regulatory element, and a 3'UTR poly(A) tail sequence.

14. (a) the tissue-specific promoter sequence comprises a TTP promoter; (b) the intron is a synthetic intron; (c) the post-transcriptional regulatory element comprises a WPRE; and (d) the 3'UTR poly(A) tail sequence comprises bGHpA; The nucleic acid molecule of claim 13.

15. The heterologous polynucleotide sequence may be selected from the group consisting of factor I (FI), factor II (FII), factor III (FIII), factor IV (FVI), factor V (FV), factor VI (FVII), factor VII (FVII), factor VIII (FVIII), factor IX (FIX), factor X (FX), factor XI (FXI), factor XII (FXII), factor XIII (FVIII), von Willebrand factor (VWF), prekallikrein, high molecular weight kininogen, fibronectin ... encoding a coagulation factor selected from the group consisting of nectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), and any combination thereof; The nucleic acid molecule of claim 1.

16. The nucleic acid molecule of claim 15, wherein the coagulation factor is FVIII.

17. 17. The nucleic acid molecule of claim 15 or 16, wherein the heterologous polynucleotide sequence encodes a full-length mature FVIII.

18. The full-length mature FVIII comprises the amino acid sequence of SEQ ID NO: 106, the FVIII comprising the A1 domain, the A2 domain, the A3 domain, the C1 domain, the C2 domain, and a partial B domain or no B domain, or the FVIII comprises the amino acid sequence of SEQ ID NO:

109.

18. The nucleic acid molecule of claim 17, comprising the amino acid sequence:

19. The coagulation factor comprises a heterologous moiety, the heterologous moiety being selected from the group consisting of albumin or a fragment thereof, an immunoglobulin Fc region, a C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), a PAS sequence, a HAP sequence, transferrin or a fragment thereof, an albumin binding portion, a derivative thereof, or any combination thereof; the FVIII further comprises the A1 domain, the A2 domain, the C1 domain, the C2 domain, an optional B domain, and a heterologous moiety, wherein the heterologous moiety is inserted immediately downstream of amino acid 745, which corresponds to mature FVIII (SEQ ID NO: 106); The FVIII further comprises an FcRn binding partner, the FcRn binding partner comprising an Fc region of an immunoglobulin constant domain; The nucleic acid sequence encoding FVIII is codon-optimized; and / or The nucleic acid molecule of any one of claims 15 to 18, wherein the nucleic acid sequence encoding FVIII is codon-optimized for expression in humans.

20. 20. The nucleic acid molecule of claim 19, wherein the heterologous moiety is linked to the N-terminus or C-terminus of FVIII or is inserted between two amino acids in FVIII.

21. The nucleic acid molecule of claim 19, wherein the nucleic acid sequence encoding the FVIII comprises the nucleotide sequence of SEQ ID NO: 107; or the nucleic acid sequence encoding the FVIII comprises the nucleotide sequence of SEQ ID NO:

71.

22. the nucleic acid molecule is formulated with a delivery agent; The nucleic acid molecule is formulated for intravenous, transdermal, intradermal, subcutaneous, pulmonary, or oral delivery, or any combination thereof; The nucleic acid molecule of claim 1.

23. 23. The nucleic acid molecule of claim 22, wherein the delivery agent comprises a lipid nanoparticle, or the delivery agent is selected from the group consisting of a liposome, a non-lipid polymer molecule, and an endosome, and any combination thereof.

24. The nucleic acid molecule of claim 22 or 23, wherein the nucleic acid molecule is formulated for intravenous delivery.

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

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

27. A pharmaceutical composition comprising the nucleic acid molecule of claim 1 and a pharma- ceutically acceptable excipient.

28. 27. A pharmaceutical composition comprising the host cell of claim 26 and a pharma- ceutically acceptable excipient.

29. 13. A kit comprising the nucleic acid molecule of claim 1 and instructions for administering the nucleic acid molecule to a subject in need thereof.

30. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is produced in an insect cell.

31. A nanoparticle delivery system comprising the nucleic acid molecule of claim 1.

32. 27. A method for producing a polypeptide, comprising culturing a host cell described in claim 26 under suitable conditions and recovering the polypeptide, wherein the polypeptide is a polypeptide having coagulation activity, and the method comprises recovering the polypeptide having coagulation activity.

33. 13. A pharmaceutical composition for use in a method for expressing a heterologous polynucleotide sequence in a subject in need thereof, said pharmaceutical composition comprising the nucleic acid molecule of claim 1.

34. 16. A pharmaceutical composition for use in a method for expressing a coagulation factor in a subject in need thereof, comprising the nucleic acid molecule of claim 15.

35. 13. A pharmaceutical composition for use in a method for treating a disease or disorder in a subject in need thereof, said pharmaceutical composition comprising the nucleic acid molecule of claim 1.

36. 16. A pharmaceutical composition for use in a method for treating a coagulation factor deficiency in a subject in need thereof, comprising the nucleic acid molecule of claim 15.

37. The nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonary, or any combination thereof; further comprising administering to the subject a second agent; the subject is a mammal; the subject is a human; administration of the nucleic acid molecule to the subject results in increased FVIII activity compared to FVIII activity in the subject prior to administration, the FVIII activity being increased by at least 2-fold; and / or The subject has a bleeding disorder.

37. The pharmaceutical composition of claim 36.

38. The pharmaceutical composition of claim 37, wherein the nucleic acid molecule is administered intravenously.

39. 38. The pharmaceutical composition of claim 37, wherein the bleeding disorder is hemophilia.

40. 40. The pharmaceutical composition of claim 39, wherein the bleeding disorder is hemophilia A.

41. 1. A pharmaceutical composition for use in a method of treating hemophilia A in a subject in need thereof, said pharmaceutical composition comprising a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding a coagulation factor, wherein the first ITR consists of the nucleotide sequence set forth in SEQ ID NO:180 and the second ITR consists of the nucleotide sequence set forth in SEQ ID NO:181; or the first ITR consists of the nucleotide sequence set forth in SEQ ID NO:183 and the second ITR consists of the nucleotide sequence set forth in SEQ ID NO:

184.

42. A pharmaceutical composition for use in a method of treating a liver metabolic disorder in a subject in need thereof, said pharmaceutical composition comprising a nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence encoding a liver-associated metabolic enzyme that is deficient in said subject, said first ITR consisting of the nucleotide sequence set forth in SEQ ID NO: 180, and said second ITR consisting of the nucleotide sequence set forth in SEQ ID NO: 181; or said first ITR consisting of the nucleotide sequence set forth in SEQ ID NO:

183. The pharmaceutical composition as described above, wherein the first ITR consists of a nucleotide sequence as set forth in SEQ ID NO:

184.

43. The gene cassette comprises a single-stranded nucleic acid; The gene cassette comprises a double-stranded nucleic acid; The liver metabolic disorder is selected from the group consisting of phenylketonuria (PKU), urea cycle disorders, lysosomal storage diseases, and glycogen storage diseases; The nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonary, or any combination thereof; further comprising administering a second agent to the subject; the subject is a mammal; and / or The subject is a human.

43. The pharmaceutical composition of claim 42.

44. 44. The pharmaceutical composition of claim 43, wherein the hepatic metabolic disorder is phenylketonuria (PKU).

45. 45. The pharmaceutical composition of claim 43 or 44, wherein the nucleic acid molecule is administered intravenously.

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