Creation of closed-end DNA using inverted terminal repeats

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 first ITR and / or the second ITR is the ITR of human bocavirus.Also disclosed is a method of using the nucleic acid molecule in gene therapy applications.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 236,215, filed August 23, 2021, which is incorporated by reference in its entirety.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the sequence listing submitted electronically in XML format (Name: SA9-481_SeqListing.xml; Size: 117,285 bytes; and Creation Date: August 22, 2022) are incorporated by reference in their entirety herein. [Background technology]

[0003] Gene therapy offers the potential for a long-term means of treating various diseases. In the past, many gene therapy treatments typically rely on the use of viral vectors. There are numerous viral agents selected for this purpose, each with significantly different properties that make them more or less suitable for gene therapy. However, the undesirable properties of some viral vectors result in concerns about clinical safety, limiting their therapeutic use. Summary of the Invention [Problem to be solved by the invention]

[0004] Adeno-associated virus (AAV) is a common gene therapy vector, but without its drawbacks. The coding sequence of the AAV genome is flanked by inverted terminal repeats (ITRs), which are required for viral replication and packaging, as well as transgene expression. The T-shaped hairpin structure of the AAV ITRs is susceptible to binding by host cell proteins that inhibit transgene expression within the AAV vector. There is a need to provide efficient and sustained expression of target sequences while circumventing the limitations of existing AAV vector technology. [Means for solving the problem]

[0005] Disclosed herein are nucleic acid molecules comprising a first inverted terminal repeat (ITR) and / or a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence, and uses thereof.

[0006] In one aspect herein, a nucleic acid molecule is provided that includes a first ITR and a second ITR flanking a gene cassette that includes a heterologous polynucleotide sequence, where the first ITR and the second ITR are bocavirus ITRs or fragments / derivatives thereof (e.g., human bocavirus type 1 ITRs). In another aspect herein, a nucleic acid molecule is provided that includes a first ITR and a second ITR, where the first ITR includes a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:1, and the second ITR includes a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:2.

[0007] In some embodiments, the first ITR comprises a polynucleotide sequence that is 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% identical to SEQ ID NO: 1. In some embodiments, the first ITR comprises a polynucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the first ITR comprises a polynucleotide sequence that is at least about 50% identical to SEQ ID NO: 1.

[0008] In some embodiments, the second ITR comprises a polynucleotide sequence that is 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% identical to SEQ ID NO: 2. In some embodiments, the second ITR comprises a polynucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the first ITR comprises a polynucleotide sequence that is at least about 50% identical to SEQ ID NO: 2.

[0009] In some embodiments, the first ITR comprises the polynucleotide sequence set forth in SEQ ID NO:1 and the second ITR comprises the polynucleotide sequence set forth in SEQ ID NO:2.

[0010] In some embodiments, the nucleic acid molecule further comprises a gene cassette comprising the heterologous polynucleotide sequence and at least one expression control sequence, such as a promoter, enhancer, intron, transcriptional termination signal, or post-transcriptional regulatory element.

[0011] In some embodiments, the gene cassette further comprises a promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter drives the expression of the heterologous polynucleotide sequence in an organ, where the organ comprises muscle, central nervous system (CNS), visual organ, liver, heart, kidney, pancreas, lung, skin, bladder, urinary tract, spleen, myeloid and lymphoid cell lineages, or any combination thereof. In some embodiments, the promoter drives the expression of the heterologous polynucleotide sequence in hepatocytes, epithelial cells, endothelial cells, cardiac myocytes, skeletal muscle cells, sinusoidal cells, afferent neurons, efferent neurons, interneurons, glial cells, astrocytes, oligodendrocytes, microglia, ependymal cells, lung epithelial cells, Schwann cells, satellite cells, photoreceptor cells, retinal ganglion cells, T cells, B cells, NK cells, macrophages, dendritic cells, or any combination thereof. In some embodiments, the promoter is located 5' to the heterologous polynucleotide sequence. In some embodiments, the promoter is a mouse transthyretin promoter (mTTR), a native human factor VIII promoter, a human alpha 1 antitrypsin promoter (hAAT), a human albumin minimal promoter, a mouse albumin promoter, a tristetraprolin (TTP) promoter, a CASI promoter, a CAG promoter, a cytomegalovirus (CMV) promoter, an alpha 1 antitrypsin (AAT) promoter, a muscle creatine kinase (MCK) promoter, a myosin heavy chain alpha (αMHC) promoter, a myoglobin (MB) promoter, a desmin (DES) promoter, a SPc5-12 promoter, a 2R5Sc5-12 promoter, a dMCK promoter, a tMCK promoter, or a phosphoglycerate kinase (PGK) promoter.

[0012] In some embodiments, the gene cassette further comprises an intron sequence. In some embodiments, the intron sequence is located 5' to the heterologous polynucleotide sequence. In some embodiments, the intron sequence is located 3' to the promoter. In some embodiments, the intron sequence comprises a synthetic intron sequence.

[0013] In some embodiments, the gene cassette further comprises a post-transcriptional regulatory element.In some embodiments, the regulatory element is located 3' to the heterologous polynucleotide sequence.In some embodiments, the regulatory element comprises a mutant woodchuck hepatitis virus regulatory element (WPRE), a microRNA binding site, a DNA nuclear targeting sequence, or any combination thereof.

[0014] In some embodiments, the gene cassette further comprises a 3'UTR poly(A) tail sequence. In some 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.

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

[0016] In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a first ITR, a gene cassette, and a second ITR, where 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.

[0017] In some 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.

[0018] In some embodiments, the gene cassette is a single-stranded nucleic acid. In some embodiments, the gene cassette is a double-stranded nucleic acid.

[0019] In some embodiments, the heterologous polynucleotide sequence encodes a therapeutic protein.

[0020] In some embodiments, the heterologous polynucleotide sequence encodes a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or any combination thereof. In some embodiments, the heterologous polynucleotide sequence encodes a clotting factor. In some embodiments, the heterologous polynucleotide sequence encodes a growth factor. In some embodiments, the heterologous polynucleotide sequence encodes a hormone. In some embodiments, the heterologous polynucleotide sequence encodes a cytokine.

[0021] In some embodiments, the heterologous polynucleotide sequence encodes a FVIII protein.

[0022] In some embodiments, the heterologous polynucleotide sequence encodes 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), GALT, OTC, CMD1A, LAMA2, or any combination thereof.

[0023] In some embodiments, the heterologous polynucleotide sequence encodes a microRNA (miRNA). In some embodiments, the miRNA downregulates the expression of target genes, including SOD1, HTT, RHO, CD38, or any combination thereof.

[0024] In some embodiments, the heterologous polynucleotide sequence encodes a coagulation factor, where the coagulation factor is Factor I (FI), Factor II (FII), Factor III (FIII), Factor IV (FIV), 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 (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), or any combination thereof.

[0025] In some embodiments, the heterologous polynucleotide sequence is codon optimized. In some embodiments, the heterologous polynucleotide sequence is codon optimized for expression in humans.

[0026] In some embodiments, the nucleic acid molecule is formulated with a delivery agent. In some embodiments, the delivery agent comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle is ionic. In some embodiments, the delivery agent comprises a liposome, a non-lipid polymer molecule, an endosome, or any combination thereof.

[0027] In some embodiments, the nucleic acid molecule is formulated for intravenous, transdermal, intradermal, intraneuronal, intraocular, intrathecal, subcutaneous, pulmonary, or oral administration, or any combination thereof. In some embodiments, the nucleic acid molecule is formulated for intravenous administration. In some embodiments, the nucleic acid molecule is formulated for administration by in situ injection. In some embodiments, the nucleic acid molecule is formulated for administration by inhalation.

[0028] In another aspect of the present specification, there is provided a vector comprising a nucleic acid molecule described herein.

[0029] In another aspect of the present specification, a host cell is provided that comprises a nucleic acid molecule described herein or a vector described herein. In some embodiments, the host cell is an insect cell.

[0030] In another aspect of the present specification, there is provided a pharmaceutical composition comprising a nucleic acid molecule described herein.

[0031] In another aspect of the present specification, there is provided a pharmaceutical composition comprising a vector described herein and a pharma- ceutically acceptable excipient.

[0032] In another aspect of the present specification, there is provided a pharmaceutical composition comprising a host cell described herein and a pharma- ceutically acceptable excipient.

[0033] In another aspect of the present specification, there is provided a kit comprising a nucleic acid molecule described herein and instructions for administering the nucleic acid molecule to a subject in need thereof.

[0034] In another aspect of the present specification, there is provided a baculovirus system for producing the nucleic acid molecules described herein.

[0035] In some embodiments, the nucleic acid molecule is produced in an insect cell.

[0036] In another aspect herein, there is provided a nanoparticle delivery system comprising a nucleic acid molecule as described herein.

[0037] In another aspect herein, there is provided a method of expressing a heterologous polynucleotide sequence in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, or a pharmaceutical composition described herein.

[0038] In another aspect herein, there is provided a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a vector described herein, or a pharmaceutical composition described herein.

[0039] In some embodiments, the nucleic acid molecule is administered intravenously, transdermally, intradermally, subcutaneously, orally, pulmonary, intraneuronally, intraocularly, intrathecally, or any combination thereof. In some embodiments, the nucleic acid molecule is administered intravenously. In some embodiments, the nucleic acid molecule is administered by in situ injection. In some embodiments, the nucleic acid molecule is administered by inhalation.

[0040] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human. [Brief description of the drawings]

[0041] [Figure 1-1] 1A-1C show schematic representations of approaches used for ceDNA production in the baculovirus system according to one embodiment of the present invention. FIG. 1A shows a schematic diagram of the One BAC approach where a single recombinant BEV encoding FVIIIXTEN and Rep genes at different loci is used for infection in Sf9 cells for ceDNA production. FIG. 1B shows a schematic diagram of the Two BAC approach where Sf9 cells are co-infected with recombinant BEV encoding FVIIIXTEN and / or Rep genes for ceDNA production. FIG. 1C shows a schematic diagram of the stable cell line approach where a FVIIIXTEN expression cassette is stably integrated into the Sf9 cell genome and rescued by infection with recombinant BEV encoding Rep gene for ceDNA production. [Figure 1-2] Continued from Figure 1-1. [Diagram 2]2A-2B are schematic representations of human FVIIIXTEN expression constructs. Figure 2A shows a linear schematic map of an expression construct according to one embodiment of the present invention, which is composed of B-domain deleted (BDD) codon-optimized human factor VIII (coFVIII) fused with XTEN 144 peptide (FVIIIXTEN) under the control of a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) with enhancer element (A1MB2), a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal. The FVIIIXTEN expression cassette is flanked by human bocavirus type 1 (HBoV1) wild-type (WT) ITRs (SEQ ID NO: 1 and SEQ ID NO: 2). FIG. 2B shows a schematic map for a Tn7 transfer vector according to one embodiment of the present invention, generated by inserting a FVIIIXTEN expression cassette (SEQ ID NO:3) into the pFastBac1 vector (Invitrogen). [Diagram 3] 3A-3C are schematic representations of replication (Rep) gene expression constructs according to embodiments of the present invention. FIG. 3A shows a linear schematic map of a synthetic DNA encoding the Sf codon-optimized HBoV1 NS1 gene under the AcMNPV polyhedrin promoter followed by the SV40 polyadenylation signal (SV40 PAS). FIG. 3B shows a schematic map of a Tn7 transfer vector according to embodiments of the present invention, generated by inserting HBoV1 NS1 synthetic DNA (SEQ ID NO: 4) into the pFastBac1 vector (Invitrogen). FIG. 3C shows a schematic map of a Cre-LoxP donor vector according to embodiments of the present invention, generated by inserting HBoV1 NS1 synthetic DNA (SEQ ID NO: 4) into the Cre-LoxP donor vector, created as described in "Baculovirus Expression System," U.S. Patent Application No. 63 / 069,073, which is incorporated herein by reference in its entirety. [Figure 4]4A-4C are schematic representations of replication (Rep) gene expression constructs according to embodiments of the present invention. FIG. 4A shows a linear schematic map of a synthetic DNA encoding the Sf codon-optimized HBoV1 NS1 gene followed by the SV40 polyadenylation signal (SV40 PAS) under the AcMNPV immediate early 1 (pIE1) promoter preceded by the AcMNPV transcriptional enhancer hr5 element. FIG. 4B shows a schematic map of a Tn7 transfer vector according to embodiments of the present invention, generated by inserting the HBoV1 NS1 synthetic DNA (SEQ ID NO: 4) into the pFastBac1 vector (Invitrogen). FIG. 4C shows a schematic map of a Cre-LoxP donor vector according to embodiments of the present invention, generated by inserting the HBoV1 NS1 synthetic DNA (SEQ ID NO: 4) into the Cre-LoxP donor vector. [Diagram 5] 5A-5D are schematic representations of replication (Rep) gene expression constructs according to an embodiment of the present invention. FIG. 5A shows a linear schematic map of synthetic DNA encoding the Sf codon-optimized HBoV1 NS1 gene followed by the SV40 polyadenylation signal (SV40 PAS) under the OpMNPV immediate early 2 (OpIE2) promoter. FIG. 5B shows a linear schematic map of synthetic DNA encoding the Sf codon-optimized HBoV1 NS1 gene followed by the SV40 polyadenylation signal (SV40 PAS) under the AcMNPV immediate early 1 (pIE1) promoter. FIG. 5C shows a schematic map of a Tn7 transfer vector according to an embodiment of the present invention, generated by inserting HBoV1 NS1 synthetic DNA (SEQ ID NO: 4) into the pFastBac1 vector (Invitrogen). FIG. 5D shows a schematic map for a Cre-LoxP donor vector according to an embodiment of the present invention, produced by inserting HBoV1 NS1 synthetic DNA (sequence number 4) under the AcMNPV immediate early 1 (pIE1) promoter into a Cre-LoxP donor vector created as described in U.S. Patent Application No. 63 / 069,073. [Figure 6A]

[0023] Figure 6 shows the generation of a recombinant baculovirus expression vector (BEV) encoding human FVIIIXTEN with HBoV1 ITRs. Figure 6A shows an agarose gel electrophoresis image of the restriction enzyme mapping of a recombinant BIVVBac bacmid encoding cloned human FVIIIXTEN with HBoV1 ITRs (BIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7). [Figure 6B]

[0023] Figure 6B is a schematic representation of a recombinant baculovirus expression vector (BEV) encoding human FVIIIXTEN together with HBoV1 ITRs. Figure 6B is a schematic representation of a recombinant BEV (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7) encoding a FVIIIXTEN expression cassette flanked by HBoV1 ITRs (SEQ ID NO:3) as indicated. [Figure 7A] 7A-7C are schematic representations of One BAC composed of human FVIIIXTEN and Rep gene expression cassette and validation studies thereof. Figures 7A-7C are agarose gel electrophoresis images of recombinant bacmid clones BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP (Figure 7A), BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)IE1.HBoV1.NS1LoxP (Figure 7B), and BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)HR5.IE1.HBoV1.NS1LoxP (Figure 7C), screened with outer / inner PCR primers (SEQ ID NO:5 and SEQ ID NO:6) as indicated by red arrows in Figures 7D-7F. [Figure 7B]7A-7C are schematic representations of One BAC composed of human FVIIIXTEN and Rep gene expression cassette and validation studies thereof. Figures 7A-7C are agarose gel electrophoresis images of recombinant bacmid clones BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP (Figure 7A), BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)IE1.HBoV1.NS1LoxP (Figure 7B), and BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)HR5.IE1.HBoV1.NS1LoxP (Figure 7C), screened with outer / inner PCR primers (SEQ ID NO:5 and SEQ ID NO:6) as indicated by red arrows in Figures 7D-7F. [Figure 7C] 7A-7C are schematic representations of One BAC composed of human FVIIIXTEN and Rep gene expression cassette and validation studies thereof. Figures 7A-7C are agarose gel electrophoresis images of recombinant bacmid clones BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP (Figure 7A), BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)IE1.HBoV1.NS1LoxP (Figure 7B), and BIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)HR5.IE1.HBoV1.NS1LoxP (Figure 7C), screened with outer / inner PCR primers (SEQ ID NO:5 and SEQ ID NO:6) as indicated by red arrows in Figures 7D-7F. [Figure 7D]7A-7D show schematic representations of One BAC composed of human FVIIIXTEN and a Rep gene expression cassette and validation studies for the same. Figure 7D shows schematic map for a recombinant baculovirus expression vector (BEV) (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP) encoding HBoV1 NS1 under the AcMNPV polyhedrin (pPolh) promoter and a FVIIIXTEN expression cassette flanked by HBoV1 ITRs, as indicated. [Figure 7E] 7A-7D show schematic representations of One BAC composed of human FVIIIXTEN and a Rep gene expression cassette and validation studies for the same. Figure 7E shows schematic map for recombinant BEV (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)IE1.HBoV1.NS1LoxP) encoding HBoV1 NS1 under the AcMNPV immediate early 1 (pIE1) promoter, flanked by HBoV1 ITRs, and a FVIIIXTEN expression cassette, as indicated. [Figure 7F] 7A-7D show schematic representations of One BAC composed of human FVIIIXTEN and a Rep gene expression cassette and validation studies thereof. Figure 7F shows schematic map for recombinant BEV (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)HR5.IE1.HBoV1.NS1LoxP) encoding HBoV1 NS1 under the AcMNPV immediate early 1 promoter preceded by the AcMNPV transcriptional enhancer hr5 element (pHR5.IE1) and a FVIIIXTEN expression cassette flanked by HBoV1 ITRs, as indicated. [Figure 8A]Figure 8A shows the construction of human FVIIIXTEN ceDNA vector using One BAC approach according to one embodiment of the present invention. Figure 8A is a schematic diagram of the One BAC approach of FVIIIXTEN ceDNA vector construction in Sf9 cells using recombinant BEV encoding HBoV1 NS1 gene under AcMNPV polyhedrin promoter and human FVIIIXTEN expression cassette flanked by HBoV1 ITR (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP). [Figure 8B] FIG. 8B shows the generation of a human FVIIIXTEN ceDNA vector using the One BAC approach according to one embodiment of the present invention. FIG. 8B shows a schematic map for AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP BEV. [Figure 8C] Figure 8C shows the construction of human FVIIIXTEN ceDNA vector using One BAC approach according to one embodiment of the present invention. Figure 8C shows the agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with titrated virus stock (P2) of (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP)BEV. The DNA bands corresponding to the sizes of FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA) are indicated by arrows. [Figure 9-1]9A-9D are schematics of recombinant baculovirus expression vectors (BEVs) containing sequences encoding HBoV1 NS1 and validation studies thereof. Figure 9A is an agarose gel electrophoresis image of restriction enzyme mapping of recombinant bacmid clones of HBoV1.NS1 under AcMNPV polyhedrin, under the immediate early 1 promoter preceded by the AcMNPV transcriptional enhancer hr5 element, or under the OpMNPV immediate early 2 promoter (BIVVBac.Polh.HBoV1.NS1Tn7, BIVVBac.HR5.IE1.HBoV1.NS1Tn7, and BIVVBac.OpIE2.HBoV1.NS1Tn7, respectively). Figure 9B shows a schematic map for AcBIVVBac.Polh.HBoV1.NS1Tn7. Figure 9C shows a schematic map for AcBIVVBac.HR5.IE1.HBoV1.NS1Tn7. Figure 9D shows a schematic map for AcBIVVBac.OpIE2.HBoV1.NS1Tn7. [Figure 9-2] Continued from Figure 9-1. [Figure 10A] Figure 10A shows the generation of human FVIIIXTEN ceDNA vector using Two BAC approach according to one embodiment of the present invention. Figure 10A is a schematic diagram of the Two BAC approach for the generation of FVIIIXTEN ceDNA vector when Sf9 cells are co-infected with recombinant BEV encoding FVIIIXTEN expression cassette flanked by HBoV1 ITR (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7) and / or encoding HBoV1 NS1 gene under AcMNPV polyhedrin promoter (AcBIVVBac.Polh.HBoV1.NS1Tn7). [Figure 10B] FIG. 10B shows the generation of a human FVIIIXTEN ceDNA vector using a Two BAC approach according to one embodiment of the present invention. FIG. 10B shows a schematic map for AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEV. [Figure 10C] Figure 10C shows the generation of human FVIIIXTEN ceDNA vector using Two BAC approach according to one embodiment of the present invention. Figure 10C shows the agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEV at constant ratio or different ratios of these at constant MOI, as indicated. Arrows indicate the DNA bands that correspond to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 11A] Figure 11A shows the construction of human FVIIIXTEN ceDNA vector using Two BAC approach according to one embodiment of the present invention. Figure 11A is a schematic diagram of Two BAC approach for the construction of FVIIIXTEN ceDNA vector when Sf9 cells are co-infected with recombinant BEV encoding FVIIIXTEN expression cassette flanked by HBoV1 ITR (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7) and / or encoding HBoV1 NS1 gene under AcMNPV polyhedrin promoter (AcBIVVBac.Polh.HBoV1.NS1Tn7) or immediate early 1 promoter preceded by AcMNPV transcription enhancer hr5 element (AcBIVVBac.HR5.IE1.HBoV1.NS1Tn7). [Figure 11B] FIG. 11B shows the generation of a human FVIIIXTEN ceDNA vector using a Two BAC approach according to one embodiment of the present invention. FIG. 11B shows a schematic map for AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.HR5.IE1.HBoV1.NS1Tn7 BEV. [Figure 11C]Figure 11C shows the construction of human FVIIIXTEN ceDNA vector using Two BAC approach according to one embodiment of the present invention. Figure 11C shows the agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEV (left image), or AcBIVVBac.HR5.IE1.HBoV1.NS1Tn7BEV (right image). Arrows indicate the DNA bands that correspond to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 12] 12A-12C show materials used in the generation of stable cell lines encoding a FVIIIXTEN expression cassette flanked by HBoV1 ITRs. FIG. 12A shows a schematic map for a plasmid encoding a neomycin resistance marker under the AcMNPV immediate early 1 (IE1) promoter preceded by the AcMNPV transcription enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal (P10 PAS). FIG. 12B shows a schematic map for a plasmid encoding an enhanced green fluorescent protein (eGFP) marker under the AcMNPV immediate early 1 (IE1) promoter preceded by the AcMNPV transcription enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal (P10 PAS). FIG. 12C shows a schematic map for a FVIIIXTEN expression cassette (SEQ ID NO: 3) flanked by HBoV1 ITRs stably integrated into the Sf9 cell genome to generate a stable cell line. [Figure 13-1]Figures 13A-13E show a workflow for the generation / purification of FVIIIXTEN ceDNA vector using the Two BAC approach according to one embodiment of the present invention. Figure 13A shows a schematic of Sf9 cell expansion and persistence (on days 0-2) where the cells were scaled up sequentially from small-scale culture flasks (0.5 L) to large-scale culture flasks (1.5 L) to achieve a cell density of 2.5-3.0 x 106 cells per mL in serum-free ESF921 medium. FIG. 13B shows a schematic of infection and duration of incubation (on days 2-6) in Sf9 large culture flasks (1.5 L) where cells were co-infected with recombinant BEV encoding a FVIIIXTEN expression cassette flanked by HBoV1 ITRs (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7) and / or encoding the HBoV1 NS1 gene under the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.HBoV1.NS1Tn7) at 0.1 and 0.01 plaque forming units (pfu) per cell, respectively. Figure 13C shows images of Plasmid Giga Prep Purification Kit and agarose gel electrophoresis (at day 6-7) with duration of treatment, where cell density and viability of infected cells were measured daily, and cells were pelleted by low speed centrifugation when cell viability reached 70-80%. FVIIIXTEN ceDNA vector was purified from infected cell pellets by PureLink™ HiPure Expi Plasmid Gigaprep Kit (Invitrogen), and aliquots were subjected to agarose gel electrophoresis to determine the productivity of FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and / or Sf9 cell genomic DNA (gDNA).Figure 13D shows images of a Bio-Rad Model 491 Prep Cell with duration of process in which the Giga-prep purified DNA was loaded onto a Preparative Agarose Gel in the Prep Cell to separate FVIIIXTEN ceDNA (approximately 8.5 kb fragment) from high molecular weight DNA, and agarose gel electrophoresis (at days 7-12). Elution fractions collected at 70-80 minute intervals from the Preparative Agarose Gel Electrophoresis were analyzed on a 0.8-1.2% agarose gel to determine the purity of FVIIIXTEN ceDNA. Figure 13E shows images of agarose gel electrophoresis in which fractions collected from the Prep Cell were combined and precipitated with 1 / 10 volume of 3M NaOAc (pH 5.5) and 3 volumes of 100% ethanol to obtain purified FVIIIXTEN ceDNA. The gel image shows the purity of FVIIIXTEN ceDNA compared to the starting material, with arrows pointing to DNA bands corresponding in size to the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 13-2] Continued from Figure 13-1. [Figure 14]14A-14B are graphical representations of plasma FVIII activity levels measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay. FIG. 14A shows a plot of plasma FVIII activity levels measured in blood samples collected at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected with 1600 or 400 μg of single-stranded FVIIIXTEN HBoV1 ITR DNA (ssDNA) per kg via fluid tail vein injection. FIG. 14B shows a plot of plasma FVIII activity levels measured in blood samples collected at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected with 80, 40, or 12 μg of FVIIIXTEN HBoV1 ITR ceDNA (ceDNA) per kg via fluid tail vein injection. Error bars represent standard deviation. [Figure 15] 15A-15C show red fluorescent (upper panels) or bright field (lower panels) microscopy images of Sf9 cells co-transfected with AcBIVVBac.Polh.HBoV1.NS1Tn7 bacmid DNA and VP80 sgRNA according to one embodiment of the present invention. FIG. 15A shows a microscopy image of cells co-transfected with AcBIVVBac.Polh.HBoV1.NS1Tn7 bacmid DNA and Cas9 alone. FIG. 15B shows a microscopy image of cells co-transfected with AcBIVVBac.Polh.HBoV1.NS1Tn7 bacmid DNA and sgRNA.VP80.T1. FIG. 15C shows a microscopy image of cells co-transfected with AcBIVVBac.Polh.HBoV1.NS1Tn7 bacmid DNA and sgRNA.VP80.T2. [Figure 16]Figures 16A-16C are diagrams showing the generation of VP80KO BEV. Figures 16A and 16B illustrate TIDE analysis for BEV clones AcBIVVBac.Polh.HBoV1.NS1ΔVP80Tn7 and AcBIVVBac.OpIE2.HBoV1.NS1ΔVP80Tn7 determining CRISPR / Cas9-induced indels. Figure 16C is an agarose gel electrophoresis image of FVIIIXTEN ceDNA vector isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1ΔVP80Tn7 BEV, or AcBIVVBac.OpIE2.HBoV1.NS1ΔVP80Tn7 BEV, as indicated. Arrows indicate DNA bands corresponding to the sizes of the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA). [Figure 17A]

[0033] Figure 17A shows the generation of human FVIIIXTEN HBoV1 ceDNA using the Two BAC approach. Figure 17A is a schematic diagram of the Two BAC approach for the generation of FVIIIXTEN ceDNA vectors. [Figure 17B] Figure 17 shows the generation of human FVIIIXTEN HBoV1 ceDNA using the Two BAC approach. Figure 17B shows the schematic map for AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEV. [Figure 17C]Figure 17C shows the generation of human FVIIIXTEN HBoV1 ceDNA using Two BAC approach. Figure 17C shows the agarose gel electrophoresis image of FVIIIXTEN ceDNA vector isolated from Sf9 cells co-infected with AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRTn7 BEV and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEV at MOI of 1.0, 2.0, 3.0, 4.0 or 5.0. Arrows indicate the DNA bands that correspond to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA). [Figure 18-1] 18A-18D are diagrams showing the generation of human FVIIIXTEN HBoV1 ceDNA vectors using the One BAC approach. FIG. 18A is a schematic diagram of the One BAC approach of FVIIIXTEN ceDNA vector generation in Sf9 cells. FIG. 18B shows a schematic map for AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1LoxP BEV. FIG. 18C is an agarose gel electrophoresis image of ceDNA isolated from HBoV1 OneBAC BEV, a clone amplified to P2 in Sf9 cells. FIG. 18D is an agarose gel electrophoresis image of ceDNA isolated from Sf9 cells infected with HBoV1 OneBAC BEV at MOIs of 0.1, 0.2, 0.3, 0.4, or 0.5. DNA bands corresponding in size to FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (gDNA) are indicated by arrows. [Figure 18-2] Continued from Figure 18-1. [Figure 19-1]19A-19C show the generation and testing of HBoV1 ssDNA and HBoV1 ceDNA in vivo. FIG. 19A is an agarose gel electrophoresis image for single stranded DNA (ssDNA) FVIIIXTEN HBoV1. FIG. 19B is an agarose gel electrophoresis image for FVIIIXTEN HBoV1 ceDNA. FIG. 19C shows FVIII expression levels normalized against percent of normal for ssFVIIIXTEN and ceFVIIIXTEN. Error bars represent standard deviation. [Figure 19-2] Continued from Figure 19-1. [Figure 20] Figures 20A-20B show the test of monomeric and multimeric forms of FVIIIXTEN HBoV1 ceDNA. Figure 20A shows the agarose gel electrophoresis image of monomeric and multimeric forms of FVIIIXTEN HBoV1 ceDNA. Figure 20B shows the FVIII expression level normalized against the percentage of normal in mice injected with monomeric and multimeric forms of FVIIIXTEN HBoV1 ceDNA. Error bars represent standard deviation. [Figure 21-1] Figures 21A-21C show the study of liver-specific mTTR and human A1AT promoters driving expression of FVIIIXTEN in HBoV1 ITR constructs. Figure 21A is a schematic diagram of FVIIIXTEN expression cassettes with liver-specific mTTR or A1AT promoters flanked by HBoV1 WT ITRs. Figure 21B is an agarose gel electrophoresis image of single-stranded DNA (ssDNA) FVIIIXTEN HBoV1 generated by restriction enzyme digestion as described. Figure 21C shows FVIII expression levels normalized to percent of normal in mice injected with mTTR or A1AT promoter constructs depicted in Figure 21A. Error bars represent standard deviation. [Figure 21-2] Continued from Figure 21-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Disclosed herein are nucleic acid molecules and uses thereof, comprising a first modified inverted terminal repeat (ITR) and / or a second modified ITR flanking a gene cassette comprising a heterologous polynucleotide sequence. In some embodiments, the first ITR and / or the second ITR are derived from human bocavirus type 1 (HBoV1).

[0043] Exemplary constructs of the present disclosure are illustrated in the accompanying figures and sequence listing. For a clear understanding of the specification and claims, the following definitions are provided below.

[0044] definition It is noted that the term "a" entity or "an" entity refers to one or more of that entity: for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Similarly, "a therapeutic protein" and "a miRNA" are understood to refer to one or more therapeutic proteins and one or more miRNAs, respectively. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0045] The term "about" is used herein to mean approximately, in the region of, roughly, or in the vicinity thereof. When used in conjunction with a numerical range, the term "about" modifies the range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10 percent upward or downward (high or low).

[0046] As used herein, "and / or" when interpreted in the alternative ("or") also refers to and includes any / all possible combinations of one or more of the associated listed items, as well as the absence of combinations.

[0047] "Nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide(s) sequence", and "polynucleotide" are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded form or within a double-stranded helix. Single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). DNA-DNA helices, DNA-RNA helices, and RNA-RNA helices, which are double-stranded, are also possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit the molecule to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence is described herein, following the usual convention, showing only the sequence along the non-transcribed strand of DNA (i.e., the strand with sequence homology to mRNA) in the 5' to 3' direction. 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 as described herein.

[0048] As used herein, "inverted terminal repeat" (or "ITR") refers to a nucleic acid subsequence located at the 5' or 3' end of a single stranded nucleic acid sequence that comprises a set of nucleotides (initial sequence) followed downstream by its reverse complement, i.e., a palindromic sequence. The intervening nucleotide sequence between the initial sequence and the reverse complement can be of any length, including zero. In one embodiment, an ITR useful in the present disclosure comprises one or more "palindromic sequences." An ITR can have any number of functions. In some embodiments, an ITR described herein forms a hairpin structure. In some embodiments, an ITR forms a T-shaped hairpin structure. In some embodiments, an ITR forms a hairpin structure other than a T-shaped, e.g., a U-shaped hairpin structure. In some embodiments, an ITR promotes the survival of a nucleic acid molecule in a cell nucleus over an extended period of time. In some embodiments, an ITR promotes the permanent survival (e.g., for the entire lifespan of a cell) of a nucleic acid molecule in a cell nucleus. In some embodiments, an ITR promotes the stability of a nucleic acid molecule in a cell nucleus. In some embodiments, the ITRs promote the retention of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs promote the persistence of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs inhibit or prevent the degradation of the nucleic acid molecule in the cell nucleus.

[0049] In one embodiment, the initial sequence and / or reverse complement of the ITR comprises from about 2 to 600 nucleotides, from about 2 to 550 nucleotides, from about 2 to 500 nucleotides, from about 2 to 450 nucleotides, from about 2 to 400 nucleotides, from about 2 to 350 nucleotides, from about 2 to 300 nucleotides, or from about 2 to 250 nucleotides. In some embodiments, the initial sequence and / or 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 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 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.

[0050] In other embodiments, the initial sequence and / or reverse complement of the ITR 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 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 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 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 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.

[0051] Thus, an "ITR" as used herein may fold back on itself to form a double-stranded segment. For example, the sequence GATCXXXXGATC includes an initial sequence of GATC and its complement (3'CTAG5') such that when folded, they form a double helix. In some embodiments, an ITR includes a continuous palindromic sequence (e.g., GATCGATC) between the initial sequence and the reverse complement. In some embodiments, an ITR includes an interrupted palindromic sequence (e.g., GATCXXXXGATC) between the initial sequence and the reverse complement. In some embodiments, the complementary portions of the continuous or interrupted palindromic sequences interact with each other to form a "hairpin loop" structure. A "hairpin loop" structure, as used herein, occurs when at least two complementary sequences on a single-stranded nucleotide molecule base pair to form a double-stranded portion. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop. In some embodiments, the ITRs retain the Rep binding element (RBE) of the wild-type ITR from which they are derived. Preservation of the RBE can be important for ITR stability and manufacturing purposes.

[0052] As used herein, the term "parvovirus" encompasses the Parvoviridae family, including, but not limited to, Parvoviruses and Dependoviruses, which are autonomously replicating. Autonomous parvoviruses include, for example, members of the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Abeparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, and Penstildensovirus. Exemplary autonomous parvoviruses include, but are not limited to, human bocavirus type 1 (HBoV1), porcine parvovirus, minute virus of mice, canine parvovirus, mink enteritis virus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus (GPV), H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, for example, Fields et al., Virology, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers).

[0053] As used herein, the term "non-AAV" encompasses nucleic acids, proteins, and viruses derived from the Parvoviridae family, excluding any adeno-associated viruses (AAVs) within 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, Abeparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, and Penstildensovirus.

[0054] 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 clades disclosed by Gao et al. (J. Virol., 78:6381 (2004)) and Morris et al. (Virol., 33:375 (2004)), as well as any other AAV now known or subsequently discovered. See, e.g., FIELDS et al., VIROLOGY, Volume 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0055] As used herein, the term "derived from" refers to a component isolated from or made using a specified molecule or organism, or information (e.g., amino acid sequence or nucleic acid sequence) derived from a specified 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 molecular species may be obtained, for example, by natural mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive a nucleotide or polypeptide may be intentionally directed, intentionally random, or a mixture of each. Mutagenesis of a nucleotide or polypeptide that creates a different nucleotide or polypeptide derived from a first nucleotide or polypeptide may be a different nucleotide or polypeptide derived from a first nucleotide or polypeptide, and the identification of the derived nucleotide or polypeptide may be made, for example, by a suitable screening method, as discussed herein. Mutagenesis of a polypeptide typically involves the manipulation of a polypeptide that encodes a polynucleotide.

[0056] A "capsid-free" or "capsid-less" vector or nucleic acid molecule refers to a vector construct that does not contain a capsid.

[0057] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons that can be translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid but is considered part of the coding region, whereas any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end that codes for the amino terminus of the resulting polypeptide and a translation stop codon at the 3' end that codes for the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present within a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Thus, a single vector may contain only a single coding region or may include two or more coding regions.

[0058] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once the growing protein chain is triggered to export across the rough endoplasmic reticulum. Those skilled in the art are aware that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide or a functional derivative of this sequence retains the ability to direct the secretion of a polypeptide operatively associated therewith. Alternatively, a heterologous mammalian signal peptide, such as human tissue plasminogen activator (TPA), or mouse β-glucuronidase signal peptide, or a functional derivative thereof, may be used.

[0059] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0060] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' of a coding region or at the origin of transcription. For example, most promoters are located upstream of the transcription start site.

[0061] As used herein, the term "gene cassette" refers to a DNA sequence capable of directing the expression of a particular polynucleotide sequence in a suitable host cell, comprising a promoter operably linked to the polynucleotide sequence of interest. A gene cassette may be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region and may include nucleotide sequences that affect transcription, RNA processing, stability, or translation of the associated coding region. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal sequence and a transcription termination sequence will typically be located 3' to the coding sequence. In some embodiments, a gene cassette comprises a polynucleotide that encodes a gene product. In some embodiments, a gene cassette comprises a polynucleotide that encodes a miRNA. In some embodiments, a gene cassette comprises a heterologous polynucleotide sequence.

[0062] A polynucleotide encoding a product, e.g., a miRNA or 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 sequence operably associated with one or more coding regions. When in operably associated, a coding region of a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a manner that expression of the gene product is under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA that encodes the gene product encoded by the coding region, and the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the gene product or the ability of the DNA template to be transcribed. Other expression control sequences other than promoters, e.g., enhancers, operators, repressors, and transcription termination signals, may also be operably associated with a coding region to direct expression of the gene product.

[0063] "Expression control sequence" refers to a regulatory nucleotide sequence, such as a promoter, enhancer, or terminator, that results in the expression of a coding sequence in a host cell. Expression control sequences generally encompass any regulatory nucleotide sequence that facilitates efficient transcription and translation of an operably linked coding nucleic acid. Non-limiting examples of expression control sequences include promoters, enhancers, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. A variety of expression control sequences are known to those skilled in the art. These include expression control sequences that function in vertebrate cells, such as, but are not limited to, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter with intron A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other expression control sequences include expression control sequences derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable expression control sequences include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters induced by interferons or interleukins). Other expression control sequences include intron sequences, post-transcriptional regulatory elements, and polyadenylation signals. Additional exemplary expression control sequences are discussed elsewhere in this disclosure.

[0064] Likewise, various translation control elements are known to those of skill in the art, including, but not limited to, ribosome binding sites, translation initiation / termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites, or IRES).

[0065] As used herein, the term "expression" refers to the process by which a polynucleotide results in a gene product, e.g., an RNA or a polypeptide. "Expression" includes, without limitation, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of an mRNA into a polypeptide. Expression results in a "gene product." As used herein, a gene product can be a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage. As used herein, the term "yield" refers to the amount of polypeptide resulting from expression of a gene.

[0066] "Vector" refers to any vehicle for cloning and / or introduction of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment is attached to effect replication of the attached segment. "Replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes vehicles for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector is accomplished by ligating a suitable polynucleotide fragment into a selected vector with complementary cohesive termini.

[0067] Vectors are engineered to encode a selectable marker or reporter that allows for the selection or identification of cells that have incorporated the vector. Expression of the selectable marker or reporter allows for the identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialaphos, sulfonamides, and the like; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, and the like. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. A selectable marker is also considered to be a reporter.

[0068] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that are or have been used as recipients of ssDNA or vectors. The term "host cell" 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 an exogenous DNA sequence. It is understood that the progeny of a single parent cell are not necessarily completely identical in shape or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations. In some embodiments, the host cell may be an in vitro host cell.

[0069] The term "selectable marker" refers to an identifying factor, typically an antibiotic resistance gene or a chemical resistance gene, that allows selection based on the effect of the marker gene, i.e., resistance to antibiotics, resistance to herbicides, colorimetric markers, enzymes, fluorescent markers, etc., where the effect is used to trace the inheritance of the nucleic acid of interest and / or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide bialaphos, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0070] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that allows identification based on the effect of the reporter gene, where the effect is used to trace the inheritance of the nucleic acid of interest, to identify cells or organisms that have inherited the nucleic acid of interest, and / or to measure induction of gene expression or transcription of the gene. Examples of reporter genes known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable marker genes are also considered reporter genes.

[0071] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters are composed of different elements in their entirety from a natural gene or from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will understand that different promoters may direct the expression of a gene in different tissues or cell types, may direct the expression of a gene at different developmental stages, or may direct the expression of a gene in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types at most times is generally referred to as a "constitutive promoter". A promoter that causes a gene to be expressed in a specific cell type is generally referred to as a "cell-specific promoter" or "tissue-specific promoter". A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter". A promoter that is induced to express a gene after exposure or treatment of cells with a drug, biomolecule, chemical, ligand, light, etc. that induces the promoter is generally referred to as an "inducible promoter" or "regulatable promoter". It is further recognized that in most cases, the exact boundaries of regulatory sequences are not fully defined, so that DNA fragments of different lengths may have the same promoter activity. Additional exemplary promoters are discussed elsewhere in this disclosure.

[0072] A promoter sequence is typically bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to incorporate the minimum number of bases or elements necessary to induce transcription at a detectable level above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0073] In some embodiments, the nucleic acid molecule comprises a tissue-specific promoter. In certain embodiments, the tissue-specific promoter drives the expression of the therapeutic protein in liver, hepatocytes, and / or endothelial cells. In a particular embodiment, the promoter comprises the TTP promoter. In a particular embodiment, the promoter comprises the mTTR promoter. In a particular embodiment, the promoter comprises the A1AT promoter.

[0074] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences of single- or double-stranded DNA or RNA, linear, circular, or supercoiled, from any source, in which multiple nucleotide sequences are joined or recombined into unique constructs that can introduce promoter fragments and DNA sequences for selected gene products, along with appropriate 3' untranslated sequences, into cells.

[0075] Eukaryotic viral vectors that may be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxviruses, such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0076] "Cloning vector" refers to a "replicon", a unit length of sequentially replicated nucleic acid, such as a plasmid, phage, or cosmid, to which another nucleic acid segment is attached to effect replication of the attached segment, and which contains an origin of replication. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria, and expression in another cell, e.g., eukaryotic cells. Cloning vectors typically contain one or more sequences used for the insertion of a nucleic acid sequence of interest into the vector and / or for the selection of cells that contain one or more multiple cloning sites.

[0077] The term "expression vector" refers to a vehicle designed to allow for the expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operable association with a regulatory region, as described above.

[0078] 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 a DNA vector transporter. As used herein, "culture", "to culture" and "to culture" 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" refers to cells that have been propagated in vitro.

[0079] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modified products of a polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. The polypeptides may be produced in any manner, including by chemical synthesis.

[0080] The term "amino acid" includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Unconventional amino acids are also within the scope of the present disclosure, including norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym., 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al., Science, 244:182 (1989); and Ellman et al., supra, may be used. Briefly, these procedures involve chemical activation of a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. Introduction of unconventional amino acids may also be accomplished using peptide chemistry reactions known in the art. As used herein, the term "polar amino acid" includes amino acids that have a net charge of zero, but have non-zero partial charges at different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids may participate in hydrophobic and electrostatic interactions. As used herein, the term "charged amino acids" includes amino acids that may have a non-zero net charge on their side chains (e.g., R, K, H, E, D). These amino acids may participate in hydrophobic and electrostatic interactions.

[0081] The present disclosure also includes fragments or variants of the polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to the polypeptide-binding domains or polypeptide-binding molecules of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for FcRn binding domains or Fc variants, coagulation activity for FVIII variants, or FVIII binding activity for VWF fragments). Polypeptide fragments include proteolytic fragments as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide-binding domains or polypeptide-binding molecules of the present disclosure include the fragments described above, and also include polypeptides in which the amino acid sequence is altered due to amino acid substitution, deletion, or insertion. Variants may be naturally occurring variants or non-naturally occurring variants. Non-naturally occurring variants are generated using mutagenesis methods known in the art. Variant polypeptides can contain conservative amino acid substitutions, deletions, or additions, or can contain non-conservative amino acid substitutions, deletions, or additions.

[0082] "Conservative amino acid substitution" refers to the amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, a family of amino acid residues with similar side chains is defined, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a stretch of amino acids is conservatively replaced with a structurally similar stretch of side chain family members that differs in order and / or composition.

[0083] The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" is readily calculated by known methods, including but not limited to those described in "Computational Molecular Biology" (Lesk, AM, ed.), Oxford University Press, New York (1988); "Biocomputing: Informatics and Genome Projects" (Smith, DW, ed.), Academic Press, New York (1993); "Computer Analysis of Sequence Data", Part I (Griffin, AM and Griffin, HG, eds.), Humana Press, New Jersey (1994); "Sequence Analysis in Molecular Biology" (Von Heijne, G., ed.), Academic Press (1987); and "Sequence Analysis Primer" (Gribskov, M. and Devereux, J., eds.), Stockton Press, New York (1991). Preferred methods of determining identity are designed to give the best match between the sequences tested. Methods of determining identity are codified in publicly available computer programs.Sequence alignment and percent identity calculations are performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing software package (DNASTAR, Inc., Madison, WI), the GCG program package (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403 (1990)); and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). In the context of this application, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein, "default values" refers to any set of values ​​or parameters that were originally loaded by the software when it was first initialized. For purposes of determining the percent identity between a query sequence (e.g., a nucleic acid sequence) and a reference sequence, only those nucleotides in the query sequence that match nucleotides in the reference sequence are used in calculating the percent identity. Thus, in determining the percent identity between a query sequence, or a specified portion thereof (e.g., nucleotides 1-522), and a reference sequence, the percent identity will be calculated by dividing the number of matched nucleotides by the total number of nucleotides in the complete query sequence.

[0084] As used herein, nucleotides corresponding to nucleotides in a particular sequence of the present disclosure are identified by alignment of the sequences of the present disclosure to maximize identity with respect to the reference sequence. The numbers used to identify equivalent amino acids in the reference sequence are based on the numbers used to identify the corresponding amino acids in the sequences of the present disclosure.

[0085] As used herein, "treat", "treatment", or "treating" refers to, for example, lessening the severity of a disease or condition; shortening the duration of the course of a disease; ameliorating one or more symptoms associated with a disease or condition; imparting a beneficial effect to a subject with a disease or condition, without necessarily curing the disease or condition; or preventing one or more symptoms associated with a disease or condition.

[0086] As used herein, "administering" refers to administering 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 via a pharma- ceutically acceptable route. The route of administration can be intravenous, e.g., intravenous injection and intravenous infusion. Additional routes of administration include, e.g., subcutaneous administration, intramuscular administration, oral administration, intranasal administration, and pulmonary administration. The nucleic acid molecules, polypeptides, and vectors can be administered as part of a pharmaceutical composition that includes at least one excipient.

[0087] As used herein, "lipid nanoparticles" refers to particles having at least one dimension on the nanometer scale (e.g., 1 nm to 1,000 nm) that contain one or more cationic lipids. In some embodiments, lipid nanoparticles are incorporated into formulations used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA), to relevant target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, lipid nanoparticles disclosed herein contain nucleic acids. Such lipid nanoparticles typically contain one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, active agents or therapeutic agents, such as nucleic acids, may be encapsulated in the lipid portion of the lipid nanoparticle, or may be encapsulated in the aqueous cavity by some or all of the lipid portion of the lipid nanoparticle, thereby protecting the active agent or therapeutic agent from enzymatic degradation or other undesirable effects induced by the host organism or host cell mechanisms, such as adverse immune responses.

[0088] As used herein, "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically acceptable and typically do not cause toxic or allergic or similar undesirable reactions, such as heartburn, dizziness, etc., when administered to humans.In some cases, the term "pharmaceutical acceptable" as used herein means approved by a regulatory agency of the United States Federal or State Government for use in animals, more particularly for use in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0089] As used herein, the phrase "subject in need thereof" includes a subject, such as a mammalian subject, who will benefit from administration of a nucleic acid molecule, polypeptide, or vector of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the subject is an individual with hemophilia. The subject may be an adult or a juvenile (e.g., under the age of 12).

[0090] As used herein, the term "therapeutic protein" refers to any polypeptide known in the art that is 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 reduces the persistence of bleeding episodes in a subject. In other words, the term "clotting factor" refers to a molecule that has procoagulant activity, i.e., a molecule that contributes to the conversion of fibrinogen into a mesh of insoluble fibrin that causes blood to coagulate (coagulate or clot). 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 its zymogen form) that can be converted to an active form. The term "clotting factor" refers to factor I (FI), factor II (FII), factor III (FIII), factor IV (FIV), 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 (FXIII), von Willebrand factor (VWF), prekallikrein, high molecular weight kininogen, fibronectin, amyloidosis, erythrocyte sedimentation, leukemia, and leukemia. Examples of antibodies that may be used include, but are not limited to, 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), zymogens thereof, activated forms thereof, or any combination thereof.

[0091] As used herein, "clotting activity" means the ability to participate in the cascade of biochemical reactions that lead to the formation of a fibrin clot and / or to reduce the severity, duration, or frequency of bleeding or bleeding episodes.

[0092] As used herein, the term "growth factor" includes any growth factor known in the art, including cytokines and hormones.

[0093] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., within a cell or polypeptide. For example, an exogenous or heterologous molecule is introduced into a cell and is present only after manipulation of the cell, e.g., by transfection or other form of genetic engineering, whereas a heterologous amino acid sequence may be present within a protein where it is not found in nature.

[0094] As used herein for comparison with the nucleotide sequence of the present disclosure, a "reference nucleotide sequence" is a polynucleotide sequence that is essentially identical to the nucleotide sequence of the present disclosure, except that the portion corresponding to the FVIII sequence is not optimized. In some embodiments, the reference nucleotide sequence for the nucleic acid molecules disclosed herein is SEQ ID NO:32.

[0095] As used herein, the term "optimized" in relation to a nucleotide sequence refers to a polynucleotide sequence that codes for a polypeptide, where the polynucleotide sequence is mutated to enhance the properties of the polynucleotide sequence. In some embodiments, the optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease binding to regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence. Examples of changes that can be made to a polynucleotide sequence to optimize the nucleotide sequence include codon optimization, G / C content optimization, removal of repeat sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis-activating elements that suppress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences near the transcription start site that enhance transcription, such as Kozak consensus sequences, removal of sequences that can form stem-loop structures, removal of destabilizing sequences, removal of CpG motifs, and combinations of two or more of these.

[0096] nucleic acid molecule Certain aspects of the present disclosure aim to overcome the deficiencies of AAV vectors for gene therapy. In particular, certain aspects of the present disclosure are directed to a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette. In some embodiments, the gene cassette encodes a therapeutic protein and / or a therapeutic miRNA. In some embodiments, the first ITR and the second ITR flank 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 disposed 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.

[0097] In one embodiment, the gene cassette is a single-stranded nucleic acid. In another embodiment, the gene cassette is a double-stranded nucleic acid. In another embodiment, the gene cassette is a closed-end double-stranded ceDNA.

[0098] In some embodiments, the nucleic acid molecule comprises: (a) a first ITR, which is an ITR from a member of the Parvoviridae family other than AAV (e.g., HBoV1 ITR); (b) a tissue-specific promoter sequence, e.g., the TTP promoter or the TTR promoter; (c) an intron, e.g., a synthetic intron; (d) a nucleotide encoding a miRNA or a therapeutic protein, e.g., a clotting factor; (e) a post-transcriptional regulatory element, e.g., a WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; and (g) a second ITR, which is an ITR from a member of the Parvoviridae family other than AAV (e.g., HBoV1 ITR). In some embodiments, the nucleic acid molecule comprises: (a) a first ITR, which is an ITR from a member of the Parvoviridae family other than AAV (e.g., HBoV1 ITR); (b) a tissue-specific promoter sequence, e.g., mTTR promoter; (c) an intron, e.g., a synthetic intron; (d) a nucleotide encoding a miRNA or a therapeutic protein, e.g., a clotting factor; (e) a post-transcriptional regulatory element, e.g., WPRE; (f) a 3'UTR poly(A) tail sequence, e.g., bGHpA; (g) a second ITR, which is an ITR from a member of the Parvoviridae family other than AAV (e.g., HBoV1 ITR). In some embodiments, the tissue-specific promoter is a human alpha 1 antitrypsin (A1AT) promoter. In some embodiments, the tissue-specific promoter comprises the nucleotide sequence of SEQ ID NO: 36.

[0099] In some embodiments herein, an isolated nucleic acid molecule is disclosed that comprises a gene cassette that comprises a nucleotide sequence that is at least about 75% identical to SEQ ID NO: 9. In some embodiments herein, a nucleic acid molecule is disclosed that comprises a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 9.

[0100] In some embodiments herein, an isolated nucleic acid molecule is disclosed that comprises a gene cassette that comprises a nucleotide sequence that is at least about 75% identical to SEQ ID NO: 33. In some embodiments herein, a nucleic acid molecule is disclosed that comprises a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 33.

[0101] In some embodiments herein, an isolated nucleic acid molecule is disclosed that includes a gene cassette that includes a nucleotide sequence that is at least about 75% identical to SEQ ID NO: 14. In some embodiments herein, a nucleic acid molecule is disclosed that includes a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 14.

[0102] In another aspect herein, an isolated nucleic acid molecule is disclosed that comprises a gene cassette expressing a factor VIII (FVIII) polypeptide, the gene cassette comprising a nucleotide sequence that is at least about 85% identical to SEQ ID NO: 35. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 90% identical to SEQ ID NO: 35. In some embodiments, the gene cassette comprises a nucleotide sequence that is at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, the nucleotide sequence is at least 50% identical to SEQ ID NO: 35.

[0103] Also disclosed herein is an isolated nucleic acid molecule comprising a gene cassette expressing a Factor VIII (FVIII) polypeptide, the gene cassette comprising the nucleotide sequence of SEQ ID NO:35.

[0104] In certain embodiments, the nucleic acid molecules disclosed herein comprise an ITR sequence derived from human bocavirus type 1 (HBoV1). In certain embodiments, the nucleic acid molecules disclosed herein comprise a first ITR that is at least about 75% identical to SEQ ID NO:1 or SEQ ID NO:2.

[0105] A. Inverted terminal repeat (ITR) Certain embodiments of the present disclosure are directed to nucleic acid molecules that include a first ITR, e.g., a 5' ITR, and a second ITR, e.g., a 3' ITR. Typically, ITRs are involved in the replication and rescue or excision of parvovirus (e.g., AAV) DNA from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; GottliebandMuzyczka, 1988). In addition, ITRs are also considered to be the minimal sequences required for the integration of AAV provirus and packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are essential for efficient replication of parvovirus genomes. It is hypothesized that the minimal canonical elements essential for ITR function are Rep binding sites and terminal separation sites plus a variable palindrome that allows hairpin formation. Palindromic nucleotide regions usually function together in cis as origins of DNA replication and packaging signals for viruses. Complementary sequences in ITRs fold into hairpin structures during DNA replication. In some embodiments, ITRs fold into T-shaped hairpin structures. In other embodiments, ITRs fold into hairpin structures other than T-shaped, such as U-shaped hairpin structures. Data suggest that the T-shaped hairpin structure of AAV ITRs can inhibit the expression of transgenes flanked by ITRs. See, for example, Zhou et al. (2017), Scientific Reports, 7:5432. By utilizing ITRs that do not form T-shaped hairpin structures, this form of inhibition is avoided. Thus, in certain aspects, polynucleotides that include non-AAV ITRs have improved transgene expression compared to polynucleotides that include AAV ITRs that form T-shaped hairpins.

[0106] As used herein, "inverted terminal repeat" (or "ITR") refers to a nucleic acid subsequence located at the 5' or 3' end of a single stranded nucleic acid sequence that comprises a set of nucleotides (initial sequence) followed downstream by its reverse complement, i.e., a palindromic sequence. The intervening nucleotide sequence between the initial sequence and the reverse complement can be of any length, including zero. In one embodiment, an ITR useful in the present disclosure comprises one or more "palindromic sequences." An ITR can have any number of functions. In some embodiments, an ITR described herein forms a hairpin structure. In some embodiments, an ITR forms a T-shaped hairpin structure. In some embodiments, an ITR forms a hairpin structure other than a T-shaped, e.g., a U-shaped hairpin structure. In some embodiments, an ITR promotes the survival of a nucleic acid molecule in a cell nucleus over an extended period of time. In some embodiments, an ITR promotes the permanent survival (e.g., for the entire lifespan of a cell) of a nucleic acid molecule in a cell nucleus. In some embodiments, an ITR promotes the stability of a nucleic acid molecule in a cell nucleus. In some embodiments, the ITRs promote the retention of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs promote the persistence of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs inhibit or prevent the degradation of the nucleic acid molecule in the cell nucleus.

[0107] Thus, an "ITR" as used herein may fold back on itself to form a double-stranded segment. For example, the sequence GATCXXXXGATC includes an initial sequence of GATC and its complement (3'CTAG5') such that when folded, they form a double helix. In some embodiments, an ITR includes a continuous palindromic sequence (e.g., GATCGATC) between the initial sequence and the reverse complement. In some embodiments, an ITR includes an interrupted palindromic sequence (e.g., GATCXXXXGATC) between the initial sequence and the reverse complement. In some embodiments, the complementary portions of the continuous or interrupted palindromic sequences interact with each other to form a "hairpin loop" structure. A "hairpin loop" structure, as used herein, occurs when at least two complementary sequences on a single-stranded nucleotide molecule base pair to form a double-stranded portion. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop.

[0108] In some embodiments, the ITR comprises a naturally occurring ITR, for example, the ITR comprises all or a portion of an ITR from a member of the Parvoviridae family. 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.

[0109] In some embodiments, the ITR comprises or consists of a portion of a naturally occurring ITR, e.g., 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; the ITR retains the functional properties of the naturally occurring ITR. In certain embodiments, the ITR comprises or consists of a fragment of a naturally occurring ITR, where the fragment comprises at least about 129 nucleotides; the ITR retains the functional properties of the naturally occurring ITR. In certain embodiments, the ITR comprises or consists of a fragment of a naturally occurring ITR, where the fragment comprises at least about 102 nucleotides; the ITR retains the functional properties of the naturally occurring ITR. In some embodiments, the ITR retains the Rep binding element (RBE) of the wild-type ITR from which it is derived.In some embodiments, an ITR retains at least one of the RBEs of the wild-type ITR from which it is derived. In some embodiments, an ITR retains at least one of the RBEs or a functional portion thereof of the wild-type ITR from which it is derived. Preservation of the RBE can be important for ITR stability and manufacturing purposes.

[0110] 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 the naturally occurring ITR; the fragment retains the functional properties of the naturally occurring ITR. In some embodiments, the first ITR and / or the second ITR are derived from wild-type HBoV1 ITR. In some embodiments, the first ITR and / or the second ITR are derived from wild-type B19 ITR. In some embodiments, the first and / or second ITR are derived from a wild-type GPV ITR.

[0111] In certain embodiments, the ITRs, when properly aligned, have a sequence similar to that of the homologous naturally occurring ITR portion, 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%, In another embodiment, the ITR comprises or consists of a sequence having at least 5%, 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 to the naturally occurring ITR; in which case 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 to the portion of the homologous naturally occurring ITR when properly aligned; in which case 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 to a portion of a homologous naturally occurring ITR; in this case, 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 to a portion of a homologous naturally occurring ITR; in this case, 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 to a portion of a homologous naturally occurring ITR; in this case, 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 to a portion of a homologous naturally occurring ITR; in this case, the ITR retains the functional properties of the naturally occurring ITR.

[0112] In some embodiments, ITR comprises ITR from AAV genome.In some embodiments, ITR is ITR of AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 and any combination thereof.In some embodiments, ITR is ITR of any AAV genome known to those skilled in the art, including natural isolate, for example natural human isolate.In certain embodiments, ITR is ITR of AAV2 genome.In another embodiment, ITR is a synthetic sequence that is engineered to comprise ITR from one or more of AAV genomes at its 5' end and 3' end.

[0113] In some embodiments, the ITRs are not derived from the AAV genome (i.e., the ITRs are derived from a virus that is not AAV). In some embodiments, the ITRs are non-AAV ITRs. In some embodiments, the ITRs are non-AAV ITRs from the Parvoviridae family, including but not limited to the following: Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Abeparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Penstildensovirus, and any combination thereof. In certain embodiments, the ITRs are derived from human bocavirus type 1 (HBoV1). In another embodiment, the ITRs are derived from erythrovirus B19 (a human virus), a parvovirus. In another embodiment, the ITRs are derived from the Muscovy Duck Parvovirus (MDPV) strain. In certain embodiments, the MDPV strain is an attenuated MDPV strain, such as MDPV FZ91-30 strain. In other embodiments, the MDPV strain is a pathogenic MDPV strain, such as MDPV YY strain. In some embodiments, the ITRs are derived from porcine parvovirus, such as porcine parvovirus U44978 strain. In some embodiments, the ITRs are derived from minute virus of mice, such as minute virus of mice U34256 strain. In some embodiments, the ITRs are derived from canine parvovirus, such as canine parvovirus M19296 strain. In some embodiments, the ITRs are derived from mink enteritis virus, such as mink enteritis virus D00765 strain. In some embodiments, the ITRs are derived from the genus Dependoparvovirus. In one embodiment, the genus Dependoparvovirus is a Dependovirus goose parvovirus (GPV) strain. In a specific embodiment, the GPV strain is an attenuated GPV strain, such as GPV 82-0321V strain. In another specific embodiment, the GPV strain is a pathogenic GPV strain, such as a GPV B strain.

[0114] The first ITR and the second ITR of the nucleic acid molecule may be derived from the same genome, for example, the genome of the same virus, or may be derived from different genomes, for example, the genomes of two or more different viral 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 can be the same, in particular, AAV2 ITR. In other embodiments, the first ITR is derived from the AAV genome and the second ITR is not derived from the AAV genome (e.g., derived from a genome other than AAV). In other embodiments, the first ITR is not derived from the AAV genome (e.g., derived from a genome other than AAV) and the second ITR is derived from the AAV genome. In yet other embodiments, neither the first ITR nor the second ITR is derived from the AAV genome (e.g., derived from a genome other than AAV). In a particular embodiment, the first ITR and the second ITR are identical.

[0115] In some embodiments, the first ITR is derived from a genome other than AAV, and the second ITR is derived from a genome other than AAV, where the first ITR and the second ITR are derived from the same genome. Non-limiting examples of viral genomes other than AAV are from the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Iteravirus, Contravirus, Abeparvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, and Penstildensovirus. In some embodiments, the first ITR is derived from a genome other than AAV, and the second ITR is derived from a genome other than AAV, where the first ITR and the second ITR are derived from different viral genomes.

[0116] In some embodiments, the first ITR is derived from an AAV genome and the second ITR is derived from human bocavirus type 1 (HBoV1). In other embodiments, the second ITR is derived from an AAV genome and the first ITR is derived from human bocavirus type 1 (HBoV1).

[0117] In some embodiments, the first ITR comprises or consists of the whole or part of the ITR from AAV genome or a genome other than AAV, and the second ITR comprises or consists of the whole or part of the ITR from AAV genome or a genome other than AAV.In some embodiments, the part of the ITR from AAV genome or a genome other than AAV is a truncated form of the ITR from naturally occurring AAV genome or a genome other than AAV.In some embodiments, the part of the ITR from AAV genome or a genome other than AAV comprises the part of the ITR from naturally occurring AAV genome or a genome other than AAV.For example, the part of the ITR from AAV genome or a genome other than AAV comprises the part of the ITR from naturally occurring AAV genome or a genome other than AAV, and at least one RBE or its functional part is conservative.

[0118] In certain embodiments, the first ITR and / or the second ITR comprises or consists of the whole or part of the ITR from HBoV1. In certain embodiments, the first ITR and / or the second ITR comprises or consists of the whole or part of the ITR from HBoV1. In some embodiments, the second ITR is the reverse complement of the first ITR. In some embodiments, the first ITR is the reverse complement of the second ITR. In some embodiments, the first ITR and / or the second ITR from HBoV1 can form a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0119] 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 the nucleotide sequence set forth in SEQ ID NO:1 or 2, wherein the first ITR and / or the second ITR retains the functional properties of the HBoV1 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 NO: 1 or 2, where the first ITR and / or the second ITR is capable of forming a hairpin structure. In certain embodiments, the hairpin structure does not comprise a T-shaped hairpin.

[0120] In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first ITR and / or the second ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the first ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1 and the second ITR comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 2.

[0121] Those skilled in the art will appreciate that any of the first ITR sequences described herein can be matched with any of the second ITR sequences described herein. In some embodiments, the first ITR sequence described herein is the 5' ITR sequence. In some embodiments, the second ITR sequence described herein is the 3' ITR sequence. In some embodiments, the second ITR sequence described herein is the 5' ITR sequence. In some embodiments, the first ITR sequence described herein is the 3' ITR sequence. Those skilled in the art will be able to determine the appropriate orientation of the first ITR and second ITR described herein for the construction of the gene cassette.

[0122] In another specific embodiment, the ITR is a synthetic sequence engineered to contain an ITR at its 5'-end and 3'-end that is not derived from the AAV genome. In another specific embodiment, the ITR is a synthetic sequence engineered to contain an ITR at its 5'-end and 3'-end that is derived from one or more non-AAV genomes. The two ITRs present in the nucleic acid molecule of the present invention can be from the same non-AAV genome or from different non-AAV genomes. In particular, the ITRs can be from the same non-AAV genome. In a specific embodiment, the two ITRs present in the nucleic acid molecule of the present invention can be the same, in particular, AAV2 ITR.

[0123] 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 such as pseudopalindromic sequences (i.e., sequences not found in nature), and combinations or modifications thereof. A "pseudopalindromic sequence" is a palindromic DNA sequence, including imperfect palindromic sequences, that share less than 80% or no nucleic acid sequence identity, including less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%, with sequences in natural AAV or non-AAV palindromic sequences that form secondary structures. Natural palindromic sequences are obtained or derived from any genome disclosed herein. Synthetic palindromic sequences can be based on any genome disclosed herein.

[0124] The palindrome may be a continuous sequence or an interrupted sequence. In some embodiments, the interrupted sequence comprises an 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 recombinase or Flp recombinase), an open reading frame for a gene product, or a combination thereof.

[0125] In some embodiments, the ITR forms a hairpin loop structure. In one embodiment, the first ITR forms a hairpin structure. In another embodiment, the second ITR forms a hairpin structure. In yet another embodiment, both the first ITR and the second ITR form a hairpin structure. In some embodiments, the first ITR and / or the second ITR do not form a T-shaped hairpin structure. In certain embodiments, the first ITR and / or the second ITR form a hairpin structure other than a T-shaped structure. In some embodiments, the hairpin structure other than a T-shaped structure comprises a U-shaped hairpin structure.

[0126] In some embodiments, the ITRs in the nucleic acid molecules described herein may be transcriptionally activating ITRs. The transcriptionally activating ITRs may comprise all or part of the wild-type ITRs that have been transcriptionally activated by the incorporation of at least one transcriptionally active element. Various types of transcriptionally active elements are suitable for use in this context. In some embodiments, the transcriptionally active element is a constitutive transcriptionally active element. Constitutive transcriptionally active elements provide sustained levels of gene transcription and are preferred when it is desired that the transgene be expressed on a sustained basis. In other embodiments, the transcriptionally active element is an inducible transcriptionally active element. Inducible transcriptionally active elements generally exhibit low activity in the absence of an inducer (or an inducing condition) and are upregulated in the presence of an inducer (or a switch to an inducing condition). Inducible transcriptionally active elements may be preferred when expression is desired only at a certain time or at a certain location, or when it is desired to titrate the expression level using an inducer. Transcriptionally active elements can also be tissue specific; that is, active only in certain tissues or cell types.

[0127] Transcriptionally active elements are incorporated into the ITRs in various ways. In some embodiments, transcriptionally active elements are incorporated 5' to any portion of the ITR or 3' to 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 separated, these elements alternate with portions of the ITR. In some embodiments, the hairpin structure of the ITR is deleted and replaced by an inverted repeat of the transcription element. This latter arrangement would create a hairpin that mimics the deleted portion of the structure. There may be multiple tandem transcriptionally active elements within the transcriptionally activating ITR, which may be adjacent or separated. In addition, protein binding sites (e.g., Rep binding sites) are also introduced into the transcriptionally active elements of the transcriptionally activating ITR. The transcriptionally active elements may include any sequence that allows the control of transcription of DNA by RNA polymerase to form RNA, and may include, for example, the transcriptionally active elements defined below.

[0128] Transcriptionally activating ITRs provide both transcriptional activation and ITR functions to a nucleic acid molecule in a relatively limited nucleotide sequence length, which effectively maximizes the length of the transgene that is carried and expressed from the nucleic acid molecule. The incorporation of transcriptionally activating elements into ITRs can be accomplished in a variety of ways. Comparison of ITR sequences and sequence requirements of transcriptionally activating elements can provide insight into the manner of encoding elements within the ITR. For example, transcriptional activity is added to an ITR through the introduction of specific changes in the ITR sequence that duplicate the functional elements of the transcriptionally activating element. There are numerous techniques in the art that efficiently add, delete, and / or change specific nucleotide sequences at specific sites (see, for example, Deng and Nickoloff (1992), Anal. Biochem., 200:81-88). Another way of creating transcriptionally activating ITRs involves the introduction of restriction sites at desired positions within the ITR. In addition, multiple transcriptionally activating elements are incorporated into transcriptionally activating ITRs using methods known in the art.

[0129] By way of example, transcriptionally activating ITRs are created by the incorporation of one or more transcriptionally active elements, such as a TATAbox, a GCbox, a CCAATbox, an Sp1 site, an Inr region, a CRE (cAMP regulatory element) site, an ATF-1 / CRE site, an APBβbox, an APBαbox, a CArGbox, a CCACbox, or any other element involved in transcription known in the art.

[0130] B. Therapeutic Proteins Certain aspects of the present disclosure are directed to nucleic acid molecules comprising a gene cassette encoding a first ITR, a second ITR, and a target sequence, the target sequence encoding 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.

[0131] Certain embodiments of the present disclosure are directed to a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a therapeutic protein, the therapeutic protein comprising 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.

[0132] 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 VIII polypeptide. As used herein, "factor VIII", abbreviated as "FVIII" throughout this application, means a functional FVIII polypeptide under its normal role in coagulation, unless otherwise specified. Thus, the term "FVIII" includes mutant polypeptides that are functional. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions are the ability to activate coagulation, act as a cofactor for factor IX, or bind Ca2 + and the ability to form a tenase complex with factor IX in the presence of phospholipids, which then converts factor X to its activated form, Xa.

[0133] As used herein, a FVIII moiety in a therapeutic protein has FVIII activity, which is 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 INR), fibrinogen test (often via the Clauss method), platelet count, platelet function test (often via PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), clotting factor assays, antiphospholipid antibodies, D-dimers, genetic tests (e.g., prothrombin mutation G20210A, which is the factor V Leiden mutation), dilute Russell's snake venom time (dRVVT), other platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0134] The aPTT test is a performance indicator that measures the efficacy of both the "intrinsic" pathway (also called the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, such as FVIII. The aPTT test is used in conjunction with the prothrombin time (PT), which measures the extrinsic pathway.

[0135] ROTEM analysis provides information about the overall dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. In thromboelastometry, the different parameters depend on many factors that affect the activity of the plasma coagulation system, platelet function, fibrinolysis, or their interactions. This assay can provide a complete picture about secondary hemostasis.

[0136] The chromogenic assay mechanism 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 p-nitroanilide (pNA) substrate, specific for factor Xa. The initial release rate of p-nitroaniline, measured at 405 nM, is directly proportional to the factor Xa activity, and therefore also to the FVIII activity in the sample. The chromogenic assay is recommended by the FVIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostatsis (ISTH). Since 1994, the chromogenic assay is also the reference method by the European Pharmacopoeia for the potency assignment of FVIII concentrates.

[0137] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a FVIII polypeptide, where the nucleotide sequence is codon-optimized. In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII driven by an mTTR promoter and a synthetic intron. In some embodiments, the gene cassette comprises a nucleotide sequence disclosed in International Application No. PCT / US2017 / 015879, which is incorporated by reference in its entirety. In some embodiments, the gene cassette is "hFVIIIco6XTEN", a gene cassette described in PCT / US2017 / 015879. In some embodiments, the gene cassette comprises SEQ ID NO: 32.

[0138] In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) fused with XTEN 144 peptide. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:9. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:33. In some embodiments, the gene cassette comprises a set of nucleotide sequences shown as SEQ ID NO:14. In some embodiments, the gene cassette has the nucleotide sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:35.

[0139] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII driven by the mTTR promoter. In some embodiments, the gene cassette further comprises an A1MB2 enhancer element. In some embodiments, the gene cassette further comprises a chimeric intron or a synthetic intron. In some embodiments, the gene cassette further comprises a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the gene cassette further comprises a bovine growth hormone polyadenylation (bGHpA) signal.

[0140] In some embodiments, the present disclosure is directed to a codon-optimized nucleic acid molecule that encodes a polypeptide with FVIII activity.In some embodiments, the polynucleotide encodes a full-length FVIII polypeptide.In other embodiments, the nucleic acid molecule encodes a B-domain deleted (BDD) FVIII polypeptide, which is deleted of all or part of the B-domain of FVIII.

[0141] In other embodiments, the nucleic acid molecules disclosed herein are further optimized by removing one or more CpG motifs and / or methylating at least one CpG motif. As used herein, "CpG motif" refers to a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine. The term "CpG motif" encompasses both methylated and unmethylated CpG dinucleotides. Unmethylated CpG motifs are common in bacterial and viral nucleic acids (e.g., plasmid DNA), but are repressed and largely methylated in vertebrate DNA. Thus, unmethylated CpG motifs prime the mammalian host to mount a rapid inflammatory response. Klinman et al. (1996), PNAS, 93:2879-2883. Exemplary methods of CpG removal are described in Yew, NS et al. (2002), Mol Ther., 5(6):731-738; and International Application No. PCT / US2001 / 010309. In some embodiments, the nucleic acid molecules disclosed herein are modified to contain a small number of CpG motifs (i.e., CpG-reduced or CpG-deleted). In one embodiment, a CpG motif located within a codon triplet for a selected amino acid is changed to a codon triplet for the same amino acid that lacks the CpG motif. In some embodiments, the nucleic acid molecules disclosed herein are optimized to reduce innate immune responses.

[0142] In a particular embodiment, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10 or a fragment thereof. In some embodiments, the nucleic acid molecule of the present disclosure encodes a FVIII polypeptide comprising a signal peptide or a fragment thereof. In other embodiments, the nucleic acid molecule encodes a FVIII polypeptide lacking a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the signal peptide comprises amino acids 1-19 of SEQ ID NO: 10.

[0143] In some embodiments, the nucleic acid molecule comprises a gene cassette encoding a first ITR, a second ITR, and a target sequence, the target sequence encoding a therapeutic protein, and the therapeutic protein comprises a growth factor. The growth factor is 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.

[0144] In some embodiments, the growth factor is adrenomedullin (AM). In some embodiments, the growth factor is angiopoietin (Ang). In some embodiments, the growth factor is an autocrine motility 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 ciliary neurotrophic factor family member. In some embodiments, the ciliary neurotrophic factor family member 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 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 a fibroblast growth factor (FGF). In some embodiments, the FGF is selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the growth factor is fetal bovine somatotrophin (FBS). In some embodiments, the growth factor is a GDNF family member. In some embodiments, the GDNF family member is selected from glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin. In some embodiments, the growth factor is growth differentiation factor 9 (GDF9). In some embodiments, the growth factor is hepatocyte growth factor (HGF).In some embodiments, the growth factor is hepatoma-derived growth factor (HDGF). In some embodiments, the growth factor is insulin. In some embodiments, the growth factor is an insulin-like growth factor. In some embodiments, the insulin-like growth factor is insulin-like growth factor 1 (IGF-1) or IGF-2. In some embodiments, the growth factor is an interleukin (IL). In some embodiments, the IL is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7. In some embodiments, the growth factor is keratinocyte growth factor (KGF). In some embodiments, the growth factor is migration stimulating factor (MSF). In some embodiments, the growth factor is macrophage stimulating protein (MSP) or hepatocyte growth factor-like protein (HGFLP). In some embodiments, the growth factor is myostatin (GDF-8). In some embodiments, the growth factor is neuregulin. In some embodiments, the neuregulin is selected from neuregulin 1 (NRG1), NRG2, NRG3, and NRG4. In some embodiments, the growth factor is a neurotrophic factor. In some embodiments, the growth factor is brain-derived neurotrophic factor (BDNF). In some embodiments, the growth factor is nerve growth factor (NGF). In some embodiments, the NGF is neurotrophic factor 3 (NT-3) or NT-4. In some embodiments, the growth factor is placental growth factor (PGF). In some embodiments, the growth factor is platelet-derived growth factor (PDGF). In some embodiments, the growth factor is renalase (RNLS). In some embodiments, the growth factor is T cell growth factor (TCGF). In some embodiments, the growth factor is thrombopoietin (TPO). In some embodiments, the growth factor is transforming growth factor. In some embodiments, the transforming growth factor is transforming growth factor alpha (TGF-α) or TGF-β. In some embodiments, the growth factor is tumor necrosis factor alpha (TNF-α). In some embodiments, the growth factor is vascular endothelial growth factor (VEGF).

[0145] C. Expression Control Sequences In some embodiments, the nucleic acid molecule or vector of the present disclosure further comprises at least one expression control sequence.For example, the isolated nucleic acid molecule of the present disclosure is operably linked to at least one expression control sequence.The expression control sequence can be, for example, a promoter sequence, or a promoter-enhancer combination.

[0146] Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters derived from cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful for the gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0147] In one embodiment, the disclosure includes expression of a transgene under the control of a tissue-specific promoter and / or enhancer. In another embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes. In certain embodiments, the promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes, sinusoidal cells, and / or endothelial cells. In a particular embodiment, the promoter or other expression control sequence selectively enhances expression of the transgene in endothelial cells. In certain embodiments, the promoter or other expression control sequence selectively enhances expression of the transgene in muscle cells, the central nervous system, the eye, the liver, the heart, or any combination thereof. Examples of liver-specific promoters include, but are not limited to, the mouse transthyretin promoter (mTTR), the native human factor VIII promoter, the human alpha 1 antitrypsin promoter (hAAT), the human albumin minimal promoter, and the mouse albumin promoter. In some embodiments, the nucleic acid molecule disclosed herein comprises the mTTR promoter. The mTTR promoter is described in Costa et al. (1986), Mol. Cell. Biol., 6:4697. The FVIII promoter is described in Figueiredo and Brownlee, 1995, J. Biol. Chem., 270:11828-11838. In some embodiments, the promoter is selected from a liver-specific promoter (e.g., alpha 1 antitrypsin (AAT) promoter), a muscle-specific promoter (e.g., muscle creatine kinase (MCK) promoter, myosin heavy chain alpha (αMHC) promoter, myoglobin (MB) promoter, and desmin (DES) promoter), a synthetic promoter (e.g., SPc5-12 promoter, 2R5Sc5-12 promoter, dMCK promoter, and tMCK promoter), or any combination thereof.

[0148] In some embodiments, transgene expression is targeted to the liver. In certain embodiments, transgene expression is targeted to hepatocytes. In other embodiments, transgene expression is targeted to endothelial cells. In a particular embodiment, transgene expression is targeted to any tissue that naturally expresses endogenous FVIII. In some embodiments, transgene expression is targeted to the central nervous system. In certain embodiments, transgene expression is targeted to neurons. In some embodiments, transgene expression is targeted to afferent neurons. In some embodiments, transgene expression is targeted to efferent neurons. In some embodiments, transgene expression is targeted to interneurons. In some embodiments, transgene expression is targeted to glial cells. In some embodiments, transgene expression is targeted to astrocytes. In some embodiments, transgene expression is targeted to oligodendrocytes. In some embodiments, transgene expression is targeted to microglia. In some embodiments, transgene expression is targeted to ependymal cells. In some embodiments, transgene expression is targeted to Schwann cells. In some embodiments, transgene expression is targeted to satellite cells. In some embodiments, transgene expression is targeted to muscle tissue. In some embodiments, transgene expression is targeted to smooth muscle. In some embodiments, transgene expression is targeted to cardiac muscle. In some embodiments, transgene expression is targeted to skeletal muscle. In some embodiments, transgene expression is targeted to the eye. In some embodiments, transgene expression is targeted to photoreceptor cells. In some embodiments, transgene expression is targeted to retinal ganglion cells.

[0149] Other promoters that are useful within the nucleic acid molecules disclosed herein include the mouse transthyretin promoter (mTTR), the native human FVIII promoter, the human alpha 1 antitrypsin promoter (hAAT), the human albumin minimal promoter, the mouse albumin promoter, the tristetraprolin (TTP) promoter, the CASI promoter, the CAG promoter, the cytomegalovirus (CMV) promoter, the alpha 1 antitrypsin (AAT) promoter, the muscle creatine kinase (MCK) promoter, the myosin heavy chain alpha (αMHC) promoter, the myoglobin (MB) promoter, the desmin (DES) promoter, the SPc5-12 promoter, the 2R5Sc5-12 promoter, the dMCK promoter, the tMCK promoter, the phosphoglycerate kinase (PGK) promoter, or the human alpha 1 antitrypsin (A1AT) promoter, or any combination thereof.

[0150] In some embodiments, the nucleic acid molecule disclosed herein comprises a transthyretin (TTR) promoter. In some embodiments, the promoter is a mouse transthyretin (mTTR) promoter. Non-limiting examples of mTTR promoters include mTTR202 promoter, mTTR202opt promoter, and mTTR482 promoter, which are disclosed in US Publication No. US2019 / 0048362, which is incorporated herein by reference in its entirety. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter. In some embodiments, the promoter is a mTTR482 promoter, which is a liver-specific modified mouse transthyretin (mTTR) promoter. Examples of mTTR482 promoters are described in Kyostio-Moore et al. (2016), Mol Ther Methods Clin Dev., 3:16006; and Nambiar B. et al. (2017), Hum Gene Ther Methods, 28(1):23-28. In some embodiments, the promoter is a liver-specific modified mouse transthyretin (mTTR) promoter comprising the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the tissue-specific promoter is a human alpha 1 antitrypsin (A1AT) promoter. In some embodiments, the tissue-specific promoter comprises the nucleotide sequence of SEQ ID NO: 36.

[0151] To achieve therapeutic efficacy, the expression level is further enhanced using one or more enhancer elements. One or more enhancers may be administered alone or in conjunction with one or more promoter elements. Typically, the expression control sequence includes multiple enhancer elements and tissue-specific promoters. In one embodiment, the enhancer includes one or more copies of the alpha-1-microglobin / bikunin enhancer (Rouet et al. (1992), J. Biol. Chem., 267:20765-20773; Rouet et al. (1995), Nucleic Acids Res., 23:395-404; Rouet et al. (1998), Biochem. J. 334:577-584; Ill et al. (1997), Blood Coagulation Fibrinolysis, 8:S23-S30). In some embodiments, the enhancer is derived from liver-specific transcription factor binding sites such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, including HNF1, (sense)-HNF3, (sense)-HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4 (antisense), along with Enh1.

[0152] In some embodiments, the enhancer element comprises one or two modified prothrombin enhancers (pPrT2), one or two alpha 1 microbikunin enhancers (A1MB2), modified mouse albumin enhancer (mEalb), Hepatitis B virus enhancer II (HE11), or CRM8 enhancer. In some embodiments, the A1MB2 enhancer is an enhancer disclosed in International Application No. PCT / US2019 / 055917. In some embodiments, the enhancer element is A1MB2. In some embodiments, the enhancer element comprises multiple copies of the A1MB2 enhancer sequence. In some embodiments, the A1MB2 enhancer is located 5' to a nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, the A1MB2 enhancer is located 5' to a promoter sequence, such as a mTTR promoter. In some embodiments, the enhancer element is an A1MB2 enhancer comprising the nucleic acid sequence of SEQ ID NO: 15.

[0153] In some embodiments, the nucleic acid molecules disclosed herein include an intron or an intron sequence. In some embodiments, the intron sequence is a naturally occurring intron sequence. In some embodiments, the intron sequence is a synthetic sequence. In some embodiments, the intron sequence is derived from a naturally occurring intron sequence. In some embodiments, the intron sequence is a hybrid synthetic intron or a chimeric intron. In some embodiments, the intron sequence is a chimeric intron consisting of a chicken beta-actin intron / rabbit beta-globin intron, modified to eliminate five existing ATG sequences to reduce false translation initiation. In some embodiments, the chimeric intron comprises the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the intron sequence is located 5' to the nucleic acid sequence encoding the FVIII polypeptide. In some embodiments, the chimeric intron is located 5' to a promoter sequence, such as the mTTR promoter.

[0154] In some embodiments, the nucleic acid molecules disclosed herein comprise a post-transcriptional regulatory element. In certain embodiments, the post-transcriptional regulatory element comprises a mutant woodchuck hepatitis virus regulatory element (WPRE). The WPRE is believed to enhance expression of a transgene delivered by a viral vector. Examples of WPREs are described in Zufferey et al. (1999), J Virol., 73(4):2886-2892; Loeb et al. (1999), Hum Gene Ther., 10(14):2295-2305. In some embodiments, the WPRE is located 3' to the nucleic acid sequence encoding the FVIII polypeptide. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO:18.

[0155] In some embodiments, the nucleic acid molecules disclosed herein comprise a transcription terminator. In some embodiments, the transcription terminator is a polyadenylation (poly(A)) sequence. Non-limiting examples of transcription terminators include those derived from bovine growth hormone polyadenylation signal (BGHpA), simian virus 40 polyadenylation signal (SV40pA), or synthetic polyadenylation signals. In one embodiment, the 3'UTR poly(A) tail comprises an actin poly(A) site. In one embodiment, the 3'UTR poly(A) tail comprises a hemoglobin poly(A) site. In some embodiments, the transcription terminator is BGHpA. An example of a BGHpA transcription terminator is described in Woychik et al. (1984), PNAS, 81:3944-3948. In some embodiments, the transcription terminator is located at the 3' end of the gene cassette encoding the nucleic acid sequence encoding the FVIII polypeptide. In some embodiments, the transcription terminator is BGHpA, which comprises the nucleic acid sequence of SEQ ID NO:19.

[0156] In some embodiments, the nucleic acid molecule disclosed herein comprises one or more DNA nuclear targeting sequences (DTS). The DTS facilitates the translocation of DNA molecules containing such sequences into the nucleus. In certain embodiments, the DTS comprises an SV40 enhancer sequence. In certain embodiments, the DTS comprises a c-Myc enhancer sequence. In some embodiments, the nucleic acid molecule comprises a DTS located between the first ITR and the second ITR. In some embodiments, the nucleic acid molecule comprises a DTS located 3' to the first ITR and 5' to the transgene (e.g., FVIII protein). In some embodiments, the nucleic acid molecule comprises a DTS located 3' to the transgene and 5' to the second ITR on the nucleic acid molecule.

[0157] In some embodiments, the nucleic acid molecules disclosed herein comprise a toll-like receptor 9 (TLR9) inhibitory sequence. Exemplary TLR9 inhibitory sequences are described, for example, in Trieu et al. (2006), Crit Rev Immunol., 26(6):527-44; Ashman et al., Int'l Immunology, 23(3):203-14.

[0158] In some embodiments, the nucleic acid molecule disclosed herein comprises a nucleic acid sequence encoding a nonstructural protein of HBoV1. "Nonstructural protein" refers to any of the six proteins expressed by HBoV1, namely NS1, NS1-70, NS2, NS3, NS4, and NP1. The nonstructural proteins are expressed by mRNA transcripts generated through alternative splicing and polyadenylation of a single viral pre-mRNA. The NS1-NS4 proteins are encoded in different regions of the same open reading frame (ORF). NS1 binds to the replication origin of HBoV1 and likely nicks the single-stranded DNA (ssDNA) of the origin during replication of the rolling hairpin. NS1 plays an important role in the expression of HBoV1 ITR-mediated vectors in eukaryotic cells. In one embodiment, an expression construct expressing HBoV1 NS1 was generated. In some embodiments, the nucleic acid molecules disclosed herein encode nonstructural proteins as described in Shen et al. (2015), J Virology, 89(19):10097-10109.

[0159] In some embodiments, the nucleic acid molecule comprises a microRNA (miRNA) binding site. In one embodiment, the miRNA binding site is for miR-142-3p. In other embodiments, the miRNA binding site is the miRNA binding site described by Rennie et al. (2016), RNA Biol., 13(6):554-560.

[0160] Production of ceDNA in baculovirus Baculoviruses are the most prominent viruses that infect insects. More than 500 baculovirus isolates have been identified, most of them from lepidopteran insects. The two most common isolates are Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV). Among expression vectors, baculoviruses stand out due to their extraordinary gene cargo capacity (up to tens of kb, some have reported up to 100 kb). This transgene capacity has been used for the generation of recombinant AAV vectors (up to 38 kb expression cassettes). However, when generating viral or non-viral vectors for gene therapy, some baculovirus expression vectors are often required to infect insect host cells. The creation of each baculovirus expression vector is time-consuming and expensive to create, which represents a significant drawback of most baculovirus expression vector systems.However, a new versatile baculovirus shuttle vector (bacmid) has been created that is specifically designed to accommodate multiple transgenes, which can be achieved by existing bacmid tools.This versatile bacmid (called "BIVVBac") is also used for the creation of rAAV vectors for in vivo gene therapy as well as the creation of any desired protein, such as recombinant protein.This bacmid expression system is further described in U.S. Patent Application No. 63 / 069,073, which is incorporated herein by reference in its entirety.

[0161] In certain embodiments, the disclosed nucleic acid molecule is produced using a baculovirus expression vector system, comprising "BIVVBac" recombinant bacmid. In certain embodiments, BIVVBac is a genetically modified AcMNPV that comprises at least two foreign sequence insertion sites. The baculovirus expression vector system, comprising bacmid that comprises at least two foreign sequence insertion sites, allows the total number of baculovirus expression vectors that need to be produced to be reduced.

[0162] In certain embodiments, the BIVVBac comprises a first foreign sequence insertion site and a second foreign sequence insertion site. The first foreign sequence insertion site and the second foreign sequence insertion site can be different when different mechanisms are used to drive the insertion of the foreign sequence (e.g., heterologous sequence, heterologous gene). The insertion of the foreign sequence can be driven by any method known in the art. For example, the foreign sequence can be inserted by transposition or by site-specific recombination. The foreign sequence insertion site is designed to be integrated into a reporter gene such that the reporter gene is disrupted upon insertion of the foreign sequence. Disruption of the reporter gene can help identify the bacmid clones into which the foreign sequence has been inserted. In such embodiments, the foreign sequence insertion site is fused in frame with the reporter gene or the reporter gene is fused in frame with the foreign sequence insertion site.

[0163] In certain embodiments, the first foreign sequence insertion site allows the insertion of the foreign sequence via transposition. In certain embodiments, the first foreign sequence insertion site comprises a preferential target site for the insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a preferential target site for the insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a preferential target site that is a junction site for a bacterial transposon. Those skilled in the art are aware of suitable bacterial transposons and their corresponding junction sites. For example, the transposon Tn7 is known for its ability to junction at high frequency into a specific site (attTn7) of a bacterial chromosome. Thus, in certain embodiments, the first foreign sequence insertion site is a preferential target site that is a junction site for a Tn7 transposon (e.g., attTn7). In some embodiments, the first foreign sequence insertion site is a preferred target site that is a junction site for a mini-Tn7 transposon (e.g., mini-attTn7, which is the minimal DNA sequence required for recognition by the Tn7 transposon and insertion of the Tn7 transposon).

[0164] In certain embodiments, the second exogenous sequence insertion site allows the insertion of exogenous sequence via site-specific recombination. In certain embodiments, the second exogenous sequence insertion site comprises a preferential target site that can mediate site-specific recombination events. A variety of site-specific recombinase techniques are known to those skilled in the art. For example, the Cre-loxP system mediates site-specific recombination via Cre recombinase, which can recognize 34 base pair DNA sequences, called loxP sites. Thus, the second exogenous sequence insertion site is a preferential target site for Cre-mediated recombination. In certain embodiments, the second exogenous sequence insertion site is a preferential target site that comprises loxP sites or their variants that can be recognized by Cre recombinase.

[0165] In some embodiments, the recombinant bacmid comprises a mutant VP80 gene, such that the bacmid exhibits reduced expression of its encoded protein. For example, disclosed herein is a baculovirus DNA backbone that comprises an inactivated VP80 gene due to an insertion and / or deletion in the VP80 gene locus. In some embodiments, the recombinant bacmid comprises the bacmid disclosed in US Patent Application No. US63 / 069,115.

[0166] In certain embodiments, ceDNA is produced using a single baculovirus expression vector. In this "OneBAC" approach, a single baculovirus expression vector (e.g., BIVVBac) encodes all the essential elements required for ceDNA production in the baculovirus system and is potentially used in any baculovirus-permissive cell line for ceDNA production. This approach is depicted in Figure 1A.

[0167] In certain embodiments, ceDNA is produced using multiple baculovirus expression vectors. In this "TwoBAC" approach, the essential elements required for ceDNA production are inserted into two different baculoviruses (e.g., two BIVVBac bacmids) and used for co-infection in potentially any cell line that is permissive for baculovirus infection. This approach is depicted in Figure 1B.

[0168] In certain embodiments, ceDNA is produced by a stable cell line. In this approach, the essential elements required for ceDNA production are inserted into any component of the baculovirus system. This approach is as depicted in Figure 1C. The stable cell line is produced by stable integration of a protein coding sequence under the control of a baculovirus gene promoter (e.g., a baculovirus constitutive gene promoter). In certain embodiments, the stable cell line is an insect stable cell line.

[0169] Methods for stable integration of nucleic acid into various host cell lines are known in the art.For example, repeated selection (e.g., via the use of a selectable marker) is used to select for cells that have integrated the nucleic acid containing the selectable marker (as well as the cap and rep genes of AAV and / or the rAAV genome).In other embodiments, the nucleic acid is site-specifically integrated into the cell line to create a producer cell line. Several site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O'Gorman, S. et al. (1991), Science, 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988), Proc. Natl. Acad. Sci., 85:5166-5170), and phi C31-att (see, e.g., Groth, A.C. et al. (2000), Proc. Natl. Acad. Sci., 97:5995-6000).

[0170] The present disclosure also provides a polypeptide encoded by the nucleic acid molecule of the present disclosure. In some embodiments, the polypeptide of the present disclosure is encoded by a vector comprising the isolated nucleic acid molecule disclosed herein. In yet other embodiments, the polypeptide of the present disclosure is produced by a host cell comprising the isolated nucleic acid molecule disclosed herein.

[0171] host cell The present disclosure also provides a host cell comprising the nucleic acid molecule or vector of the present disclosure. As used herein, the term "transformation" is used broadly to refer to the introduction of DNA into a recipient host cell, resulting in a change in the genotype and, as a result, in the change of the recipient cell.

[0172] "Host cell" refers to a cell that is transformed with a vector constructed using recombinant DNA methods and encoding at least one heterologous gene. The host cell of the present disclosure is preferably of mammalian origin; most preferably of human or murine origin. Those skilled in the art are considered to be capable of preferentially determining the particular host cell line that is best suited for their purpose. Exemplary host cell lines include, but are not limited to, CHO, DG44, and DUXB11 (Chinese hamster ovary cell line, DHFR deleted), HELA (human cervical carcinoma), CVI (monkey kidney cell line), COS (a derivative of CVI cells with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3x63-Ag8.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), PER.C6®, NS0, CAP, BHK21, and HEK293 (human kidney). In a particular embodiment, the host cell is selected from the group consisting of CHO cells, HEK293 cells, BHK21 cells, PER.C6® cells, NS0 cells, CAP cells, and any combination thereof. In some embodiments, the host cell of the present disclosure is derived from an insect. In a particular embodiment, the host cell is an SF9 cell. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or published literature.

[0173] Introduction of the nucleic acid molecules or vectors of the present disclosure into host cells can be accomplished by a variety of techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact viruses. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pages 470-472, "Vectors," edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate for the production of light and heavy chains, and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0174] The host cells containing the isolated nucleic acid molecule or vector of the present disclosure are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium containing nutrients required for cell growth. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain calf serum or fetal calf serum (FCS). In one embodiment, the medium is substantially free of IgG. The growth medium will generally select for cells containing the DNA construct, for example, by drug selection or deficiency of essential nutrients, complemented by a selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM / F12). In one embodiment, the medium is CDoptiCHO (Invitrogen, Carlsbad, Calif.). In another embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.) Selection of an appropriate medium for the particular cell line used is within the level of one of ordinary skill in the art.

[0175] An embodiment of the present disclosure provides a method for cloning a nucleic acid molecule described herein, comprising inserting a nucleic acid molecule capable of complex secondary structures into a suitable vector and introducing the resulting vector into a suitable bacterial host strain. As is known in the art, complex secondary structures of nucleic acids (e.g., long palindromic regions) can be unstable and difficult to clone in bacterial host strains. For example, nucleic acid molecules of the present disclosure that include a first ITR and a second ITR (e.g., parvovirus ITRs other than AAV, e.g., HBoV1 ITR) can be difficult to clone using conventional methods. Long DNA palindromic sequences inhibit DNA replication and are unstable in the genomes of E. coli, Bacillus, Streptococcus, Streptomyces, Saccharomyces cerevisiae, mice, and humans. These effects result from the formation of hairpin or cruciform structures by intrastrand base pairing. In E. coli, inhibition of DNA replication can be significantly overcome in SbcC or SbcD mutants. SbcD is the nuclease subunit and SbcC is the ATPase subunit of the SbcCD complex. The E. coli SbcCD complex is an exonuclease complex that contributes to blocking the replication of long palindromic sequences. The SbcCD complex is a core with ATP-dependent double-stranded DNA exonuclease activity and ATP-independent single-stranded DNA endonuclease activity. SbcCD can collapse replication forks by recognizing DNA palindromic sequences and attacking the resulting hairpin structures.

[0176] In certain embodiments, suitable bacterial host strains are unable to degrade cruciform DNA structures. In certain embodiments, suitable bacterial host strains comprise disruption in the SbcCD complex. In some embodiments, disruption in the SbcCD complex comprises gene disruption in the SbcC gene and / or in the SbcD gene. In certain embodiments, disruption in the SbcCD complex comprises gene disruption in the SbcC gene. In the art, various bacterial host strains are known that comprise gene disruption in the SbcC gene. For example, without limitation, bacterial host strain PMC103 comprises the genotypes sbcC, recD, mcrA, ΔmcrBCF; bacterial host strain PMC107 comprises the genotypes recBC, recJ, sbcBC, mcrA, ΔmcrBCF; bacterial host strain SURE comprises the genotypes recB, recJ, sbcC, mcrA, ΔmcrBCF, umuC, uvrC. Thus, in some embodiments, the method of cloning a nucleic acid molecule described herein comprises inserting a nucleic acid molecule capable of complex secondary structures into a suitable vector and introducing the resulting vector into the host strain PMC103, PMC107, or SURE. In certain embodiments, the method of cloning a nucleic acid molecule described herein comprises inserting a nucleic acid molecule capable of complex secondary structures into a suitable vector and introducing the resulting vector into the host strain PMC103.

[0177] Suitable vectors are known in the art.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.

[0178] Thus, the present disclosure provides a method of cloning a nucleic acid molecule comprising inserting a nucleic acid molecule capable of complex secondary structures into a suitable vector and introducing the resulting vector into a bacterial host strain 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:1 or 2, or a functional derivative thereof.

[0179] Production of FVIII Polypeptides The present disclosure also provides a polypeptide encoded by the nucleic acid molecule of the present disclosure. In some embodiments, the polypeptide of the present disclosure is encoded by a vector comprising the isolated nucleic acid molecule disclosed herein. In yet other embodiments, the polypeptide of the present disclosure is produced by a host cell comprising the isolated nucleic acid molecule of the present disclosure.

[0180] A variety of methods are available for recombinantly producing FVIII proteins from the optimized nucleic acid molecules of the present disclosure. A polynucleotide with a desired sequence may be produced by de novo solid-phase DNA synthesis or may be produced through PCR-based mutagenesis of an already produced polynucleotide. Oligonucleotide-mediated mutagenesis is one method for producing substitutions, insertions, deletions, or changes (e.g., codon changes) in a nucleotide sequence. For example, the starting DNA is modified by hybridizing an oligonucleotide encoding a desired mutation with a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, incorporating the oligonucleotide primer. In one embodiment, genetic engineering, such as primer-based PCR mutagenesis, is sufficient to incorporate the changes defined herein to produce a polynucleotide of the present disclosure.

[0181] For production of recombinant proteins, the optimized polynucleotide sequences of the present disclosure encoding FVIII proteins are inserted into an appropriate expression vehicle, i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence, or, in the case of RNA viral vectors, the necessary elements for replication and translation.

[0182] The polynucleotide sequence of the present disclosure is inserted into a vector in the correct reading frame. The expression vector is then transfected into an appropriate target cell to express the polypeptide. Transfection methods known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al., 1978, Cell, 14:725) and electroporation (Neumann et al., 1982, EMBO J., 1:841). A variety of host-expression vector systems are utilized to express the FVIII protein described herein in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including a mammalian cell (e.g., HEK293 cell, PER.C6® cell, CHO cell, BHK cell, Cos cell, HeLa cell). The polynucleotide sequence of the present disclosure may also encode a signal sequence that allows secretion of the FVIII protein. One skilled in the art will understand that when the FVIII protein is translated, the signal sequence is cleaved by the cell to form the mature protein. A variety of signal sequences are known in the art, such as the native factor VII signal sequence, the native factor IX signal sequence, and the mouse IgK light chain signal sequence. Alternatively, if no signal sequence is incorporated, the FVIII protein is recovered by lysing the cells.

[0183] The FVIII protein of the present disclosure is synthesized in transgenic animals such as rodents, goats, sheep, pigs or cows. The term "transgenic animals" refers to non-human animals that incorporate foreign genes into their genome. This gene is present in germ line tissues and is passed from parent to offspring. Exogenous genes are introduced into single-cell embryos (Brinster et al., 1985, Proc. Natl. Acad. Sci. USA, 82:4438). Methods for producing transgenic animals are known in the art, including transgenic animals that produce immunoglobulin molecules (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA, 78:6376; ​​McKnight et al., 1983, Cell, 34:335; Brinster et al., 1983, Nature, 306:332; Ritchie et al., 1984, Nature, 312:517; Baldassarre et al., 2003, Theriogenology, 59:831; Robl et al., 2003, Theriogenology, 59:107; Malassagne et al., 2003, Xenotransplantation 10(3):267).

[0184] The expression vector may code for a tag that allows easy purification or identification of recombinantly produced proteins. Examples include, but are not limited to, pUR278 (Ruther et al., 1983, EMBO J., 2:1791), a vector in which the coding sequence of the FVIII protein described herein is ligated into the vector in frame with the lac Z coding region, so that hybrid proteins are produced; pGEX vectors are used to express proteins with glutathione S-transferase (GST) tags. These proteins are usually soluble and easily purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vector contains a cleavage site (e.g., PreCission Protease (Pharmacia, Peapack, NJ)) for easy removal of the tag after purification.

[0185] For the purposes of this disclosure, numerous expression vector systems are employed. These expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Expression vectors may include expression control sequences, including but not limited to promoters (e.g., naturally associated or heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors may also utilize DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), cytomegalovirus (CMV), or SV40 virus. Other expression vectors involve the use of polycistronic systems with internal ribosome binding sites.

[0186] Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362) that permits detection of cells transformed with the desired DNA sequences. Cells that have integrated the DNA into their chromosomes are selected by introducing one or more markers that permit selection of transfected host cells. The marker may confer prototrophy to an auxotrophic host, may confer resistance to biocides (e.g., antibiotics), or may confer resistance to heavy metals such as copper. The selection marker gene may be directly linked to the DNA sequence to be expressed, or may be introduced into the same cell by cotransformation.

[0187] An example of a vector useful for expressing an optimized FVIII sequence is NEOSPLA (US Pat. No. 6,159,730). This vector contains the cytomegalovirus promoter / enhancer, mouse beta globin major promoter, SV40 origin of replication, bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, dihydrofolate reductase gene and leader sequence. This vector has been found to result in extremely high levels of antibody expression upon integration of variable and constant region genes, followed by transfection of cells and selection in G418-containing medium and methotrexate amplification. Vector systems are also taught in US Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system results in high expression levels, for example, >30 pg per cell per day. Other exemplary vector systems are disclosed, for example, in US Pat. No. 6,413,777.

[0188] In other embodiments, the polypeptide of the present disclosure is expressed using a polycistronic construct. In these expression systems, multiple gene products of interest, such as multiple polypeptides of multimeric binding proteins, are produced from a single polycistronic construct. These systems are advantageous because they use internal ribosome entry sites (IRES) to produce relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein.

[0189] More generally, once a vector or DNA sequence encoding a polypeptide is prepared, the expression vector is introduced into a suitable host cell. That is, the host cell is transformed. The introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled in the art, as discussed above. The transformed cell is grown under conditions suitable for the production of FVIII polypeptide, and assayed for the synthesis of FVIII polypeptide. Exemplary assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0190] In describing processes for the isolation of polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to denote the source of the polypeptide, unless expressly specified otherwise. In other words, recovery of the polypeptide from "cells" can mean recovery from spun down whole cells, or it can mean the cell culture containing both the medium and the suspended cells.

[0191] The host cell line used for protein expression is preferably of mammalian origin, since the isolated nucleic acid of the present disclosure is optimized for expression in human cells; most preferably of human or mouse origin.Exemplary host cell lines are described above.In one embodiment of the method for producing a polypeptide with FVIII activity, the host cell is HEK293 cell.In another embodiment of the method for producing a polypeptide with FVIII activity, the host cell is CHO cell.

[0192] The gene encoding the polypeptide of the present disclosure can also be expressed in non-mammalian cells, such as bacteria or yeast or plant cells. In this regard, it will be appreciated that a variety of unicellular microorganisms other than mammals, i.e., microorganisms capable of growth in culture or fermentation, such as bacteria, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the polypeptide typically becomes part of inclusion bodies. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0193] Alternatively, the optimized nucleotide sequences of the present disclosure are incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., US 5,741,957; Rosen, US 5,304,489; and Meade et al., US 5,849,992). A suitable transgene comprises a coding sequence for a polypeptide operably linked to a promoter and enhancer derived from a mammary gland-specific gene, such as casein or beta-lactoglobulin.

[0194] In vitro production allows for scale-up to provide large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art, including homogenous suspension cultures, for example in airlift or continuous stirred reactors, or immobilized or encapsulated cell cultures, for example in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and / or desired, the solution of the polypeptide is purified by conventional chromatographic methods, for example, gel filtration chromatography, ion exchange chromatography, chromatography through DEAE-cellulose, or (immuno) affinity chromatography, for example after the preferential biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein. Optionally, an affinity tag sequence (e.g., His(6) tag) may be attached to or incorporated into the polypeptide sequence to facilitate downstream purification.

[0195] Once expressed, the FVIII protein is purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, HPLC purification, gel electrophoresis, and the like (see generally, Scopes, "Protein Purification," Springer-Verlag, NY (1982)). Substantially pure proteins having at least about 90-95% homogeneity are preferred for pharmaceutical uses, with 98-99% or more homogeneity being most preferred.

[0196] Pharmaceutical Compositions A composition containing an isolated nucleic acid molecule of the present disclosure, a polypeptide having FVIII activity encoded by the nucleic acid molecule, a vector, or a host cell may contain a suitable pharma- ceutically acceptable carrier. For example, the composition may contain excipients and / or adjuvants that facilitate processing of the active compound into a product designed for delivery to a site of action.

[0197] The pharmaceutical composition is formulated for parenteral administration (i.e., intravenous, subcutaneous, or intramuscular) by bolus injection. The formulation for injection is presented in unit dosage form, for example, in ampoules or multi-dose containers with added preservatives. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous medium, and may contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable medium, for example, pyrogen-free water.

[0198] Preparations suitable for parenteral administration also include aqueous solutions of the active compound in water-soluble form, for example, in water-soluble salt form. In addition, suspensions of the active compound as appropriate oily injection suspensions are also administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and dextran. Optionally, the suspension may also contain a stabilizer. Liposomes are also used to encapsulate the molecules of the present disclosure for delivery to cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like. In some embodiments, the composition includes an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride. In other embodiments, the composition includes a pharma- ceutically acceptable substance, such as a humectant, or minor amounts of auxiliary substances, such as humectants or emulsifiers, preservatives or buffers, which enhance the shelf life or efficacy of the active ingredient.

[0199] The compositions of the present disclosure may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solids, and solids. The preferred form depends on the mode of administration and therapeutic application.

[0200] The composition is formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of the above-listed ingredients as required, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active ingredient into a sterile medium containing a basic dispersion medium and other required ingredients from the above-listed ingredients. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained by using a coating such as lecithin, or by maintaining the required particle size in the case of dispersions, or by using surfactants. Prolonged absorption of injectable compositions can be achieved by incorporating an agent that delays absorption, such as monostearate salts and gelatin, into the composition.

[0201] The active ingredient is formulated with controlled release formulation or device.The examples of such formulation and device include implant, transdermal patch and microencapsulated delivery system.Biodegradable polymer, biocompatible polymer, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid are used.The method for producing such formulation and device is known in the art.See, for example, "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0202] Injectable depot preparations are made by forming microencapsulated matrices of drugs in biodegradable polymers such as polylactide-polyglycolide.Depending on the drug-to-polymer ratio and the nature of the polymer used, the drug release rate is controlled.Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides.Injectable depot preparations are also made by encapsulating drugs in liposomes or microemulsions.

[0203] The composition may also incorporate an auxiliary active compound. In one embodiment, the chimeric protein of the present disclosure is formulated with another coagulation factor, or a variant, fragment, analog, or derivative thereof. For example, the coagulation factor includes, but is not limited to, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor, or recombinant soluble tissue factor (rsTF), or an activated form of any of the foregoing. The coagulation factor of the hemostatic agent may also include an antifibrinolytic agent, such as epsilon-aminocaproic acid, tranexamic acid.

[0204] Dosage regimen is adjusted to obtain the desired optimal response. For example, a single bolus may be administered, or a number of divided doses may be administered over time, and the dose may be reduced or increased accordingly as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in unit dosage form. For example, see "Remington's Pharmaceutical Sciences" (Mack Pub.Co., Easton, Pa., 1980).

[0205] In addition to the active compound, liquid dosage forms may contain inactive ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofururyl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.

[0206] Non-limiting examples of suitable pharmaceutical carriers are also described in "Remington's Pharmaceutical Sciences" by EW Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, etc. The composition may also contain a pH buffering agent and a humectant or emulsifier.

[0207] For oral administration, the pharmaceutical composition may take the form of a tablet or capsule, which is prepared by conventional means. The composition may also be prepared as a liquid, for example, a syrup or suspension. The liquid may contain a suspending agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifying agent (lecithin or gum acacia), a non-aqueous medium (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and a preservative (e.g., methyl-p-hydroxybenzoate or propyl-p-hydroxybenzoate, or sorbic acid). The preparation may also contain flavorings, colorings, and sweetening agents. Alternatively, the composition may be provided as a dry product for constitution with water or another suitable vehicle.

[0208] For buccal administration, the composition may take the form of tablets or lozenges following conventional protocols.

[0209] For inhalation administration, the compound for use according to the present disclosure is conveniently delivered in the form of a nebulized aerosol, with or without excipients, or in the form of an aerosol spray from a pressurized pack or nebulizer, optionally with a propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide, or other suitable gas.In the case of a pressurized aerosol, the dosage unit is determined by providing a valve that delivers a metered amount.Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator are formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0210] Pharmaceutical compositions can also be formulated for rectal administration as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0211] In one embodiment, the pharmaceutical composition comprises a polypeptide having factor VIII activity, an optimized nucleic acid molecule encoding a polypeptide having factor VIII activity, a vector comprising the nucleic acid molecule, or a host cell comprising the vector and a pharma- ceutically acceptable carrier. In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration, and oral administration. Parenteral administration can be intravenous or subcutaneous administration.

[0212] Treatment In some aspects, the present disclosure is directed to a method of treating a disease or condition in a subject in need thereof, the method comprising administering a nucleic acid molecule, vector, polypeptide, or pharmaceutical composition disclosed herein.

[0213] In some embodiments, the present disclosure is directed to a method of treating a bleeding disorder. In some embodiments, the present disclosure is directed to a method of treating hemophilia A.

[0214] The isolated nucleic acid molecule, vector, or polypeptide may be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, for example, via oral, sublingual, buccal, sublingual, intranasal, rectal, vaginal, or pulmonary routes. The coagulation factor protein may be implanted within or linked to a biopolymeric solid support that allows for sustained release of the chimeric protein to the desired site.

[0215] In one embodiment, the administration route of the isolated nucleic acid molecule, vector, or polypeptide is parenteral.As used herein, the term "parenteral" includes intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intrarectal administration, or intravaginal administration.In some embodiments, the isolated nucleic acid molecule, vector, or polypeptide is administered intravenously.Although all of these administration forms are expressly contemplated to be within the scope of the present disclosure, the form for administration will be injectable solution, particularly for intravenous or intraarterial injection or instillation.

[0216] The effective dose of the composition of the present disclosure for treating a condition varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other medicines administered, and whether the treatment is a preventive or therapeutic treatment.Usually, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated.Treatment dosages are titrated using routine methods known to those skilled in the art that optimize safety and efficacy.

[0217] The nucleic acid molecules, vectors, or polypeptides of the disclosure are optionally administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic treatment).

[0218] As used herein, administration of the disclosed isolated nucleic acid molecule, vector, or polypeptide with or in combination with adjunctive therapy refers to sequential administration or application, simultaneous administration or application, co-administration or application, co-administration or application, or parallel administration or application of the therapy and the disclosed polypeptide. Those skilled in the art will recognize that the administration or application of various components of the combined therapy regimen is timed to enhance the efficacy of treatment. Those skilled in the art (e.g., physicians) will be able to easily identify an effective combined therapy regimen based on the selected adjunctive therapy and the teachings of this specification without undue experimentation.

[0219] It will be further appreciated that the isolated nucleic acid molecules, vectors, or polypeptides of the present disclosure may be used with or in combination with one or more drugs (e.g., to provide a combination therapeutic regimen). Exemplary drugs to be combined with the polypeptides or polynucleotides of the present disclosure include drugs that represent the current standard of care for the particular disorder being treated. Such drugs may be chemicals or biopharmaceuticals in nature. The term "biopharmaceutical" or "biopharmaceutical agent" refers to any pharmacologic active agent made from living organisms and / or their products that is intended for use as a therapeutic agent.

[0220] The amount of drugs used in combination with the polynucleotides or polypeptides of the present disclosure may vary from subject to subject and may be administered according to what is known in the art. See, for example, Bruce A Chabner et al., "Antineoplastic Agents," GOODMAN and GILMAN, "PHARMACOLOGICAL BASIS OF THERAPEUTICS," pp. 1233-1287 (Joel G. Hardman et al., eds., 9th ed., 1996). In another embodiment, amounts of such drugs are administered that are consistent with standard of care.

[0221] In one embodiment, also disclosed herein is a kit comprising the nucleic acid molecule disclosed herein and instructions for administering the nucleic acid molecule to a subject in need thereof. In another embodiment, disclosed herein is a baculovirus system for producing the nucleic acid molecule provided herein. The nucleic acid molecule is produced in insect cells. In another embodiment, provided is a nanoparticle delivery system for an expression construct. The expression construct comprises the nucleic acid molecule disclosed herein.

[0222] Gene Therapy In some embodiments, the nucleic acid molecule disclosed herein is used in gene therapy.The optimized FVIII nucleic acid molecule disclosed herein is used in any context where expression of FVIII is required.In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2.In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1.

[0223] For example, somatic cell gene therapy is being explored as a possible treatment for hemophilia A. Gene therapy is a particularly attractive treatment for hemophilia due to its potential to cure the disease through sustained endogenous production of FVIII after a single administration of a vector. Hemophilia A is well suited to a gene replacement approach because its clinical symptoms are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200 ng / ml) in plasma.

[0224] In one aspect, the nucleic acid molecules described herein are used in AAV gene therapy. AAV can infect many mammalian cells. See, for example, Tratschin et al. (1985), Mol. Cell Biol., 5:3251-3260; and Grimm et al. (1999), Hum. Gene Ther., 10:2445-2450. rAAV vectors carry the nucleic acid sequence that codes for a gene of interest, or a fragment thereof, under the control of a regulatory sequence that directs the expression of the gene product in cells. In some embodiments, rAAV is formulated with a carrier and additional components suitable for administration.

[0225] In another aspect, the nucleic acid molecules described herein are used in lentivirus gene therapy. Lentiviruses are RNA viruses whose viral genome is RNA. When lentivirus infects a host cell, the genomic RNA is reverse transcribed into a DNA intermediate and is highly efficiently integrated into the chromosomal DNA of the infected cell. In some embodiments, lentiviruses are formulated with carriers and additional components suitable for administration. In another aspect, the nucleic acid molecules described herein are used in adenovirus therapy. A review of the use of adenovirus for gene therapy can be found, for example, in Wold et al. (1985), Curr Gene Ther., 13(6):421-33. In another aspect, the nucleic acid molecules described herein are used in non-viral gene therapy. The optimized FVIII protein of the present disclosure is produced in vivo in a mammal, e.g., a human patient, and a gene therapy approach may be therapeutically beneficial for the treatment of bleeding diseases or disorders selected from the group consisting of bleeding coagulation disorders, hemarthrosis, intramuscular bleeding, oral bleeding, bleeding into muscle, oral bleeding, trauma, traumatic head, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding into the retropharyngeal space, bleeding into the retroperitoneal space, and bleeding into the iliopsoas sheath. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves the administration of an optimized FVIII-encoding nucleic acid operably linked to an appropriate expression control sequence. In certain embodiments, these sequences are incorporated into a viral vector. The viral vector suitable for such gene therapy includes adenoviral vector, lentiviral vector, baculoviral vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, and adeno-associated virus (AAV) vector.The viral vector can be a replication-defective viral vector.In another embodiment, the adenoviral vector is deleted its E1 gene or E3 gene.In another embodiment, the sequence is also incorporated into a non-viral vector, which is known to those skilled in the art.

[0226] In another embodiment, the nucleic acid molecules disclosed herein are used for specific modification of genetic information (e.g., genome) of an organism. As used herein, the term "modification" or "modification of genetic information" refers to any change in the genome of a cell. In the context of treating a genetic disorder, modification can include, but is not limited to, insertion, deletion, and / or correction.

[0227] In some embodiments, the alteration may also include knocking in, knocking out, or knocking down of a gene. As used herein, the term "knock-in" refers to the addition of a DNA sequence or a fragment thereof to a genome. Such DNA sequence to be knocked in may include the entire gene, or the entire gene or gene may include the regulatory sequences associated with the gene or any part or fragment thereof. For example, a cDNA encoding a wild-type protein is inserted into the genome of a cell carrying a mutant gene. A knock-in strategy does not require replacing a defective gene in whole or in part. In some cases, a knock-in strategy may further involve replacing an existing sequence with a prepared sequence, for example, replacing a mutant allele with a wild-type copy. The term "knock-out" refers to the loss of a gene or gene expression. For example, a gene is knocked out by the deletion or addition of a nucleotide sequence that results in a disruption of the reading frame. As another example, a gene is knocked out by replacing a part of the gene with a non-involved sequence. As used herein, "knock-down" refers to the reduction of expression of a gene or its gene product(s). Gene knockdown may result in attenuation of protein activity or function, or in reduced or abolished protein levels.

[0228] In some embodiments, the nucleic acid sequences disclosed herein are used for genome editing. Genome editing generally refers to a process of modifying the nucleotide sequence of a genome, preferably in a precise or predetermined manner. Examples of genome editing methods described herein include methods using site-directed nucleases to cleave deoxyribonucleic acid (DNA) at precise target positions in the genome, thereby creating single-stranded or double-stranded DNA breaks at specific positions in the genome. Such breaks can be repaired and are regularly repaired by natural endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), as recently reviewed in Cox et al. (2015), Nature Medicine, 21(2):121-31. These two major DNA repair processes comprise a family of alternative pathways. NHEJ directly connects the DNA ends resulting from the double-stranded break, but in some cases involves the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR utilizes homologous or donor sequences as templates for inserting a defined DNA sequence into the breakpoint. Homologous sequences can be present in endogenous genomes, such as sister chromatids. Alternatively, donor can be exogenous nucleic acid, such as plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, duplex oligonucleotide, or virus, which has a large region of homology with the locus that is cut by nuclease, but also contains additional sequences or sequence changes, including deletions, that are integrated into the cut target locus. The third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as "alternative NHEJ", whose genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cut site. MMEJ may use a small number of base-pair homologous sequences flanking the DNA break site to drive a more favorable repair outcome by joining the DNA ends, but recent reports have further elucidated the molecular mechanisms of this process (see, e.g., Cho and Greenberg (2015), Nature, 518, 174-76).In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the DNA break site.

[0229] Each of these genome editing mechanisms is used to create the desired genome modification.The step in genome editing process can be to create one DNA break or two DNA breaks as double-strand breaks or two single-strand breaks in the target locus as the adjacent site of intended mutation.This is achieved through the use of site-directed polypeptides, such as CRISPR endonuclease system.

[0230] In another embodiment, the nucleic acid molecule described herein is used in lipid nanoparticle (LNP) mediated delivery of FVIII ceDNA.To prevent the degradation of nucleic acid in plasma and facilitate the uptake of oligonucleotide into cells, lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides are used.Such lipid nanoparticles are used to deliver the nucleic acid molecule described herein to subjects.

[0231] The present disclosure provides a method of increasing expression of a polypeptide with FVIII activity in a subject, comprising administering to a subject in need thereof an isolated nucleic acid molecule of the present disclosure, wherein expression of the polypeptide is increased relative to a reference nucleic acid molecule comprising SEQ ID NO: 6. The present disclosure also provides a method of increasing expression of a polypeptide with FVIII activity in a subject, comprising administering to a subject in need thereof a vector of the present disclosure, wherein expression of the polypeptide is increased relative to a vector comprising the reference nucleic acid molecule.

[0232] All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

[0233] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0234] Working Example Having provided the foregoing disclosure, a further understanding can be obtained by reference to the examples provided herein, which are intended for purposes of illustration only and are not intended to be limiting. EXAMPLES

[0235] Approaches for generating ceDNA In the baculovirus-insect cell system, recombinant BEV delivers a gene of interest under a strong promoter, resulting in a transcription complex that is essential for viral replication in insect cells. The baculovirus-insect cell system provides the flexibility to insert a transgene of interest into the baculovirus genome and / or into the insect cell genome in the form of a stable cell line. Taking advantage of these advantages of the baculovirus-insect cell system, three different approaches for ceDNA production were designed to provide a wide range of options according to ease of scalability.

[0236] 1.OneBAC To explore the use of the OneBac approach for transgene expression, an optimized FVIIIXTEN expression cassette was inserted into the mini-attTn7 site in the polyhedrin locus in BIVVBac with parvovirus ITRs via Tn7 transposition, and an ITR-specific replication (Rep) gene expression cassette was inserted into the LoxP site in the EGT locus in the same backbone via Cre-LoxP recombination. Recombinant BEV was then produced and used for infection in Sf9 cells to generate FVIIIXTEN ceDNA, as depicted in Figure 1A. As described below, different promoters were used to control Rep expression levels to demonstrate the concept of the OneBac approach for ceDNA generation.

[0237] 2.TwoBAC: To explore the use of the TwoBac approach for transgene expression, an optimized FVIIIXTEN expression cassette was inserted via Tn7 transposition into the mini-attTn7 site in the polyhedrin locus in two different BIVVBac bacmids together with parvovirus ITR and ITR-specific replication (Rep) gene expression cassettes. Recombinant BEV was then produced and used for co-infection in Sf9 cells to generate FVIIIXTEN ceDNA, as depicted in Figure 1B. As described in the following experiments, the challenges associated with the Two BAC approach were explored using two baculoviruses with different multiplicity of infection (MOI) ratios and fine-tuning of Rep expression levels to obtain reproducible ceDNA productivity.

[0238] 3. Stable cell lines: To explore the use of a stable cell line approach for transgene expression, a stable cell line was generated with an optimized FVIIIXTEN expression cassette with parvovirus ITRs. An ITR-specific replication (Rep) gene expression cassette was inserted into the mini-attTn7 site in the polyhedrin locus in the BIVVBac bacmid via Tn7 transposition. Recombinant Rep.BEV was then generated and used for infection into FVIIIXTEN stable cell lines, generating FVIIIXTEN ceDNA, as depicted in Figure 1C. As described in the following experiment, the challenges associated with the stable cell line approach were explored by enriching FVIIIXTEN transfectants via FACS cell sorting, using GFP as a proxy to speed up the process of generating stable cell lines. EXAMPLES

[0239] FVIIIXTEN HBoV1 ITR expression construct Human bocavirus type 1 (HBoV1), an autonomous parvovirus, is a helper virus that supports the replication of wild-type adeno-associated virus type 2 (AAV2). The use of AAV and non-AAV parvovirus ITRs for FVIIIXTEN ceDNA production has been demonstrated in the baculovirus system (see, e.g., U.S. Patent Application No. 63 / 069,073). HBoV1 ITRs have a unique size and morphology compared to other parvovirus ITRs. HBoV1 5' (REH) ITR is 140 bp long (SEQ ID NO: 1) and forms a "U" shaped hairpin with perfect base pairing, whereas the 3' (LEH) ITR is 200 bp long (SEQ ID NO: 2) and forms a loop with a three-pronged branch point, making HBoV1 ITRs asymmetric, which is notably different from the terminal regions of other parvovirus ITRs (Figure 2A).

[0240] HboV1 ITRs were explored for use in generating FVIIIXTEN ceDNA. It was hypothesized that asymmetric ITRs could enhance long-term sustained expression by stabilizing the transgene. To test this hypothesis, a DNA construct was synthesized via GenScript® (Piscataway, NJ) containing B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) containing XTEN 144 peptide (FVIIIXTEN) under the control of a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) with enhancer element (A1MB2), a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), a bovine growth hormone polyadenylation (bGHpA) signal, and adjacent human HBoV1 5'ITR / 3'ITR to generate the set of nucleic acid sequences shown as SEQ ID NO:3 (Figure 2A). This synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector to generate the pFastBac.mTTR.FVIIIXTEN.HBoV1.ITR transfer vector (FIG. 2B). This vector was then transformed into BIVVBacDH10B The recombinant BEV, AcBIVVBac.Polh.GPV.Rep, was transformed into E. coli. Tn7 was prepared. EXAMPLES

[0241] HBoV1 NS1 (non-structural) expression construct HBoV1 NS1 Tn7 transfer vector HBoV1 is known to express five nonstructural proteins, namely NS1, NS2, NS3, NS4, and NP1, from mRNA transcripts generated through alternative splicing and polyadenylation of a single viral pre-mRNA. The NS1-NS4 proteins are encoded within different regions of the same open reading frame (ORF). NS1 consists of an origin binding / endonuclease domain (OBD), a helicase domain, and a putative transactivation domain (TAD) at the N-terminus, middle, and C-terminus, respectively. NS1 binds to the HBoV1 replication origin and likely nicks the single-stranded DNA (ssDNA) of the origin during replication of the rolling hairpin.

[0242] To explore the role of NS1 in ITR-mediated vector production in eukaryotic cells and to "rescue" the HBoV1 ITR-flanked FVIIIXTEN ceDNA vector genome from Sf9 cells, an HBoV1 NS1 expression construct was generated and inserted into BIVVBac to generate recombinant BEV expressing HBoV1 NS1 in Sf9 cells.

[0243] To generate the expression vector, the coding sequence of HBoV1 NS1 was obtained from the HBoV1 genome (GenBank Accession Number: JQ923422) and codon-optimized for the Sf cell genome prior to synthesis via GenScript® to generate the nucleic acid sequence set shown as SEQ ID NO: 4. The synthetic HBoV1 NS1 DNA was then cloned into the pFastBac1 (Invitrogen) vector (FIG. 3A) under the control of the AcMNPV polyhedrin promoter to generate the pFastBac.Polh.HBoV1.NS1 transfer vector (FIG. 3B). The synthetic HBoV1 NS1 DNA was also cloned into the pFastBac1 (Invitrogen) vector (FIG. 4A) under the control of the immediate early 1 (IE1) promoter preceded by the AcMNPV transcription factor hr5 element to generate the pFastBac.HR5.IE1.HBoV1.NS1 transfer vector (FIG. 4B). The synthetic HBoV1 NS1 DNA was also cloned into the pFastBac1 (Invitrogen) vector (Figure 5A) under the OpMNPV immediate early 2 (IE2) promoter to generate the pFastBac.OpIE2.HBoV1.NS1 transfer vector (Figure 5C). DH10B The recombinant BEV was transformed into E. coli: AcBIVVBac.Polh.HBoV1.NS1, respectively. Tn7 ,AcBIVVBac.HR5.IE1.HBoV1.NS1 Tn7 , or AcBIVVBac.OpIE2.HBoV1.NS1 Tn7 was prepared.

[0244] These recombinant BEVs were then used for coinfection with FVIIIXTEN BEV (TwoBAC) in Sf9 cells to generate FVIIIXTEN ceDNA vectors.

[0245] HBoV1 NS1 Cre-LoxP donor vector To explore the use of the OneBac approach for the generation of ceDNA, the HBoV1 NS1 gene was inserted into the LoxP site in a recombinant BIVVBac encoding a FVIIIXTEN expression cassette at the Tn7 site in the polyhedrin locus. The rationale for inserting both of these genes at these sites was to avoid interference from the inverted terminal repeats (ITRs) that flank FVIIIXTEN together with the LoxP sequences, which are also palindromic repeats.

[0246] In addition, to address the challenges associated with the OneBAC system described above, different promoters of baculovirus genes expressed at different times and levels during the infection cycle were examined to control the expression levels of HBoV1 NS1 within OneBAC encoding both FVIIIXTEN HBoV1 ITR / NS1.

[0247] Synthetic Sf codon-optimized HBoV1 NS1 DNA was cloned into a Cre-LoxP donor vector (described in U.S. Patent Application No. 63 / 069,073) under the control of the AcMNPV polyhedrin promoter (FIG. 3A) or the immediate early 1 promoter preceded by the AcMNPV transcriptional enhancer hr5 element (FIG. 4A) and not (FIG. 5B) to generate the Cre-LoxP donor vectors pCL.Polh.HBoV1.NS1 (FIG. 3C), pCL.HR5.IE1.HBoV1.NS1 (FIG. 4C), and pCL.IE1.HBoV1.NS1 (FIG. 5D), respectively. These constructs are designated by "pCL," the prefix for "plasmid Cre-LoxP" (see FIGS. 3C, 4C, 5D). The resulting Cre-LoxP donor vector was then inserted at the LoxP sites into the BIVVBac bacmid (FIG. 6B), which encodes the FVIIIXTEN HBoV1 ITRs at the Tn7 site, as described below. EXAMPLES

[0248] FVIIIXTEN HBoV1 ITR Baculovirus Expression Vector (BEV) To generate a recombinant BEV encoding the FVIIIXTEN expression cassette together with the HBoV1 ITRs (Figure 2A), we first transformed the BIVVBac DH10B E. coli (described in US Patent Application Serial No. 63 / 069,073) was highly transformed with the Tn7 transfer vector, pFastBac.mTTR.FVIIIXTEN.HBoV1.ITR (FIG. 2B). Transformants were selected with kanamycin, gentamicin, X-Gal, and IPTG. Site-specific transfer of the FVIIIXTEN expression cassette and gentamicin resistance gene at the mini-attTn7 insertion site in BIVVBac disrupted LacZα (fused in-frame with mini-attTn7), resulting in white E. coli colonies upon X-Gal-mediated double antibiotic selection. Recombinant bacmid DNA was isolated from white E. coli colonies by alkaline lysis miniprep method and digested with restriction enzymes to determine the exact gene structure. Restriction mapping results showed the predicted fragments for each recombinant bacmid, suggesting site-specific transfer of the FVIIIXTEN expression cassette with the HBoV1 ITR into the polyhedrin locus of BIVVBac (Figure 6A). Further confirmation was obtained by PCR amplifying the region spanning the predicted insertion site using primers internal and external to the transfer plasmid and sequencing the resulting amplicons (data not shown).

[0249] The correct recombinant bacmid encoding the FVIIIXTEN expression cassette together with the HBoV1 ITR was maxiprep-purified and transfected into Sf9 cells using Cellfectin® (Invitrogen) transfection reagent according to the manufacturer's instructions. Four to five days after transfection, progeny baculovirus were harvested and plaque-purified in Sf9 cells as previously described. Jarvis et al. (2014), Methods Enzymol., 536:149-163. Recombinant BEV, AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR Tn7 Six plaque-purified RFP+ clones from (Figure 6B) were cultured at 0.5 × 10 per mL in ESF-921 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a T25 flask. 6 The BEV clones were amplified to P1 (first passage) in Sf9 cells seeded with 10 ... EXAMPLES

[0250] FVIIIXTEN HBoV1 ITR+HBoV1 NS1 Baculovirus Expression Vector (BEV) To investigate whether BIVVBac can be used to house multiple transgenes, a family of derivative vectors was created that encode two transgene expression cassettes under the control of different promoters: 1) FVIIIXTEN HBoV1 ITR, and 2) HBoV1 NS1. These BEVs were generated in two steps. First, the FVIIIXTEN expression cassette with the HBoV1 ITR was inserted into the mini-attTn7 site in the polyhedrin locus in BIVVBac via Tn7 transposition as described above. The resulting bacmid, BIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR (FIG. 6B), was then used to insert the HBoV1 NS1 expression cassette into the LoxP site in the EGT locus via in vitro Cre-LoxP recombination using Cre recombinase (New England Biolabs).

[0251] In step 1, a Cre-LoxP donor vector encoding HBoV1 NS1 under the AcMNPV polyhedrin promoter (Figure 3C) or the immediate early 1 (IE1) promoter preceded by the AcMNPV transcriptional enhancer hr5 element (Figure 4C) and without (Figure 5D) was inserted into the BIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR bacmid (Figure 6B). Recombination reactions were transformed into DH10B E. coli and transformants were selected with kanamycin, gentamicin, and ampicillin. Triple antibiotic resistant colonies were screened by restriction enzyme mapping and / or by PCR amplifying the region spanning the predicted insertion site using primers internal and external to the transfer plasmid (Figures 7A, 7B, and 7D) and sequencing the resulting amplicon sequences.

[0252] The correct recombinant bacmids encoding both transgene cassettes were maxiprep-purified and transfected into Sf9 cells using Cellfectin® (Invitrogen) transfection reagent. Four to five days after transfection, progeny baculoviruses were harvested and plaque-purified in Sf9 cells. Each recombinant BEV (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1) was transfected with 100% PBS. LoxP Figure 7D: AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)IE1.HBoV1.NS1 LoxP 7E; and AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)HR5.IE1.HBoV1.NS1 LoxP Six plaque-purified RFP+ and GFP+ clones from Fig. 7F were cultured at 0.5 × 10 per mL in ESF921 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a T25 flask. 6 The BEV clones were amplified to P1 (first passage) in Sf9 cells seeded with 10 ... EXAMPLES

[0253] Generation of FVIIIXTEN HBoV1 ITR ceDNA vector from OneBAC OneBAC BEV encoding both the FVIIIXTEN HBoV1 ITR and HBoV1 NS1 genes (Figures 7D-7F) was examined for FVIIIXTEN ceDNA production in Sf9 cells. Approximately 2.5 x 10 cells per mL were cultured. 6 were infected with titrated working stocks (P2) of each BEV at a multiplicity of infection (MOI) of 0.1, 0.5, 1.0, 2.0, or 3.0 plaque-forming units (pfu) per cell (Figure 8A). Cells were suspended in 50 mL of serum-free ESF-921 medium and then incubated for 72-96 h or until cell viability reached 60-70% in a 28°C shaking incubator. Approximately 96 h after infection, infected cells were harvested and pellets were processed for isolation of FVIIIXTEN ceDNA vector by PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. Final elution fractions were analyzed on 0.8-1.2% agarose gel electrophoresis to determine FVIIIXTEN ceDNA vector productivity.

[0254] AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITR)Polh.HBoV1.NS1, encoding FVIIIXTEN together with HBoV1 ITRs and polyhedrin-driven HBoV1-NS1 LoxP Agarose gel analysis for BEV (Figure 8B) is shown in Figure 8C. Results showed DNA bands corresponding to the size of FVIIIXTEN HBoV1 ITR (approximately 8.5 kb) ceDNA at all doses tested, with productivity increasing with increasing MOI.

[0255] This result was the opposite of the ceDNA productivity obtained with AAV2 ITR OneBAC, where a decrease in productivity was observed along with an increase in viral load. Without being bound by theory, HBoV1-NS1 protein may have a unique binding mechanism and endonuclease activity at the terminal separation site of HBoV1 ITR for DNA replication, which may be due to the significantly different structures of REH and LEH ITRs (Figure 2A).

[0256] In conclusion, these experiments demonstrated that the One BAC approach demonstrates the concept of ceDNA generation from a single recombinant BEV encoding FVIIIXTEN together with the HBoV1 ITR and NS1 transgenes. It also demonstrates the feasibility and functionality of multiple transgenes inserted at different loci within a baculovirus shuttle vector (BIVVBac) and its potential use for the generation of recombinant AAV vectors in the baculovirus-insect cell system. EXAMPLES

[0257] HBoV1 NS1 (non-structural) baculovirus expression vector (BEV) The only structurally characterized parvovirus NS1 N-terminal nuclease domain is derived from AAV2 Rep, which binds to consecutive tetra-oligonucleotide repeats at the origin of replication (Ori). However, such tetra-oligonucleotide repeats are specific to AAV and are absent in HBoV1. Indeed, the LEH (3' ITR) of the HBoV1 genome forms a loop with a three-way branch point, whereas the REH (5' ITR) is a hairpin with perfect base pairing (Figure 2A), and the LEH and REH are conserved in bocaviruses and significantly different from the terminal regions of the AAV and parvovirus B19 (B19V) genomes. These findings suggest that the recognition of Ori by NS1 in HBoV1 is significantly different from that in AAV. Furthermore, AAV is not known to cause human disease, and it is a dependant virus because the replication of the virus requires a helper virus such as herpesvirus or adenovirus. HBoV1 NS1 shares only 14% sequence identity with AAV Rep. HBoV1-NS1 contains a positively charged surface that is a putative binding site for Ori, and it has been demonstrated that it directly supports the replication of HBoV DNA, similar to the common rolling-hairpin mechanism proposed for parvoviruses.

[0258] HBoV1-NS1 is believed to be essential for ITR-mediated vector production in eukaryotic cells. To explore the potential "rescue" of ITR-flanked FVIIIXTEN vector genomes from Sf9 cells, or FVIIIXTEN BEV, recombinant BEVs encoding HBoV1-NS1 were generated under different baculovirus promoters to optimize the expression level of NS1 in Sf9 cells.

[0259] To generate these BEVs, BIVVBac DH10B E. coli (see U.S. Patent Application No. 63 / 069,073) was highly transformed with the Tn7 transfer vectors pFastBac.Polh.HBoV1-NS1 (FIG. 3B), pFastBac.HR5.IE1.HBoV1-NS1 (FIG. 4B), or pFastBac.OpIE2.HBoV1.NS1 (FIG. 5C). Transformants were selected with kanamycin, gentamicin, X-Gal, and IPTG. Site-specific transfer of the HBoV1-NS1 expression cassette and gentamicin resistance gene at the mini-attTn7 insertion site in BIVVBac disrupted LacZα (fused in-frame with mini-attTn7) and resulted in white E. coli colonies upon X-Gal-mediated double antibiotic selection. Therefore, recombinant bacmid DNA was isolated from white E. coli colonies by alkaline lysis miniprep method and digested with restriction enzymes to determine the exact gene structure. Restriction enzyme mapping results showed the predicted fragments for each recombinant bacmid, suggesting site-specific transposition of HBoV1-NS1 within the polyhedrin locus of BIVVBac (Figure 9A). Further confirmation was obtained by PCR amplifying the region spanning the predicted insertion site using primers internal and external to the transfer plasmid and sequencing the resulting amplicon (data not shown).

[0260] Sf9 cells were transfected with the correct recombinant bacmids confirmed to encode Polh.HBoV1-NS1, HR5.IE1.HBoV1-NS1, or OpIE2-HBoV1-NS1 using Cellfectin® (Invitrogen) transfection reagent according to the manufacturer's instructions. Four to five days after transfection, progeny baculoviruses were harvested and plaque purified in Sf9 cells as previously described. Jarvis et al. (2014), Methods Enzymol., 536:149-163. Each recombinant BEV, AcBIVVBac.Polh.HBoV1-NS1 Tn7 (Figure 9B), AcBIVVBac.HR5.IE1.HBoV1.NS1 Tn7 (Figure 9C), and AcBIVVBac.OpIE2.HBoV1.NS1 Tn7 Six plaque-purified RFP+ clones from (Figure 9D) were cultured at 0.5 × 10 per mL in ESF-921 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a T25 flask. 6 The viruses were amplified to P1 (first passage) in Sf9 cells seeded with 1000 μg / ml of each vector. Four to five days after infection, all clones showed a progression of infection as determined by the number of RFP+ cells for each BEV clone, suggesting that the virus was capable of normal replication and that the insertion of HBoV1-NS1 in the baculovirus genome had no deleterious effect on the generation of progeny viruses.

[0261] The highest RFP+ clones were selected for further amplification in Sf9 cells to generate working BEV stocks (P2). The titrated virus stocks were then used for co-infection with FVIIIXTEN BEV in stable cell lines for the TwoBAC system or FVIIIXTEN HBoV1 ITR for generation of ceDNA vectors. EXAMPLES

[0262] Construction of FVIIIXTEN HBoV1 ITR ceDNA vector from TwoBAC To explore the Two BAC approach for transgene expression, clonal recombinant BEVs encoding FVIIIXTEN HBoV1 ITRs together with polyhedrin-driven HBoV1-NS1 BEVs were co-infected in Sf9 cells at different MOIs of 1:10 and 1:5, or at different MOIs of 0.3, 1.0, 3.0, and 5.0 pfu per cell for FVIIIXTEN ceDNA vector production (Figure 10A). 6 AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR cells were seeded in 50 mL of serum-free ESF-921 medium at MOIs of 0.1, 0.3, 0.5, 1.0, 3.0, and 5.0 pfu per cell. Tn7 Titrated working stocks of BEV (P2) were prepared with either 0.01, 0.03, 0.05, 0.1, 0.3, 0.5 pfu per cell at a constant ratio of 1:10 or 0.02, 0.06, 0.1, 0.2, 0.6, 1.0 pfu per cell at a constant ratio of 1:5, AcBIVVBac.Polh.HBoV1-NS1. Tn7 The cells were co-infected with BEV. Similarly, cells were also co-infected at ratios of 1:1, 1:2, 1:5, or 1:10 with a constant MOI of 0.3, 1.0, 3.0, or 5.0 pfu per cell (Figure 10B). In each case, the virus inoculum was not removed and the cells were incubated in a shaking incubator at 28°C until cell viability reached 60-70%. Approximately 96 hours after infection, infected cells were harvested and pellets were processed for isolation of FVIIIXTEN ceDNA vectors by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions. The final elution fractions were analyzed on 0.8-1.2% agarose gel electrophoresis to determine ceDNA productivity.

[0263] As expected, agarose gel analysis showed that different conditions showed varying degrees of FVIIIXTEN ceDNA productivity. However, TwoBAC co-infected at MOI of 3.0 pfu per cell showed increasing levels of FVIIIXTEN ceDNA productivity with increasing virus load ratio, with 1:10 being the highest virus load ratio compared to other conditions tested (Figure 10C). High virus load appears to improve FVIIIXTEN HBoV1 ITR ceDNA productivity, which is consistent with the observations in OneBAC BEV (see Example 6). This further suggests the requirement of high levels of HBoV1-NS1 for HBoV1-ITR-dependent FVIIIXTEN ceDNA replication in Sf9 cells.

[0264] The results from OneBAC or TwoBAC indicate that the replication level of HBoV1-NS1 has a profound effect on the productivity of FVIIIXTEN ceDNA in the baculovirus system.

[0265] As an alternative to testing several different co-infection conditions discussed above, we explored other ways to improve FVIIIXTEN ceDNA productivity by utilizing different promoters of baculovirus genome. Baculovirus gene promoters are divided into immediate early promoter, early promoter, late promoter, and very late promoter according to their transcription initiation in the infection cycle. Among them, as the name indicates, immediate early (ie) gene promoters are turned on immediately after virus infection and remain active throughout the infection cycle. However, late gene promoters such as polyhedrin or very late gene promoters remain silent until the virus reaches the late stage of infection.

[0266] To take advantage of this broad selection of promoters from the baculovirus genome, the immediate early 1 (IE1) promoter was examined for HBoV1-NS1. The IE1 promoter was preceded by a transcriptional enhancer hr5 element, which has been shown to increase expression levels in Sf9 cells. This resulted in the creation of a recombinant BEV encoding HBoV1-NS1 under the control of the AcMNPV immediate early 1 (IE1) promoter preceded by the AcMNPV transcriptional enhancer hr5 element, as depicted in Figure 9C.

[0267] Based on the results obtained in Figure 10C, Sf9 cells were co-infected with BEV encoding FVIIIXTEN HBoV1 ITR and hr5.IE1-driven HBoV1-NS1 at different MOIs by keeping the ratio constant at 1:10. As a positive control, polyhedrin-driven HBoV1-NS1 BEV was incorporated, which was also examined within the same set of experiments. More specifically, approximately 2.0 x 10 Sf9 cells per mL were co-infected with BEV encoding FVIIIXTEN HBoV1 ITR and hr5.IE1-driven HBoV1-NS1 at different MOIs by keeping the ratio constant at 1:10. 6 and AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR at MOIs of 0.1, 0.3, 0.5, 1.0, 3.0, and 5.0 pfu per cell. Tn7 A titrated working stock (P2) of BEV (Figure 10B) was prepared with AcBIVVBac.Polh.HBoV1-NS1 at MOIs of 0.01, 0.03, 0.05, 0.1, 0.3, and 0.5 pfu per cell at a constant ratio of 1:10. Tn7 BEV, or AcBIVVBac.hr5.IE1.HBoV1-NS1 Tn7 The remaining procedures were carried out as described above (see Example 6).

[0268] The final eluted fractions were analyzed on 0.8-1.2% agarose gel electrophoresis to determine ceDNA productivity. Co-infection with polyhedrin-driven HBoV1-NS1 showed increasing levels of FVIIIXTEN ceDNA productivity with increasing MOI, further confirming the reproducibility of the Two BAC approach for ceDNA generation. Surprisingly, however, co-infection with hr5.IE1-driven HBoV1-NS1 showed barely detectable levels of FVIIIXTEN ceDNA and no clear increase in productivity with increasing MOI was observed, as observed in Figure 10C.

[0269] This data suggests that early expression of HBoV1-NS1 may not be critical for rescuing FVIIIXTEN ceDNA with HBoV1 ITRs. Instead, high expression levels at later stages of infection are required for efficient rescue and productivity of FVIIIXTEN ceDNA with HBoV1 ITRs. These results further confirm the requirement of high levels of HBoV1-NS1 for HBoV1-ITR-dependent FVIIIXTEN ceDNA replication in Sf9 cells.

[0270] In conclusion, these experiments demonstrated that the Two BAC approach provides a proof of concept for generating ceDNA from two recombinant BEVs encoding FVIIIXTEN with HBoV1 ITR and / or NS1 transgenes. These experiments also support the importance of optimal MOI ratios and / or promoters to achieve high productivity of FVIIIXTEN ceDNA in Sf9 cells. EXAMPLES

[0271] Stable cell lines for FVIIIXTEN HBoV1 ITR We hypothesized that the insect cell genome could potentially be modified to generate ceDNA for therapeutic applications after baculovirus infection. To test this hypothesis, we synthesized plasmids encoding a neomycin resistance marker (pUC57.HR5.IE1.NeoR.P10PAS: SEQ ID NO: 7) (FIG. 12A) or an enhanced green fluorescent protein (eGFP) (pUC57.HR5.IE1.eGFP.P10PAS: SEQ ID NO: 8) (FIG. 12B) under the control of the AcMNPV immediate early (ie1) promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal from GenScript® (Piscataway, NJ).

[0272] These plasmids were co-transfected into Sf9 cells with a plasmid encoding FVIIIXTEN with HBoV1 ITR (Sf.mTTR.FVIIIXTEN.HBoV1.ITR) (FIG. 12C) using a modified calcium phosphate transfection method. 24 hours after transfection, cells were visualized under a fluorescent microscope to determine transfection efficiency, and the results showed >80% GFP+ cells, suggesting a high transfection efficiency. 72 hours after transfection, cells were selected with G418 antibiotic (Sigma Aldrich) suspended at a final concentration of 1.0 mg / mL in complete TNMFH medium (Grace's Insect Medium supplemented with 10% FBS + 0.1% Pluronic F68). After about one week of selection, about 50% of transformed cells were recovered, suggesting that the neomycin resistance marker was stably integrated into the cell population. Viable cells were removed from the selection medium, fed with fresh complete TNMFH medium, and grown to confluence. Confluent cells were progressively expanded into larger culture vessels as adherent cultures as they continued to divide. Polyclonal cell populations were then adapted to suspension culture by growth in shake flasks for one passage in complete TNMFH medium, and in shake flasks for one passage in 10% FBS supplemented ESF-921 medium. Finally, cells were adapted to serum-free ESF-921 in shake flasks as suspension cultures. These shake flask cultures were routinely maintained in serum-free ESF-921 medium with passages every 4 days, and cell growth was monitored. EXAMPLES

[0273] Purification of FVIIIXTEN ceDNA In the baculovirus-insect cell system, recombinant BEV delivers a gene of interest under a strong promoter, resulting in a transcription complex that is essential for viral replication in insect cells. Typically, the baculovirus DNA genome is replicated in the nucleus to produce tens of millions of progeny virus particles, each containing the full-length DNA genome. It has been demonstrated that baculovirus genomic DNA is co-purified with ceDNA during DNA isolation from insect cells using plasmid DNA-based purification methods, such as silica gel columns. Commercially available plasmid DNA kit columns are generally not designed to separate DNA based on their molecular weight, so typically all forms of DNA present in the sample bind to these columns. Furthermore, the binding capacity of high molecular weight DNA may be different from low molecular weight DNA, and anion exchange-based kit columns are not optimized based on the binding efficiency of different sizes of DNA.

[0274] We hypothesized that the high molecular weight DNA (>20 kb) observed in the ceDNA preps was likely baculovirus genomic DNA and / or Sf9 cell genomic DNA that co-purified with the low molecular weight FVIIIXTEN ceDNA (approximately 8.5 kb) (see, e.g., Figures 8C, 10C, and 11C).

[0275] Previously, we employed an indirect approach to reduce baculovirus DNA by knocking out baculovirus capsid genes, such as VP80, which are required for the production of infectious progeny viruses. This approach showed a significant reduction in baculovirus DNA in ceDNA preps obtained from knockout BEV (see U.S. Patent Application No. 63 / 069,115). Although this approach was efficient in reducing baculovirus DNA contamination, it was not able to reduce cellular genomic DNA, which is present in significant amounts (about 60%) of the total DNA obtained from infected cell pellets.

[0276] Thus, we have employed a straightforward approach to separate FVIIIXTEN ceDNA from unwanted DNA and demonstrated efficient yield of purified FVIIIXTEN (>95% purity) from total DNA preps derived from infected cell pellets. This novel approach utilizes preparative electrophoresis, which is widely used to separate different protein molecules according to their size and charge. See, for example, Michov, B. (2020), "Electrophoresis", Berlin, Boston: De Gruyter, pp. 405-424. For example, Bio-Rad Model 491 prep cell or other such units are used to separate complex molecules based on their size.

[0277] The overall workflow of ceDNA purification is shown in Figure 13, where the process begins with the scale-up of Sf9 cell culture in serum-free insect cell culture medium from 0.5 L to 1.5 L or larger volumes (Figure 13A). Typically, approximately 1.3 x 10 cells per mL are purified. 6 After 2 days of incubation, a seeding density of approximately 2.5 x 10 cells per mL is desired. 6 Once a cell density of 1000 ng / ml is reached, cells are infected with OneBAC BEV or TwoBAC BEV (depending on the approach used for ceDNA generation) at an optimized MOI and incubated in a shaking incubator at 28 °C until viability reaches approximately 60-70%, which typically takes approximately 4 days (Figure 13B). Once viability reaches approximately 70%, cells are harvested and processed for total DNA purification by anion exchange chromatography kit column, such as the PureLink HiPure Expi Plasmid Gigaprep Purification Kit (Invitrogen), following the manufacturer's instructions. An aliquot of the purified DNA material is run on a 0.8-1.2% agarose gel to determine DNA productivity and integrity (Figure 13C).

[0278] The purified material is then loaded into a pharmaceutical agarose gel electrophoresis unit assembled according to the manufacturer's instructions and containing 0.5% pharmaceutical agarose gel and 0.25% stacking agarose gel. Samples are run at 4°C at low voltage (constant at about 40 volts) with a buffer recirculation flow rate of about 50 mL / min and an elution buffer flow rate of 50 μL / min for 6-7 days, with each fraction collected after 70-80 minutes in the fraction collection chamber. After sequential elution electrophoresis, 20 μL of each fraction is cross-referenced on a 0.8-1.2% agarose gel electrophoresis to determine the purity of the FVIIIXTEN ceDNA (Figure 13D). The desired fractions are combined and precipitated with 3M NaOAc, pH 5.5 and 100% EtOH at -20°C for 1-2 hours. Finally, the precipitated FVIIIXTEN ceDNA was pelleted at high speed and washed once with 70% EtOH before resuspension in TE buffer, pH 8.0. The purified FVIIIXTEN ceDNA was again run on a 0.8-1.2% agarose gel electrophoresis to confirm purity and integrity before injection into animals for in vivo efficacy studies (Figure 13E). EXAMPLES

[0279] In vivo efficacy of FVIIIXTEN HBoV1 ITR ssFVIIIXTEN HBoV1 ITR (single stranded DNA) We hypothesized that hairpins formed within the ITR regions would allow for long-term, sustained, high-level transgene expression. To probe the functionality of the HBoV1 ITRs in vivo, single-stranded DNA (ssDNA) containing codon-optimized human FVIIIXTEN with preformed HBoV1 ITRs was synthesized using hFVIIIR593C. + / +hFVIIIR593C / HemA mice were examined. These mice contain a human FVIII-R593C transgene designed with a mouse albumin (Alb) promoter driving the expression of a modified human coagulation factor VIII (FVIII) cDNA carrying a mutation frequently observed in patients with mild hemophilia A. These mice also carry a knockout of the FVIII gene and are deficient for endogenous FVIII protein. These double mutant mice tolerate injections of human FVIII and have no FVIII activity. These double mutant mice produce only trace amounts of inhibitory antibodies after treatment with human FVIII and lack FVIII-responsive T or B cells. + / + The / HemA mice are further described in Bril et al. (2006), Thromb. Haemost., 95(2):341-7.

[0280] Single chain FVIIIXTEN (ssFVIIIXTEN) with preformed HBoV1 ITR was generated by denaturing the double stranded DNA fragment products (FVIII expression cassette and plasmid backbone) of PvuII digestion at 95°C, followed by cooling at 4°C to allow the palindromic ITR sequences to fold. ssFVIIIXTEN was then injected systemically via hydrodynamic tail vein injection at 10 μg or 40 μg per mouse, equivalent to 400 μg or 1600 μg / kg, respectively. Plasma samples were collected from injected mice at 7-day intervals over a period of 5.5 months. Plasma FVIII activity was measured by Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0281] Plasma FVIII activity normalized to percent normal for animals injected with ssFVIIIXTEN is shown in FIG. 14A. Results showed a dose-dependent response over the course of 5.5 months in HemA mice, with FVIII expression at supraphysiological levels (>1000% of normal levels) at all doses tested. However, an initial drop in FVIII expression was observed by day 56, followed by stabilization of levels by day 168, suggesting sustained expression of ssFVIIIXTEN flanked by HBoV1 ITRs from the liver of injected animals. Thus, these results validate the functionality of HBoV1 ITRs in vivo for sustained expression of FVIIIXTEN over a long period of time.

[0282] ceFVIIIXTEN HBoV1 ITR (closed-end DNA) To verify the functionality of the HBoV1 ITRs within the ceDNA, ceFVIIIXTEN purified from infected Sf9 cell pellets was transfected with hFVIIIR593C via hydrodynamic tail vein injection as described above. + / + In / HemA mice, FVIII was systemically injected at 0.3 μg, 1.0 μg, or 2.0 μg per mouse, which is equivalent to 12 μg, 40 μg, and 80 μg / kg, respectively. Plasma samples from the injected mice were collected at 7-day intervals, and FVIII activity was measured by the chromogenic assay described above.

[0283] Plasma FVIII activity normalized to percent normal for animals injected with ceFVIIIXTEN is shown in FIG. 14B. The results of this study showed a dose-dependent response in HemA mice, with supraphysiological levels of FVIII expression (>500% of normal levels) observed at the highest dose tested up to 56 days after injection. Interestingly, similar expression levels were also achieved when mice were injected with ssFVIIIXTEN at 1600 μg / kg, at least 20-fold higher dose than ceFVIIIXTEN (80 μg / kg) (FIGS. 14A-14B). This data suggests that ceDNA results in higher levels of FVIII expression compared to the ssDNA form.

[0284] In conclusion, these in vivo studies validate the functionality of the HBoV1 ITRs in either ssDNA or ceDNA form and support their use to generate functional ceDNA encoding transgenes of interest in the baculovirus-insect cell system. EXAMPLES

[0285] Improved ceDNA vector purity using CRISPR Cas-mediated knockout of VP80 in HBoV1 NS1 BEV HBoV1 NS1 expressed under AcMNPV polyhedrin promoter was indeed able to rescue FVIIIXTEN flanked by HBoV1 ITR, proving the concept of ceDNA production by HBoV1 ITR in baculovirus system. However, significant levels of baculovirus DNA (vDNA) contamination were observed in ceDNA preps, probably due to the high viral load compared to AAV2 Rep-BEV required to achieve high ceDNA productivity. The high molecular weight DNA (>20 kb) observed in these ceDNA preps (Figure 8C, Figure 10C) was likely baculovirus genomic DNA that was co-purified with low molecular weight ceDNA (approximately 8 kb).

[0286] In order to reduce the contamination of baculovirus DNA in ceDNA preps, an indirect approach was carried out to knock out VP80, an essential gene in the baculovirus genome that is required to produce infectious virus particles in insect cells (Sf9). Using the Alt-R CRISPR-Cas9 system (see US Patent Application No. 63 / 069,115), VP80 was knocked out in all three NS1 BEVs (Figures 9B, 9C, and 9D). This approach potentially reduces the number of progeny virus particles, ultimately reducing the contamination of baculovirus DNA in ceDNA preparations.

[0287] CRISPR-Cas9 knocks out AcMNPV VP80 gene: Recombinant BEV encoding HBoV1 NS1 under the AcMNPV polyhedrin promoter (Figure 9B) or the OpMNPV OpIE2 promoter (Figure 9C) were selected to knock out the vp80 gene using the CRISPR-Cas9 system as previously described (see, e.g., International Application No. PCT / US2021 / 047202).

[0288] Briefly, two crRNAs targeting the coding sequence were designed and used to generate functional sgRNAs using the Alt-R CRISPR-Cas9 system (Integrated DNA Technology™) according to the manufacturer's instructions. Each sgRNA was then transfected into Sf9 cells using Cellfectin® (Invitrogen™) transfection reagent along with SpCas9 nuclease and the bacmid DNA, AcBIVVBac.Polh.HBoV1.NS1. Tn7 , or AcBIVVBac.OpIE2.HBoV1.NS1 Tn7 and 0.5 × 10 cells per mL in serum-free ESF-921 medium in a T25 flask. 6Four to five days after transfection, cells were visualized under a fluorescent microscope, and the results showed approximately 10% RFP+ cells for both sgRNA targets. Exemplary fluorescent microscope images of infected cells are shown in FIG. 15. Cas9 alone, AcBIVVBac.Polh.HBoV1.NS1 Tn7 Cells infected with BEV showed progressive infection as expected (Figure 15A), whereas cells treated with sgRNA.VP80.T1 (Figure 15B) or sgRNA.VP80.T2 (Figure 15C) showed restricted infection to individual cells, likely due to knockout of VP80.

[0289] To determine the indels induced by each sgRNA, progeny baculovirus were harvested and plaque purified in the complementing Sf.39K.VP80 cell line as previously described (Jarvis et al. (2014), Methods Enzymol. 536:149-163). Five to six days after infection, twelve plaque-purified RFP+ clones were cultured at 0.5 × 10 per mL in ESF-921 medium supplemented with 10% FBS in a T25 flask. 6AcMNPV vp80 was amplified to P1 in Sf.39K.VP80 cells seeded with 10 ... The resulting sequences were analyzed by the TIDE (tracking of indels by decomposition) program (tide.deskgen.com) using default settings to determine indels induced by each sgRNA. TIDE analysis was performed on AcBIVVBac.Polh.HBoV1.NS1 treated with sgRNA.T1. Tn7 A frameshift mutation in BEV clone 4, with the highest degree (97.1%) (15 bp) deletion in the vp80 coding sequence (Figure 16A), was observed in AcBIVVBac.OpIE2.HBoV1.NS1 Tn7 A frameshift mutation in BEV clone 4 was shown to result in the highest (37.4%) (4 bp) / (26.9%) (3 bp) deletion (Figure 16B) within the vp80 coding sequence, with no detectable insertions. Each clone was amplified to P2 to generate a BEV working stock, followed by titration in Sf.39K.VP80 cells as previously described (Jarvis et al. (2014), Methods Enzymol., 536:149-163). The titrated working stock of vp80KO BEV was then used for co-infection in the TwoBAC system for the generation of FVIIIXTEN HBoV1 ceDNA vectors.

[0290] Generation of human FVIIIXTEN ceDNA using vp80KO BEV: cells approximately 2.0×10 6 AcBIVVBac.FVIIIXTEN.HBoV1.ITR cells were seeded in 100 mL of serum-free ESF-921 medium at MOIs of 1.0, 2.0, 3.0, 4.0, and 5.0 pfu per cell. Tn7 BEV, and AcBIVVBac.Polh.HBoV1.NS1ΔVP80 Tn7 BEV, or AcMNPV.OpIE2.HBoV1.NS1ΔVP80 Tn7 Titrated PP1P2 stocks of BEV were co-infected. In each case, the virus inoculum was not removed and the cells were incubated in a shaking incubator at 28°C until cell viability reached 60-70%. Approximately 96 hours after infection, infected cells were harvested and pellets were processed for isolation of FVIIIXTEN HBoV1 ceDNA by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions.

[0291] The final elution fractions were analyzed by 0.8-1.2% agarose gel electrophoresis to determine ceDNA productivity and purity. Agarose gel analysis showed very low to undetectable contamination of high molecular weight (>20 kb) baculovirus DNA (vDNA) in vp80KO BEV expressing HBoV1 NS1 under AcMNPV polyhedrin promoter or OpMNPV OpIE2 promoter (Figure 16C). This suggests that the vp80KO approach can reduce contaminating baculovirus DNA and simultaneously improve FVIIIXTEN HboV1 ceDNA yield when cells are co-infected at MOI of 2.0, 3.0 or 4.0 pfu per cell (Figure 11C, Figure 16C). EXAMPLES

[0292] Generation of FVIIIXTEN HBoV1 ceDNA vector from the TwoBAC system Genetic instability is one of the major concerns in the field of baculovirology, especially over several passages of recombinant baculovirus in Sf9 cells. In addition, baculovirus genome contains several homologous regions (hr) that are prone to recombination over passages in Sf9 cells, potentially losing transgenes in recombinant BEV. Inverted terminal repeats (ITRs) are also palindromic repeat sequences, and considering the large size of baculovirus DNA, they are potentially recombined at different loci in the baculovirus genome. Therefore, to determine the genetic stability of recombinant BEV that encodes FVIIIXTEN gene under liver-specific mTTR promoter together with HBoV1 WT ITR, BEV was serially amplified by infecting Sf9 cells with MOI of 0.1 pfu per cell as previously described. Jarvis et al. (2014), Methods Enzymol., 536:149-163. The resulting recombinant BEV was examined for the generation of FVIIIXTEN HBoV1 ceDNA using the TwoBAC system (see constructs depicted in Figures 17A and 17B).

[0293] cells approximately 2.0×10 6 AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR cells were seeded in 100 mL of serum-free ESF-921 medium at MOIs of 1.0, 2.0, 3.0, 4.0, and 5.0 pfu per cell. Tn7 BEV, and AcBIVVBac.Polh.HBoV1.NS1 Tn7 Titrated working stocks of BEV (P3 or P4) were co-infected. In each case, the virus inoculum was not removed and the cells were incubated in a shaking incubator at 28 °C until cell viability reached 60-70%. Approximately 96 h postinfection, infected cells were harvested and pellets were processed for isolation of FVIIIXTEN HBoV1 ceDNA by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions.

[0294] The final elution fractions were analyzed by 0.8-1.2% agarose gel electrophoresis to determine ceDNA productivity. Agarose gel analysis shown in Figure 17C showed similar levels of FVIIIXTEN HBoV1 ceDNA productivity for BEVs at P3 or P4 (and P5; data not shown), suggesting that the recombinant BEVs encoding FVIIIXTEN HBoV1 ITRs are genetically stable upon subsequent high passage passaging in Sf9 cells. EXAMPLES

[0295] Generation of FVIIIXTEN HBoV1 ceDNA vector from the OneBAC line The HBoV1 OneBAC system has been shown to produce FVIIIXTEN HBoV1 ceDNA vectors in Sf9 cells (see, for example, FIG. 8C). However, proof of concept was achieved using polyclonal recombinant BEVs. To support large-scale production, BEV clones need to be generated. Therefore, in this study, HBoV1 OneBAC polyclonal BEVs were plaque purified and amplified in Sf9 cells (see FIG. 18A). These clonal OneBAC BEVs were then screened for the production of FVIIIXTEN HBoV1 ceDNA vectors in Sf9 cells.

[0296] Plaque purification and amplification of recombinant HBoV1 OneBAC BEV was performed as previously described (Jarvis et al. (2014), Methods Enzymol. 536:149-163). Approximately 1.0 × 10 Sf9 cells per mL were cultured in 100 mL of ESF-921 medium supplemented with 10% fetal bovine serum. 6Six plaque-purified clones were amplified to P2 by infecting 10 x 100 cells and incubated for 4-5 days or until cell viability reached 60-70% in a shaking incubator at 28°C. Four to five days after infection, cell-free supernatants were harvested and kept as P2 working stock, and cell pellets were processed for isolation of FVIIIXTEN HBoV1 ceDNA by PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions.

[0297] The final elution fractions were analyzed by 0.8-1.2% agarose gel electrophoresis to determine the productivity of FVIIIXTEN HBoV1 ceDNA vector. Figure 18C shows the agarose gel analysis for HBoV1 OneBAC (construct depicted in Figure 18B) encoding FVIIIXTEN together with HBoV1 ITR and polyhedrin-driven HBoV1-NS1. The results showed that the degree of HBoV1 ceDNA productivity varied for different clones, with clone 2 and clone 4 being high producers of HBoV1 ceDNA compared to the other clones tested (Figure 18C). This result shows the variability in different baculovirus clones obtained from the same stock solution and highlights the importance of using clonal recombinant BEV for large-scale ceDNA production.

[0298] To determine the optimal productivity of the clone, HBoV1 OneBAC BEV, approximately 2.0 × 10 cells were cultured at 4 °C for 1 h. 6were infected with titrated working stocks (P2) of HBoV1 OneBAC BEV clone 5 at MOIs of 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 pfu per cell. In each case, the virus inoculum was not removed and the cells were incubated in a shaking incubator at 28 °C until cell viability reached 60-70%. Approximately 96 h after infection, infected cells were harvested and pellets were processed for isolation of FVIIIXTEN HBoV1 ceDNA by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions. Final elution fractions were analyzed by 0.8-1.2% agarose gel electrophoresis to determine FVIIIXTEN HBoV1 ceDNA productivity.

[0299] Agarose gel analysis showed DNA bands corresponding to the size of FVIIIXTEN HBoV1 ceDNA (approximately 8.5 kb) at all doses tested, and productivity increased with increasing MOI. This result was the opposite of the ceDNA productivity obtained with AAV2 ITR OneBAC, where a decrease in productivity was observed with increasing viral load. This HBoV1 One BAC approach demonstrates the concept of ceDNA generation from a single recombinant BEV encoding FVIIIXTEN together with HBoV1 ITR and NS1 transgenes. It also shows the feasibility and functionality of multiple transgenes inserted at different loci within a baculovirus shuttle vector (BIVVBac). EXAMPLES

[0300] In vivo efficacy of FVIIIXTEN HBoV1 ssDNA versus FVIIIXTEN HBoV1 ceDNA ssFVIIIXTEN HBoV1 ITR (single stranded DNA) We hypothesized that hairpins formed within the HBoV1 ITR region would drive long-term, sustained transgene expression at high levels. To validate the functionality of the HBoV1 ITR in vivo, single-stranded DNA (ssDNA) containing codon-optimized human FVIIIXTEN (ssFVIIIXTEN) with the preformed HBoV1 ITR was transformed into hFVIIIR593C + / + This was investigated in / HemA mice.

[0301] ssFVIIIXTEN with preformed HBoV1 ITRs was generated by denaturing the double-stranded DNA (dsDNA) fragment products (FVIII expression cassette and plasmid backbone) of PmlI digestion at 95°C, followed by cooling at 4°C to allow the palindromic ITR sequences to fold. The resulting ssFVIIIXTEN was verified by 0.8-1.2% agarose gel electrophoresis. Gel analysis showed half the size of dsDNA for ssFVIIIXTEN, suggesting efficient hairpin formation (Figure 19A). ssFVIIIXTEN was injected systemically via hydrodynamic tail vein injection at 10 μg or 40 μg per mouse, equivalent to 400 μg or 1600 μg / kg, respectively. Plasma samples were collected from injected mice at 7-day intervals over a period of 5.5 months. Plasma FVIII activity was measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0302] Plasma FVIII activity normalized to percent normal for animals injected with ssFVIIIXTEN is shown in FIG. 19C. Results showed a dose-dependent response over the course of 5.5 months in HemA mice, with FVIII expression at supraphysiological levels (>1000% of normal levels) in the high-dose cohort. However, an initial drop in FVIII expression was observed by day 56, followed by stabilization of levels by day 140, suggesting sustained expression of ssFVIIIXTEN flanked by HBoV1 ITRs from the liver. These results validate the functionality of HBoV1 ITRs in vivo for sustained expression of FVIIIXTEN over a long period of time.

[0303] ceFVIIIXTEN HBoV1 ITR (closed-end DNA) There is a major structural difference between closed-end DNA (ceDNA) and single-stranded DNA (ssDNA), the former being double-stranded and the latter being single-stranded, respectively. This difference may affect the expression level as well as the stability of the nucleic acid molecule. This study shows the functionality of HBoV1 ITR in ceDNA form in vivo. To investigate this, ceFVIIIXTEN was obtained using the TwoBac approach described in Example 8, purified from infected Sf9 cell pellets, and the quality was determined by 0.8-1.2% agarose gel electrophoresis. Agarose gel analysis showed a purity of >90% for ceFVIIIXTEN, with no detectable contaminating DNA (Figure 19B).

[0304] The resulting ceFVIIIXTEN was administered via hydrodynamic tail vein injection to hFVIIIR593C + / +In / HemA mice, ceFVIIIXTEN was injected systemically at 0.3 μg, 1.0 μg, or 2.0 μg per mouse, which is equivalent to 12 μg, 40 μg, and 80 μg / kg, respectively. Plasma samples from the injected mice were collected at 7-day intervals, and FVIII activity was measured by the chromogenic assay described above. Plasma FVIII activity normalized to percent of normal for animals injected with ceFVIIIXTEN is shown in FIG. 19C.

[0305] The results showed a dose-dependent response in HemA mice, with FVIII expression at supraphysiological levels (>500% of normal levels) at the highest dose tested for ceFVIIIXTEN (80 μg / kg). For ceDNA, the highest level of FVIII expression was half that achieved by ssFVIIIXTEN at 1600 μg / kg. However, ssDNA was administered at a much higher dose to achieve these high levels of FVIII expression. ceDNA appears to provide a higher level of FVIII expression per dose. For example, the FVIII expression levels for ssDNA at 400 μg / kg and ceDNA at 40 μg / kg were comparable (Figure 19C). These in vivo studies validate the functionality of the HBoV1 ITRs in either ssDNA or ceDNA form and demonstrate that the HBoV1 ITRs can be used to generate functional ceDNA encoding a transgene of interest in a baculovirus-insect cell system. EXAMPLES

[0306] In vivo efficacy of FVIIIXTEN HBoV1 monomeric ceDNA versus FVIIIXTEN HBoV1 multimeric ceDNA Recombinant AAV genomes have been shown to persist in episomes, and their episomal presence appears to correlate with long-term transgene expression. These genomes are thought to arise via a monomeric circularization process, resulting in AAV head-to-tail circular genomes. Over time, however, the monomeric circular intermediates decay in favor of high molecular weight circular concatamers. Further details are disclosed in Duan et al. (1998), J Virol. 72(11), 8568-8577. Currently, little is known about the presence of closed-end DNA (ceDNA) in episomes and the benefits of monomeric ceDNA forms over concatameric ceDNA forms in vivo.

[0307] This study is + / + A study was conducted to determine the impact of the monomeric form of ceDNA versus the multimeric form in vivo by examining both forms of FVIIIXTEN HBoV1 ceDNA (ceFVIIIXTEN) via hydrodynamic tail vein injection in / HemA mice.

[0308] Monomeric and multimeric forms of ceFVIIIXTEN were produced by PAGE purification as previously described (see International Application No. PCT / US2021 / 047218). The quality of the concatameric forms of ceFVIIIXTEN was determined by 0.8-1.2% agarose gel electrophoresis, and the results showed that the majority of the molecular species were monomeric or multimeric forms of ceFVIIIXTEN (Figure 20A). Purified monomeric or multimeric ceFVIIIXTEN was purified from hFVIIIR593C. + / + / HemA mice were injected systemically at 40 μg / kg via hydrodynamic tail vein injection. Plasma samples were collected from the injected mice at 7-day intervals for approximately 3 months. Plasma FVIII activity was measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0309] Plasma FVIII activity normalized to percent normal for animals injected with ceFVIIIXTEN is shown in Figure 20B. Results showed no significant difference in FVIII expression levels between the monomeric or multimeric forms of ceFVIIIXTEN over the course of three months. This data suggests that both the monomeric and multimeric forms of ceFVIIIXTEN have comparable potency and stability in vivo. EXAMPLES

[0310] In vivo efficacy of FVIIIXTEN HBoV1 mTTR ssDNA versus FVIIIXTEN HBoV1 A1AT ssDNA The FVIIIXTEN expression cassette used in the above-disclosed experiments contains mTTR promoter and enhancer elements (V2.0; FIG. 1). This promoter is mouse liver-specific, but its liver-specific expression has not been studied in large animal models or human subjects. Therefore, in this study, the V3.0 FVIIIXTEN expression cassette (SEQ ID NO: 35) was created by replacing the mTTR promoter and enhancer elements with the human liver-specific alpha 1 antitrypsin (A1AT) promoter (SEQ ID NO: 36) in the V2.0 expression cassette (FIG. 1).

[0311] To verify the functionality of the mTTR promoter in vivo versus the A1AT promoter, single-stranded DNA (ssDNA) containing codon-optimized human FVIIIXTEN (ssFVIIIXTEN) with preformed HBoV1 ITRs was synthesized using the hFVIIIR593C + / +The results were examined in hFVIIIR593C / HemA mice (Figure 21A). ssFVIIIXTEN with preformed HBoV1 ITRs was generated by denaturing the double-stranded DNA (dsDNA) fragment products of PmlI digestion (mTTR expression cassette or A1AT FVIII expression cassette and plasmid backbone) at 95°C, followed by cooling at 4°C to allow the palindromic ITR sequences to fold. The resulting ssFVIIIXTEN was collated by 0.8-1.2% agarose gel electrophoresis. Gel analysis showed half the size of dsDNA for ssFVIIIXTEN, suggesting efficient hairpin formation (Figure 21B). ssFVIIIXTEN was cloned into hFVIIIR593C / HemA mice (Figure 21B). + / + 10 μg per mouse was injected systemically into / HemA mice via hydrodynamic tail vein injection. Plasma samples were collected from the injected mice at 7-day intervals over a period of 5.5 months. Plasma FVIII activity was measured by the Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions.

[0312] Plasma FVIII activity, normalized to percent of normal, for animals injected with ssFVIIIXTEN is shown in Figure 21C. These results showed comparable levels of FVIII expression up to 21 days post-injection, consistent with hFVIIIR593C. + / + These results suggest that there is no significant difference in FVIIIXTEN levels expressed by the mTTR promoter or the A1AT promoter in the / HemA mouse animal model.

[0313] array

[0314] [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Claims

1. A nucleic acid molecule comprising a first inverted terminal repeat (ITR) and a second ITR flanking a gene cassette comprising a heterologous polynucleotide sequence, A nucleic acid molecule, wherein the first ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:1, and the second ITR comprises a polynucleotide sequence that is at least about 75% identical to SEQ ID NO:

2.

2. promoter; intron sequences; post-transcriptional regulatory elements; 3'UTR poly(A) tail sequence; and / or Enhancer sequence The nucleic acid molecule of claim 1, further comprising:

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises, from 5' to 3', a first ITR, a gene cassette, and a second ITR, and 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.

4. The nucleic acid molecule of claim 3, wherein 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.

5. The nucleic acid molecule of claim 4, wherein the gene cassette comprises the nucleotide sequence of SEQ ID NO: 3, 9, 14, 33, or 35.

6. 10. The nucleic acid molecule of claim 1, wherein the heterologous polynucleotide sequence encodes a therapeutic protein, a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, or any combination thereof.

7. The nucleic acid molecule of claim 1 , wherein the heterologous polynucleotide sequence encodes a microRNA (miRNA).

8. The nucleic acid molecule of claim 1 formulated with a delivery agent.

9. 9. The nucleic acid molecule of claim 8, wherein the delivery agent comprises a lipid nanoparticle (LNP), a liposome, a non-lipid polymer molecule, an endosome, or any combination thereof.

10. 10. The nucleic acid molecule of claim 1, formulated for administration intravenously, transdermally, intradermally, intraneurally, intraocularly, intrathecally, subcutaneously, intrapulmonaryly, or orally, or any combination thereof; or by in situ injection; or by inhalation.

11. A vector comprising the nucleic acid molecule of any one of claims 1 to 10.

12. A host cell comprising the nucleic acid molecule of any one of claims 1 to 10 or the vector of claim 11.

13. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 10; the vector of claim 11 and a pharmaceutically acceptable excipient; or the host cell of claim 12 and a pharmaceutically acceptable excipient.

14. A baculovirus system for producing a nucleic acid molecule according to any one of claims 1 to 10.

15. A nucleic acid molecule for use in treating a bleeding disorder, the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:

3.

16. 16. The nucleic acid molecule of claim 15, wherein the disorder is hemophilia A.

17. a sequence encoding HBoV1 Rep, wherein the inserted HBoV1 Rep sequence disrupts the reading frame of a reporter gene or a functional portion thereof; and / or Heterologous sequences comprising nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 3, 9, 14, 33, or 35. A recombinant bacmid comprising:

18. A set of recombinant bacmids comprising a first bacmid and a second bacmid, The first bacmid contains a sequence encoding Rep, and the inserted Rep disrupts the reading frame of a reporter gene or a functional portion thereof; A set of recombinant bacmids, wherein the second bacmid comprises a heterologous sequence comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 9, 14, 33, or 35.

19. A stable cell line comprising the nucleic acid sequence of SEQ ID NO: 3, 9, 14, 33, or 35, wherein the nucleic acid sequence is stably integrated into the genome of the stable cell line.

20. 1. A method for generating a closed-ended DNA (ceDNA) molecule, comprising: Infecting insect cells with a recombinant baculovirus comprising the bacmid of claim 17 or a set of recombinant bacmids of claim 18; or 20. A step of introducing a baculovirus encoding Rep proteins into the stable cell line of claim 19. A method comprising:

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