Baculovirus Expression System

Abstract

Provided herein is a baculovirus expression vector for the production of a desired protein.In one embodiment, the desired protein is a closed-end DNA (ceDNA) molecule that comprises wild-type and / or truncated inverted terminal repeats derived from the genome of a member of the Parvoviridae virus family.

Description

[Technical field]

[0001] Related Applications This application claims priority to International Application No. PCT / US2021 / 047218, filed August 23, 2021, and U.S. Provisional Application No. 63 / 310,038, filed February 14, 2022, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the Sequence Listing, submitted electronically in an ASCII text file (Name: 732841_SA9-474BPC_ST26.xml; Size: 86.2KB; and Date Created: August 17, 2022), are incorporated herein by reference in their entirety. [Background technology]

[0003] Gene therapy offers the potential for a permanent means of treating a variety of diseases. Recent developments in gene therapy treatment employ the use of viral or non-viral vectors. Currently, several techniques are used for the production of gene therapy vectors.

[0004] For example, the baculovirus expression vector system (BEVS) is an established system for the production of gene therapy vectors. In this system, insect host cells are infected with recombinant baculoviruses. The insect host cells provide the necessary protein processing machinery to produce the products encoded by the recombinant baculoviruses. BEVS has been used to produce products for many different applications, such as vaccines, therapeutic proteins, protein crystallography, and products for basic and applied research. When making viral vectors for gene therapy, several baculovirus expression vectors are often used to infect insect host cells. The generation of each baculovirus expression vector is time-consuming and increases the cost of production. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for improved baculovirus expression vector systems that avoid the limitations of existing systems. [Means for solving the problem]

[0006] Provided herein is a baculovirus expression vector system comprising a baculovirus shuttle vector (bacmid) and / or a stable cell line designed to produce a therapeutic product. Provided herein is a baculovirus expression vector system comprising a recombinant bacmid comprising one or more foreign sequence insertion sites and one or more donor vectors capable of mediating the insertion of a foreign sequence (e.g., a heterologous gene) into the foreign sequence insertion site.

[0007] In one embodiment, provided herein is a bacmid comprising a bacterial replicon; a selectable marker sequence; a first reporter gene comprising a first selective target site for insertion of a transposon; a second reporter gene operably linked to a baculovirus-inducible promoter; and a second selective target site capable of mediating a site-specific recombination event.

[0008] In certain exemplary embodiments, the bacterial replicon is a low copy number replicon. In certain exemplary embodiments, the low copy number replicon is a mini-F replicon.

[0009] In certain exemplary embodiments, the selectable marker sequence comprises an antibiotic resistance gene, hi certain exemplary embodiments, the antibiotic resistance gene is a kanamycin resistance gene.

[0010] In certain exemplary embodiments, the first reporter gene encodes an enzyme capable of metabolizing a chromogenic substrate. In certain exemplary embodiments, the enzyme is LacZα or a functional part thereof. In certain exemplary embodiments, the chromogenic substrate is Blue-gal or X-gal.

[0011] In certain exemplary embodiments, the first selective target site for the insertion of transposon does not disrupt the reading frame of the first reporter gene.In certain exemplary embodiments, the first selective target site is the attachment site for bacterial transposon.In certain exemplary embodiments, the first selective target site is the attachment site for T7 transposon.In certain exemplary embodiments, the transposon is T7 transposon.

[0012] In certain exemplary embodiments, the second reporter gene encodes a fluorescent protein. In certain exemplary embodiments, the fluorescent protein is a red fluorescent protein.

[0013] In certain exemplary embodiments, the baculovirus-inducible promoter is the 39K promoter.

[0014] In certain exemplary embodiments, the second selective target site comprises a LoxP site or a variant thereof.

[0015] In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

[0016] In another embodiment, provided herein is a bacmid comprising a mini-F replicon; an antibiotic resistance gene; a LacZα gene or a functional portion thereof comprising an attachment site for a T7 transposon; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0017] In another embodiment, provided herein is a recombinant bacmid comprising: a bacterial replicon; a first selectable marker sequence; a heterologous sequence inserted into a first reporter gene, where the inserted heterologous sequence disrupts the reading frame of the first reporter gene; a second reporter gene operably linked to a baculovirus-inducible promoter; and selective target sites capable of mediating site-specific recombination events.

[0018] In certain exemplary embodiments, the heterologous sequence comprises a heterologous gene. In certain exemplary embodiments, the heterologous gene comprises a sequence that encodes a protein.

[0019] In certain exemplary embodiments, the heterologous sequence further comprises an expression control sequence, hi certain exemplary embodiments, the expression control sequence is operably linked to the protein coding sequence.

[0020] In certain exemplary embodiments, the expression control sequence comprises a baculovirus promoter.In certain exemplary embodiments, the baculovirus promoter is an immediate early, early, late or very late gene promoter.In certain exemplary embodiments, the baculovirus promoter is selected from the group consisting of polyhedrin promoter; immediate early 1 promoter; and immediate early 2 promoter.

[0021] In certain exemplary embodiments, the expression control sequence includes a polyadenylation signal.

[0022] In certain exemplary embodiments, the protein is a Rep protein isolated from the genome of a member of the viral family Parvoviridae. In certain exemplary embodiments, the protein is a parvovirus Rep protein. In certain exemplary embodiments, the parvovirus Rep protein is selected from the group consisting of B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

[0023] In certain exemplary embodiments, the heterologous sequence comprises a second selectable marker sequence. In certain exemplary embodiments, the second selectable marker sequence comprises a gentamicin resistance gene.

[0024] In certain exemplary embodiments, the bacterial replicon is a mini-F replicon. In certain exemplary embodiments, the first selectable marker sequence comprises a kanamycin resistance gene. In certain exemplary embodiments, the first reporter gene encodes LacZα or a functional portion thereof. In certain exemplary embodiments, the second reporter gene encodes a red fluorescent protein. In certain exemplary embodiments, the baculovirus-inducible promoter is a 39K promoter. In certain exemplary embodiments, the second selective target site comprises a LoxP site or a variant thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

[0025] In another aspect, provided herein is a recombinant bacmid comprising: a heterologous sequence inserted into a mini-attTn7 site, the heterologous sequence encoding Rep, wherein the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; and a LoxP site or a variant thereof.

[0026] In another aspect, provided herein is a recombinant bacmid comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a mini-attTn7 site, the heterologous sequence encoding Rep, wherein the inserted Rep disrupts the reading frame of a LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0027] In another aspect, provided herein is a recombinant bacmid comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a mini-attTn7 site, the heterologous sequence encoding B19 Rep, wherein the inserted B19 Rep disrupts the reading frame of a LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0028] In another aspect, provided herein is a recombinant bacmid comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a mini-attTn7 site, wherein the heterologous sequence encodes GPV Rep, and wherein the inserted GPV Rep disrupts the reading frame of a LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0029] In another embodiment, the following: Provided herein is a recombinant bacmid comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a mini-attTn7 site, the heterologous sequence encoding AAV2 Rep, wherein the inserted AAV2 Rep disrupts the reading frame of a LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0030] In another aspect, provided herein is a nucleic acid vector comprising: a first origin of replication for propagating a nucleic acid vector in a first bacterial strain, the first origin of replication being a conditional origin of replication; a second origin of replication for propagating a nucleic acid vector in a second bacterial strain; a multiple cloning site for insertion of a heterologous sequence; a selectable marker sequence; a reporter gene; and a selective target site capable of mediating a site-specific recombination event.

[0031] In certain exemplary embodiments, the first origin of replication is conditional on the presence of the π-protein. In certain exemplary embodiments, the first origin of replication is R6Kγ.

[0032] In certain exemplary embodiments, the first bacterial strain comprises a π-protein.

[0033] In certain exemplary embodiments, the second origin of replication is pUC57.

[0034] In certain exemplary embodiments, the selectable marker sequence comprises an antibiotic resistance gene, hi certain exemplary embodiments, the antibiotic resistance gene is an ampicillin resistance gene.

[0035] In certain exemplary embodiments, the reporter gene encodes a fluorescent protein, hi certain exemplary embodiments, the fluorescent protein is green fluorescent protein.

[0036] In certain exemplary embodiments, the selective target sites comprise LoxP sites or variants thereof. In certain exemplary embodiments, the site-specific recombination events are mediated by Cre recombinase.

[0037] In another aspect, provided herein is a nucleic acid vector comprising: a first origin of replication for propagating a nucleic acid vector in a first bacterial strain, the first origin of replication being a conditional origin of replication; a second origin of replication for propagating a nucleic acid vector in a second bacterial strain; a multiple cloning site comprising a heterologous sequence; a selectable marker sequence; a reporter gene; and a selective target site capable of mediating a site-specific recombination event.

[0038] In certain exemplary embodiments, the heterologous sequence comprises a heterologous gene. In certain exemplary embodiments, the heterologous gene comprises a sequence that encodes a protein.

[0039] In certain exemplary embodiments, the heterologous sequence further comprises an expression control sequence. In certain exemplary embodiments, the expression control sequence is operably linked to the protein-coding sequence. In certain exemplary embodiments, the expression control sequence comprises a tissue-specific promoter. In certain exemplary embodiments, the tissue-specific promoter is a tristetraprolin (TTP) or mouse transthyretin (mTTR) promoter. In certain exemplary embodiments, the expression control sequence comprises a polyadenylation signal. In certain exemplary embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal. In certain exemplary embodiments, the expression control sequence comprises a post-transcriptional regulatory element. In certain exemplary embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0040] In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a clotting factor. In certain exemplary embodiments, the clotting factor is factor VIII (FVIII). In certain exemplary embodiments, the clotting factor is FVIII-XTEN.

[0041] In certain exemplary embodiments, the heterologous sequence comprises a 5' inverted terminal repeat (ITR). In certain exemplary embodiments, the heterologous sequence comprises a 3' inverted terminal repeat (ITR). In certain exemplary embodiments, the 5'ITR and the 3'ITR are derived from a parvovirus. In certain exemplary embodiments, the parvovirus is selected from the group consisting of B19, GPV, HBoV1, and AAV2. In certain exemplary embodiments, the 5'ITR is a wild-type or variant 5'ITR derived from B19. In certain exemplary embodiments, the 5'ITR is a wild-type or variant 5'ITR derived from GPV. In certain exemplary embodiments, the 5'ITR is a wild-type or variant 5'ITR derived from AAV2. In certain exemplary embodiments, the 5'ITR is a wild-type or variant 5'ITR derived from HBoV1. In certain exemplary embodiments, the variant 5'ITR is a truncated 5'ITR. In certain exemplary embodiments, the 3'ITR is a wild-type or variant 3'ITR derived from B19. In certain exemplary embodiments, the 3'ITR is a wild-type or variant 3'ITR derived from GPV. In certain exemplary embodiments, the 3'ITR is a wild-type or variant 3'ITR derived from AAV2. In certain exemplary embodiments, the variant 3'ITR is a truncated 3'ITR. In certain exemplary embodiments, the 3'ITR is a wild-type 3'ITR derived from HBoV1.

[0042] In certain exemplary embodiments, the first origin of replication is conditional on the presence of the π-protein. In certain exemplary embodiments, the first origin of replication is R6Kγ.

[0043] In certain exemplary embodiments, the first bacterial strain comprises a π-protein.

[0044] In certain exemplary embodiments, the second origin of replication is pUC57.

[0045] In certain exemplary embodiments, the selectable marker sequence comprises an antibiotic resistance gene, hi certain exemplary embodiments, the antibiotic resistance gene is an ampicillin resistance gene.

[0046] In certain exemplary embodiments, the reporter gene encodes a fluorescent protein, hi certain exemplary embodiments, the fluorescent protein is green fluorescent protein.

[0047] In certain exemplary embodiments, the selective target sites comprise LoxP sites or variants thereof. In certain exemplary embodiments, the site-specific recombination events are mediated by Cre recombinase.

[0048] In another embodiment, provided herein is a recombinant bacmid comprising: a first heterologous sequence inserted into a first reporter gene that disrupts the reading frame of the first reporter gene; a first selective target site capable of mediating a site-specific recombination event; a multiple cloning site comprising a second heterologous sequence; and a second selective target site capable of mediating a site-specific recombination event.

[0049] In certain exemplary embodiments, the first heterologous sequence comprises a first heterologous gene. In certain exemplary embodiments, the first heterologous sequence comprises an expression control sequence operably linked to a protein-encoding sequence.

[0050] In certain exemplary embodiments, the expression control sequence comprises a baculovirus promoter. In certain exemplary embodiments, the baculovirus promoter is an immediate early, early, late, or very late promoter. In certain exemplary embodiments, the baculovirus promoter is selected from the group consisting of a polyhedrin promoter, an immediate early 1 promoter, and an immediate early 2 promoter.

[0051] In certain exemplary embodiments, the protein is a Rep protein isolated from the genome of a member of the viral family Parvoviridae. In certain exemplary embodiments, the protein is a parvovirus Rep protein. In certain exemplary embodiments, the parvovirus Rep protein is selected from the group consisting of B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

[0052] In certain exemplary embodiments, the second heterologous sequence comprises a second heterologous gene. In certain exemplary embodiments, the second heterologous sequence comprises an expression control sequence operably linked to the protein-coding sequence. In certain exemplary embodiments, the expression control sequence comprises a tissue-specific promoter, a polyadenylation signal, and / or a post-transcriptional regulatory element. In certain exemplary embodiments, the tissue-specific promoter is a tristetraprolin (TTP) or mouse transthyretin (mTTR) promoter. In certain exemplary embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal. In certain exemplary embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0053] In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a clotting factor. In certain exemplary embodiments, the clotting factor is factor VIII (FVIII). In certain exemplary embodiments, the clotting factor is FVIII-XTEN.

[0054] In certain exemplary embodiments, the second heterologous sequence comprises a 5' inverted terminal repeat (ITR). In certain exemplary embodiments, the second heterologous sequence comprises a 3' inverted terminal repeat (ITR). In certain exemplary embodiments, the 5'ITR is derived from the genome of a first member of the virus family Parvoviridae, and the 3'ITR is derived from the genome of a second member of the virus family Parvoviridae. In certain exemplary embodiments, the first and second members are the same. In certain exemplary embodiments, the first and second members are different. In certain exemplary embodiments, the 5'ITR and the 3'ITR are derived from a parvovirus selected from the group consisting of B19, GPV, and AAV2. In certain exemplary embodiments, the 5'ITR is a wild-type or truncated 5'ITR derived from B19. In certain exemplary embodiments, the 5'ITR is a wild-type or truncated 5'ITR derived from GPV. In certain exemplary embodiments, the 5'ITR is a wild-type or truncated 5'ITR from AAV2. In certain exemplary embodiments, the 5'ITR is a wild-type or truncated 5'ITR from HBoV1. In certain exemplary embodiments, the 3'ITR is a wild-type or truncated 3'ITR from B19. In certain exemplary embodiments, the 3'ITR is a wild-type or truncated 3'ITR from GPV. In certain exemplary embodiments, the 3'ITR is a wild-type or truncated 3'ITR from AAV2. In certain exemplary embodiments, the 5'ITR is a wild-type 3'ITR from HBoV1.

[0055] In certain exemplary embodiments, the recombinant bacmid further comprises a bacterial replicon. In certain exemplary embodiments, the bacterial replicon is a mini-F replicon.

[0056] In certain exemplary embodiments, the recombinant bacmid further comprises one or more selectable marker sequences. In certain exemplary embodiments, the one or more selectable marker sequences comprise one or more antibiotic resistance genes. In certain exemplary embodiments, the one or more antibiotic resistance genes are selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, and a gentamicin resistance gene.

[0057] In certain exemplary embodiments, the first reporter gene encodes LacZα or a functional portion thereof.

[0058] In certain exemplary embodiments, the recombinant bacmid further comprises at least a second and a third reporter gene.In certain exemplary embodiments, the second and the third reporter gene each encode a fluorescent protein.In certain exemplary embodiments, the fluorescent protein is green fluorescent protein or red fluorescent protein.

[0059] In certain exemplary embodiments, the first and second selective target sites comprise LoxP sites or variants thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

[0060] In another embodiment, provided herein is a bacmid that comprises the following: a sequence encoding Rep inserted into a mini-attTn7 site, wherein the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a multiple cloning site that comprises a heterologous sequence, the heterologous sequence comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from a first genome of a member of the viral family Parvoviridae; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from a second genome of a member of the viral family Parvoviridae.

[0061] In certain exemplary embodiments, the first and second genomes of the virus family Parvoviridae are the same. In certain exemplary embodiments, the first and second genomes of the virus family Parvoviridae are different.

[0062] In certain exemplary embodiments, Rep is derived from a first or second genome of a member of the viral family Parvoviridae.

[0063] In another aspect, provided herein is a bacmid that comprises the following: a sequence encoding B19 Rep inserted into a mini-attTn7 site, wherein the inserted B19 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a multiple cloning site that comprises heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from B19; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from B19.

[0064] In another aspect, provided herein is a bacmid that comprises the following: a sequence encoding GPV Rep inserted into a mini-attTn7 site, wherein the inserted GPV Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a multiple cloning site that comprises heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from GPV; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from GPV.

[0065] In another aspect, provided herein is a bacmid that comprises the following: a sequence encoding AAV2 Rep inserted into a mini-attTn7 site, wherein the inserted AAV2 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a multiple cloning site comprising heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from AAV2; a protein coding sequence; one or more expression control sequences operably linked to the protein coding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from AAV2.

[0066] In another aspect, provided herein is a bacmid that comprises the following: a sequence encoding AAV2 HBoV1 Rep inserted into a mini-attTn7 site, wherein the inserted HBoV1 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; and a multiple cloning site comprising a heterologous sequence, the heterologous sequence comprising, from 5' to 3': a wild-type 5' inverted terminal repeat derived from HBoV1; a sequence encoding a protein; one or more expression control sequences operably linked to the sequence encoding the protein; and a wild-type 3' inverted terminal repeat derived from HBoV1.

[0067] In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a clotting factor. In certain exemplary embodiments, the clotting factor is factor VIII (FVIII). In certain exemplary embodiments, the clotting factor is FVIII-XTEN.

[0068] In another aspect, a host cell is provided that comprises a bacmid as described herein. In another aspect, a host cell is provided that comprises a recombinant bacmid as described herein. In another aspect, a host cell is provided that comprises a vector as described herein.

[0069] In another aspect, provided herein is a method of producing a recombinant bacmid described herein, the method comprising: introducing into a bacterial cell a donor nucleic acid molecule comprising a bacmid described herein; and a bacterial replicon operably linked to a transposon comprising a heterologous sequence described herein; incubating the bacterial cell under conditions in which transposition occurs; and isolating the recombinant bacmid from the bacterial cell.

[0070] In another aspect, provided herein is a method of producing a recombinant bacmid described herein, the method comprising introducing into a bacterial cell a recombinant bacmid described herein; a vector described herein; and Cre recombinase; incubating the bacterial cell under conditions in which site-specific recombination occurs; and isolating the recombinant bacmid from the bacterial cell.

[0071] In another aspect, provided herein is a method for producing a recombinant baculovirus, comprising transfecting an insect cell under suitable conditions with a recombinant bacmid described herein.

[0072] In another aspect, provided herein is a method of generating a closed-end DNA (ceDNA) molecule, the method comprising transfecting an insect cell with a recombinant bacmid described herein under conditions suitable to generate a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under conditions suitable to generate the ceDNA molecule.

[0073] In another aspect, provided herein is a method of generating a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated B19 inverted terminal repeat, the method comprising transfecting an insect cell with a recombinant bacmid described herein comprising a wild-type or truncated B19 inverted terminal repeat under conditions suitable for making a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under conditions suitable for generating the ceDNA molecule.

[0074] In another aspect, provided herein is a method for generating a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated GPV inverted terminal repeat, the method comprising transfecting an insect cell with a recombinant bacmid described herein comprising a wild-type or truncated GPV inverted terminal repeat under conditions suitable for generating a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under conditions suitable for generating the ceDNA molecule.

[0075] In another aspect, provided herein is a method for generating a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated AAV2 inverted terminal repeat, the method comprising transfecting an insect cell with a recombinant bacmid described herein comprising a wild-type or truncated AAV2 inverted terminal repeat, under conditions suitable for generating a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under conditions suitable for generating the ceDNA molecule.

[0076] In another aspect, provided herein is a method for generating a closed-end DNA (ceDNA) molecule comprising a wild-type HBoV1 inverted terminal repeat, the method comprising transfecting an insect cell with a recombinant bacmid described herein comprising a wild-type HBoV1 inverted terminal repeat under conditions suitable for generating a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under conditions suitable for generating the ceDNA molecule.

[0077] In another embodiment, provided herein is a plasmid comprising a nucleic acid sequence that includes, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from the genome of a first member of the viral family Parvoviridae; a protein coding sequence; one or more expression control sequences operably linked to the protein coding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from the genome of a second member of the viral family Parvoviridae.

[0078] In some embodiments, disclosed herein is a set of recombinant bacmids, comprising a first bacmid and a second bacmid, wherein the first bacmid comprises a sequence encoding Rep inserted into a mini-attTn7 site, the inserted Rep disrupting the reading frame of a reporter gene or a functional portion thereof; and the second bacmid comprises a heterologous sequence, wherein the heterologous sequence comprises, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat (ITR) from a first genome of a member of the virus family Parvoviridae; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat (ITR) from a second genome of a member of the virus family Parvoviridae. In some embodiments, the 5'ITR and 3'ITR are derived from a parvovirus selected from the group consisting of B19, GPV, HBoV1, and AAV2.

[0079] In certain exemplary embodiments, the one or more expression control sequences include a tissue-specific promoter, a polyadenylation signal, and / or a post-transcriptional regulatory element. In certain exemplary embodiments, the tissue-specific promoter is a tristetraprolin (TTP) or mouse transthyretin (mTTR) promoter. In certain exemplary embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal. In certain exemplary embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0080] In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a clotting factor. In certain exemplary embodiments, the clotting factor is factor VIII (FVIII). In certain exemplary embodiments, the clotting factor is FVIII-XTEN.

[0081] In certain exemplary embodiments, the first and / or second member is selected from the group consisting of B19, GPV, HBoV1, and AAV2. In certain exemplary embodiments, the first and second members are the same. In certain exemplary embodiments, the first and second members are different.

[0082] In another aspect, provided herein is a stable cell line comprising a nucleic acid sequence described herein, wherein the nucleic acid sequence is stably integrated into the genome of the stable cell line.

[0083] In certain exemplary embodiments, the stable cell line is a stable insect cell line. In certain exemplary embodiments, the stable insect cell line is Sf9.

[0084] In another aspect, provided herein is a method for generating a closed-end DNA (ceDNA) molecule containing wild-type or truncated inverted terminal repeats, the method comprising introducing a recombinant bacmid described herein into a stable cell line described herein.

[0085] In another aspect, provided herein is a method for generating a closed-end DNA (ceDNA) molecule containing wild-type or truncated inverted terminal repeats, the method comprising introducing into a host cell the following: a plasmid described herein; and a recombinant bacmid described herein.

[0086] In certain exemplary embodiments, the host cell is an insect cell. In certain exemplary embodiments, the insect cell is Sf9. [Brief description of the drawings]

[0087] [Figure 1]Figures 1A-1D are schematic diagrams of recombinant bacmids according to one embodiment of the present invention. Figure 1A shows a schematic diagram of bMON14272 encoding a kanamycin resistance marker (KanR), a mini-attTn7 insertion site fused in frame to LacZα, and a mini-F origin of replication (mini-F). Figure 1B shows a schematic linear diagram of a synthetic transfer vector containing a gene encoding red fluorescent protein (RFP) in the presence of the AcMNPV 39K promoter (p39K) followed by an ets polyadenylation signal (etsPAS), a LoxP recombination site, and an EGT flanking gene sequence. Figure 1C shows a schematic diagram of the recombinant bacmid (BIVVBac) encoding a kanamycin resistance marker (KanR), a mini-attTn7 insertion site fused in frame with LacZα, a mini-F origin of replication (mini-F), a gene encoding a red fluorescent protein (RFP) gene in the presence of the AcMNPV 39K promoter (p39K) followed by an ets polyadenylation signal (etsPAS), and LoxP recombination sites. Figure 1D shows a schematic diagram of the E. coli strain (BIVVBacDH10B) harboring the BIVVBac bacmid and a Tn7 helper plasmid encoding the transposase. [Diagram 2] Figures 2A-2B are schematic diagrams of a Cre-LoxP donor vector according to one embodiment of the present invention. Figure 2A shows a schematic linear diagram of a synthetic DNA containing a gene encoding enhanced GFP in the presence of the AcMNPV immediate early 1 (pIE1) promoter preceded by the AcMNPV transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal (p10PAS), a LoxP recombination site (LoxP), an ampicillin antibiotic resistance gene, a multiple cloning site (MCS), ColE1, and an R6Kγ origin of replication (Ori). Figure 2B shows a schematic map of a Cre-LoxP donor vector according to one embodiment of the present invention, which was generated by inserting the synthetic DNA (Figure 2A) into a pUC57 vector. [Figure 3-1]3A-3F are schematic diagrams of replication (Rep) protein expression constructs according to embodiments of the present invention. FIG. 3A shows a schematic linear map of synthetic DNA encoding the B19 Rep gene in the presence of the AcMNPV polyhedrin or immediate early 1 (ie1) 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 the B19.Rep synthetic DNA (FIG. 3A) into the pFastBac1 vector (Invitrogen). FIG. 3C shows a schematic linear map of synthetic DNA encoding the GPV Rep78 and Rep52 insert genes in the presence of the AcMNPV polyhedrin promoter followed by the SV40 polyadenylation signal (SV40 PAS). The modified mRNA transcript (messenger RNA) is shown as a wavy line with a non-canonical start codon (CUG) for Rep78, a canonical start codon (AUG) for Rep52, and a stop codon (UAA). Figure 3D shows a schematic map of a Tn7 transfer vector according to an embodiment of the present invention, which was generated by inserting GPV.Rep synthetic DNA (Figure 3C) into a pFastBac1 vector (Invitrogen). Figure 3E shows a schematic linear map of synthetic DNA encoding the AAV2 Rep78 and Rep52 insert genes in the presence of the AcMNPV polyhedrin promoter followed by the SV40 polyadenylation signal (SV40 PAS). The modified mRNA transcript (mRNA) is shown as a wavy line with a non-canonical start codon (CUG) for Rep78, a canonical start codon (AUG) for Rep52, and a stop codon (UAA). FIG. 3F shows a schematic map of a Tn7 transfer vector according to an embodiment of the invention, generated by inserting the AAV2.Rep synthetic DNA (FIG. 3E) into the pFastBac1 vector (Invitrogen). [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 4A]Schematic diagram of human FVIIIco6XTEN expression construct. Figure 4A shows a schematic linear map of the expression construct encoding codon-optimized human factor VIII (FVIIIco6) containing the XTEN144 peptide (FVIIIco6XTEN) under the control of the liver-specific TTPp promoter, the woodchuck posttranscriptional regulatory element (WPRE), and the bovine growth hormone polyadenylation (bGHpA) signal. The hFVIIIco6XTEN expression cassette is flanked by symmetric truncated (Δ) ITRs, symmetric wild-type (WT) ITRs, and asymmetric (Asy) ITRs of B19 (Δ135, WT), GPV (Δ162, WT, and Asy), or AAV2 (WT, Asy1, Asy2, Asy3), respectively. The ITR sequences shown can be found in Table 1. [Figure 4B] Schematic diagram of human FVIIIco6XTEN expression construct. Figure 4B shows a schematic linear map of the Cre-LoxP donor vector encoding the hFVIIIco6XTEN expression cassette flanked by the ITRs of B19, GPV, or AAV2 as described in Figure 4A cloned into the Cre-LoxP donor vector as described in Figure 2B. [Figure 5-1]Figures 5A-5G show schematic diagrams of recombinant baculovirus expression vectors (BEVs) containing sequences encoding Rep, as well as validation studies thereof. Figure 5A shows an agarose gel electrophoresis image of restriction enzyme mapping of recombinant BIVVBac bacmids encoding B19.Rep, GPV.Rep, or AAV2.Rep (BIVVBac.IE1.B19.RepTn7, BIVVBac.Polh.GPV.RepTn7, and BIVVBac.Polh.AAV2.RepTn7, respectively) compared to the parental BIVVBac and bMON14272 bacmids. Figure 5B shows a schematic map of AcBIVVBac.IE1.B19.RepTn7. Figure 5C shows a schematic map of AcBIVVBac.Polh.GPV.RepTn7. Figure 5D shows a schematic map of AcBIVVBac.Polh.AAV2.RepTn7. Figure 5E shows B19.REP immunoblotting in plaque purified clones (clones 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#5, Cl#6) of recombinant AcBIVVBac.IE1.B19.RepTn7 BEV with polyclonal anti-B19 NS1 (1:2500) as primary antibody and IRDye@680RD donkey anti-rabbit LI-COR (1:10,000) as secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as a standard and is shown on the left. Figure 5F shows GPV.REP immunoblotting in plaque purified clones of recombinant AcBIVVBac.Polh.GPV.RepTn7BEV (clones 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#5, Cl#6) with polyclonal anti-GPV REP peptide antibody (1:500) as the primary antibody and IRDye@680RD donkey anti-rabbit LI-COR (1:10,000) as the secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as a standard and is shown on the left.Figure 5G shows AAV2.REP immunoblotting on plaque-purified clones of recombinant AcBIVVBac.Polh.AAV2.RepTn7BEV with monoclonal anti-AAV2 Rep (1:500) as the primary antibody and IRDye@800CW donkey anti-mouse LI-COR (1:10,000) as the secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as a standard and is shown on the left. [Figure 5-2] Continued from Figure 5-1. [Figure 6-1] Figures 6A-6C are schematic diagrams of BEVs containing sequences encoding Rep and hFVIIIco6XTEN. Figure 6A shows a schematic map of recombinant BEV (AcBIVVBac(B19.Rep)FVIII.B19.ITRsLoxP) encoding a B19.Rep expression cassette and a hFVIIIco6XTEN expression cassette flanked by B19 ITRs as indicated. Figure 6B shows a schematic map of recombinant BEV (AcBIVVBac(GPV.Rep)FVIII.GPV.ITRsLoxP) encoding a GPV.Rep expression cassette and a hFVIIIco6XTEN expression cassette flanked by GPV ITRs as indicated. Figure 6C shows a schematic map of the recombinant BEV (AcBIVVBac(AAV2.Rep)FVIII.AAV2.ITRsLoxP) encoding the AAV2.Rep expression cassette and the hFVIIIco6XTEN expression cassette flanked by AAV2 ITRs. The ITR sequences shown in Figures 6A-6C can be found in Table 1. [Figure 6-2] Continued from Figure 6-1. [Figure 7]Figures 7A-7C show the generation of human FVIIIco6XTEN ceDNA vector using the baculovirus expression vector system according to one embodiment of the present invention. Figure 7A is a schematic map of recombinant BEV (AcBIVVBac(GPV.Rep)FVIII.GPV.Asy.ITRsLoxP) encoding GPV.Rep and hFVIIIco6XTEN expression cassettes flanked by GPV asymmetric ITRs. The ITR sequences shown can be found in Table 1. Figure 7B is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with plaque purified clones of AcBIVVBac(GPV.Rep)FVIII.GPV.Asy.ITRsLoxPBEV (Clones 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#5, Cl#6). The DNA band corresponding to the size of hFVIIIco6XTEN is indicated by an arrow. Figure 7C is an agarose gel electrophoresis image of the ceDNA vector derived from recombinant AcBIVVBac(GPV.Rep)FVIII.GPV.Asy.ITRsLoxP BEV clone #4 (as shown in Figure 7B) loaded at different amounts. The DNA band corresponding to the size of hFVIIIco6XTEN is indicated by an arrow. [Figure 8] Figures 8A-8B show the materials used in generating stable human FVIIIco6XTEN encoding insect cell lines. Figure 8A shows a schematic map of the plasmid encoding the neomycin resistance marker in the presence of the AcMNPV immediate early 1 (ie1) promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal. Figure 8B shows a schematic map of the hFVIIIco6XTEN expression cassette flanked by B19, GPV, or AAV2 ITRs that are stably integrated into the Sf9 cell genome to generate stable cell lines. A list of the stable cell lines generated is shown in Table 5, as well as the sequences of the ITRs in Table 1. [Figure 9]Figures 9A-9C are agarose gel electrophoresis images showing the production of human FVIIIco6XTEN ceDNA vectors from stable cell lines. Figure 9A is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf cell lines-1 and -2, each flanked by symmetric truncated (B19Δ135) or symmetric wild-type (B19.WT) ITRs and encoding hFVIIIco6XTEN. Figure 9B is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf cell lines-3, -4, and -5, each flanked by symmetric truncated (GPVΔ162), symmetric wild-type (GPV.WT), or asymmetric (GPV.Asy) ITRs and encoding hFVIIIco6XTEN. Figure 9C is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf cell lines-6, -7, -8, and -9 encoding hFVIIIco6XTEN flanked by symmetric wild type (AAV2.WT), asymmetric 1 (AAV2.Asy1), asymmetric 2 (AAV2.Asy2), or asymmetric 3 (AAV2.Asy3), respectively. The stable cell lines used in Figures 9A-9C are shown in Table 5, and the sequences of the ITRs are shown in Table 1. [Figure 10] 1 is a plot showing hFVIII activity measured by Chromogenix Coatest® SP Factor VIII chromogenic assay. ceDNA isolated from a stable cell line is transfected into Huh7 cells, and cell-free supernatants are collected 48 and 72 hours after transfection and tested for hFVIII detection by chromogenic assay. The solid line represents the ceDNA sample, and the dotted line represents the plasmid DNA sample used as a control. [Figure 11-1]11A-11F are schematic diagrams of replication (Rep) protein expression constructs according to embodiments of the present invention. FIG. 11A shows a schematic linear map of synthetic DNA encoding the B19 Rep gene in the presence of the OpMNPV immediate early (OpIE2) promoter followed by the SV40 polyadenylation signal (SV40 PAS). FIG. 11B shows a schematic map of a transient expression plasmid made by replacing the IE1 promoter with OpIE2 in the pFastBac.IE1.B19.Rep construct shown in FIG. 3B. FIG. 11C shows a schematic linear map of synthetic DNA encoding the GPV Rep78 and Rep52 insert genes in the presence of the OpMNPV immediate early (OpIE2) promoter followed by the SV40 polyadenylation signal (SV40 PAS). The modified mRNA transcript (messenger RNA) is shown as a wavy line with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) of Rep52, and the stop codon (UAA). Figure 11D shows a schematic map of the transient expression plasmid made by replacing the polyhedrin promoter with OpIE2 in the pFastBac.Polh.GPV.Rep construct shown in Figure 3D. Figure 11E shows a schematic linear map of the synthetic DNA encoding the AAV2 Rep78 and Rep52 insert genes in the presence of the OpMNPV immediate early (OpIE2) promoter followed by the SV40 polyadenylation signal (SV40 PAS). The modified mRNA transcript (messenger RNA) is shown as a wavy line with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) of Rep52, and the stop codon (UAA). FIG. 11F shows a schematic map of the transient expression plasmid generated by replacing the polyhedrin promoter with OpIE2 in the pFastBac.Polh.AAV2.Rep construct shown in FIG. 3F. [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 12]Figures 12A-12C are schematic diagrams of modified FVIIIXTEN expression cassettes with parvoviral ITRs, liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482), and (V2.0) codon-optimized BDDco-FVIIIXTEN transgene (V2.0 FVIIIXTEN) (SEQ ID NO: 19) according to embodiments of the present invention. Figure 12A shows a schematic linear map of modified FVIIIXTEN expression cassettes flanked by AAV2 WT ITRs (SEQ ID NO: 5, SEQ ID NO: 6). Figure 12B shows a schematic linear map of modified FVIIIXTEN expression cassettes flanked by B19 WT (SEQ ID NO: 2) or B19 minimal (SEQ ID NO: 16). Figure 12C shows a schematic linear map of modified FVIIIXTEN expression cassettes flanked by GPVΔ120 (SEQ ID NO: 17) or GPVΔ186 (SEQ ID NO: 18). [Figure 13] Figures 13A-13C are schematic diagrams of approaches used for ceDNA production in the baculovirus system according to one embodiment of the present invention. Figure 13A shows a schematic diagram of the One BAC approach, in which a single recombinant BEV encoding V2.0 FVIIIXTEN and Rep genes at different loci was used to infect Sf9 cells for ceDNA production. Figure 13B shows a schematic diagram of the 2BAC approach, in which Sf9 cells were co-infected with recombinant BEV encoding V2.0 FVIIIXTEN and / or Rep genes for ceDNA production. Figure 13C shows a schematic diagram of the stable cell line approach, in which the FVIIIXTEN expression cassette was stably integrated into the Sf9 cell genome and rescued by infecting recombinant BEV encoding Rep genes for ceDNA production. [Figure 14]Figures 14A-14C show the production of human FVIIIXTEN ceDNA vector from AAV2 One BAC according to one embodiment of the present invention. Figure 14A is a schematic diagram of the One BAC approach (AcBIVVBac(mTTR.FVIIIXTEN.AAV2.WT.ITRs)Polh.AAV2.RepLoxP) of FVIIIXTEN ceDNA vector production in Sf9 cells using a recombinant BEV encoding the AAV2 Rep gene in the presence of AcMNPV polyhedrin promoter and a human FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by AAV2 WT ITRs. Figure 14B shows a schematic map of AcBIVVBac(mTTR.FVIIIXTEN.AAV2.WT.ITRs)Polh.AAV2.RepLoxP BEV. Figure 14C is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with titered viral stock (P2) of AcBIVVBac(mTTR.FVIIIXTEN.AAV2.WT.ITRs)Polh.AAV2.RepLoxP BEV. DNA bands corresponding in size to FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 15]Figures 15A-15C show the production of human FVIIIXTEN ceDNA vector from B19 One BAC according to one embodiment of the present invention. Figure 15A is a schematic diagram of the One BAC approach (AcBIVVBac(mTTR.FVIIIXTEN.B19.WT.ITRs)Polh.B19.NS1LoxP) of FVIIIXTEN ceDNA vector production in Sf9 cells using a recombinant BEV encoding the B19 NS1 gene in the presence of AcMNPV polyhedrin promoter and a human FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by B19 WT ITRs. Figure 15B shows a schematic map of AcBIVVBac(mTTR.FVIIIXTEN.B19.WT.ITRs)Polh.B19.NS1LoxP BEV. Figure 15C is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with titered viral stock (P2) of AcBIVVBac(mTTR.FVIIIXTEN.B19.WT.ITRs)Polh.B19.NS1LoxP BEV. DNA bands corresponding in size to FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 16]Figures 16A-16C show the production of human FVIIIXTEN ceDNA vectors from AAV2 Two BAC according to one embodiment of the present invention. Figure 16A is a schematic diagram of the Two BAC approach for the production of FVIIIXTEN ceDNA vectors in which Sf9 cells were co-infected with recombinant BEV encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by AAV2 WT ITRs (AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITRsTn7) and / or recombinant BEV encoding the AAV2 Rep gene in the presence of the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.AAV2.RepTn7). Figure 16B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITRsTn7 and AcBIVVBac.Polh.AAV2.RepTn7 BEVs. Figure 16C shows an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected at different MOIs with the indicated AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITRsTn7 and AcBIVVBac.Polh.AAV2.RepTn7 BEVs. DNA bands corresponding to the sizes of the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 17]Figures 17A-17C show the production of human FVIIIXTEN ceDNA vector from B19 Two BAC according to one embodiment of the present invention. Figure 17A is a schematic diagram of the Two BAC approach for FVIIIXTEN ceDNA vector production, in which Sf9 cells are co-infected with recombinant BEV (AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITRsTn7) encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by B19 WT ITRs and / or recombinant BEV (AcBIVVBac.Polh.B19.NS1Tn7) encoding the B19 NS1 gene in the presence of the AcMNPV polyhedrin promoter. Figure 17B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITRsTn7 and AcBIVVBac.Polh.B19.NS1Tn7 BEVs. Figure 17C shows an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected at different MOIs with the indicated AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITRsTn7 and AcBIVVBac.Polh.B19.NS1Tn7 BEVs. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 18]Figures 18A-18C show the generation of human FVIIIXTEN ceDNA vector from GPV Two BAC according to one embodiment of the present invention. Figure 18A is a schematic diagram of the Two BAC approach for FVIIIXTEN ceDNA vector production, in which Sf9 cells were co-infected with recombinant BEV (AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITRsTn7) encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by GPVΔ120 ITRs and / or recombinant BEV (AcBIVVBac.Polh.GPV.NS1Tn7) encoding the GPV NS1 gene in the presence of the AcMNPV polyhedrin promoter. Figure 18B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITRsTn7 and AcBIVVBac.Polh.GPV.NS1Tn7 BEV. Figure 18C shows an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected at different MOIs with the indicated AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITRsTn7 and AcBIVVBac.Polh.GPV.NS1Tn7 BEV. DNA bands corresponding to the sizes of the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 19]Figures 19A-19C show the production of human FVIIIXTEN ceDNA vectors from AAV2 stable cell lines according to one embodiment of the present invention. Figure 19A shows a schematic diagram of the stable cell line approach of FVIIIXTEN ceDNA vector production, where a stable cell line encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by AAV2 WT ITRs is infected with a recombinant BEV (AcBIVVBac.Polh.AAV2.RepTn7) encoding the AAV2 Rep gene in the presence of the AcMNPV polyhedrin promoter. Figure 19B shows a schematic map of AcBIVVBac.Polh.AAV2.RepTn7 BEV. Figure 19C shows an agarose gel electrophoresis image of ceDNA vectors isolated from AAV2 stable cell lines infected at different MOIs with the illustrated AcBIVVBac.Polh.AAV2.RepTn7 BEV. DNA bands corresponding in size to the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 20]Figures 20A-20C show the production of human FVIIIXTEN ceDNA vector from B19 stable cell line according to one embodiment of the present invention. Figure 20A shows a schematic diagram of the stable cell line approach of FVIIIXTEN ceDNA vector production, where a stable cell line encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by B19 minimal ITRs is infected with a recombinant BEV (AcBIVVBac.Polh.B19.NS1Tn7) encoding the B19 NS1 gene in the presence of AcMNPV polyhedrin promoter. Figure 20B shows a schematic map of AcBIVVBac.Polh.B19.NS1Tn7 BEV. Figure 20C shows an agarose gel electrophoresis image of ceDNA vector isolated from B19 stable cell line infected at different MOIs of AcBIVVBac.Polh.B19.NS1Tn7 BEV as shown. DNA bands corresponding in size to the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 21]Figures 21A-21C show the production of human FVIIIXTEN ceDNA vectors from GPV stable cell lines according to one embodiment of the present invention. Figure 21A shows a schematic diagram of the stable cell line approach of FVIIIXTEN ceDNA vector production, where a stable cell line encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN and flanked by GPV Δ120 ITRs is infected with a recombinant BEV (AcBIVVBac.Polh.GPV.NS1Tn7) encoding the GPV NS1 gene in the presence of the AcMNPV polyhedrin promoter. Figure 21B shows a schematic map of the AcBIVVBac.Polh.GPV.NS1Tn7 BEV. Figure 21C shows an agarose gel electrophoresis image of ceDNA vectors isolated from GPV stable cell lines infected at different MOIs of AcBIVVBac.Polh.GPV.NS1Tn7 BEV as shown. DNA bands corresponding in size to the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Figure 22]Figures 22A-22E show the workflow of FVIIIXTEN ceDNA vector production and purification using the Two BAC approach according to one embodiment of the present invention. Figure 22A shows a schematic of cell growth and persistence (days 0-2), where cells are successively scaled up from small cell (0.5 L) to large culture (1.5 L) flasks to achieve a cell density of 2.5-3.0x106 / mL in serum-free ESF921 medium. FIG. 22B shows a schematic of infection and duration of incubation (days 2-6) in a large culture (1.5 L) flask of Sf9 where cells are co-infected with recombinant BEV encoding a FVIIIXTEN expression cassette containing V2.0 FVIIIXTEN flanked by HBoV1 ITRs (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRsTn7) and / or a recombinant BEV encoding the HBoV1 NS1 gene in the presence of the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.HBoV1.NS1Tn7) at an MOI of 0.1 and 0.01 plaque forming units (pfu) / cell, respectively. Figure 22C shows images of Plasmid Giga Prep Purification Kit and agarose gel electrophoresis over the duration of treatment (days 6-7), where cell density and viability of infected cells were measured daily and cells were pelleted by low speed centrifugation after cell viability reached 70-80%. FVIIIXTEN ceDNA 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 (genomic DNA). FIG. 22D shows images of Bio-Rad model 491 Prep cells and agarose gel electrophoresis over the duration of treatment (days 7-12) in which Giga-prep purified DNA was loaded onto a preparative agarose gel in Prep cells to separate FVIIIXTEN ceDNA (approximately 8.5 kb fragment) from high molecular weight DNA.To determine the purity of FVIIIXTEN ceDNA, elution fractions taken at 70-80 min intervals from the preparative agarose gel electrophoresis were analyzed in 0.8-1.2% agarose gels. Figure 22E shows an image of an agarose gel electrophoresis in which fractions taken from Prep cells were collected and precipitated with 1 / 10 volume of 3 M 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 indicating DNA bands corresponding in size to the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA). [Diagram 23] Figure 23 shows a graphical representation of plasma FVIII activity levels measured by Chromogenix Coatest® SP Factor VIII chromogenic assay.Figure 23 shows a graphical plot of plasma FVIII activity levels measured in blood samples taken at different intervals from hFVIIIR593C+ / + / HemA mice systemically injected by tail vein hydrodynamic injection with 80, 40, or 12 μg / kg FVIIIXTEN HBoV1 (wtHBoV1) or AAV2 (wtAAV2) ITRs ceDNA. [Figure 24]Figures 24A-24C show the production of FVIIIXTEN ceDNA vectors using the One BAC approach according to one embodiment of the present invention. Figure 24A is a schematic diagram of the One BAC approach of FVIIIXTEN ceDNA vector production in Sf9 cells using a recombinant BEV (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS1LoxP) encoding the HBoV1 NS1 gene in the presence of AcMNPV polyhedrin promoter and a human FVIIIXTEN expression cassette flanked by HBoV1 ITRs. Figure 24B shows a schematic map of the AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS11LoxP BEV. Figure 24C is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with titered viral stock (P2) of (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS11LoxP)BEV. DNA bands corresponding in size to FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. [Diagram 25]Figures 25A-25C show the production of FVIIIXTEN ceDNA vectors using the Two BAC approach according to one embodiment of the present invention. Figure 25A is a schematic diagram of the Two BAC approach of FVIIIXTEN ceDNA vector production, where Sf9 cells are co-infected with recombinant BEV (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRsTn7) encoding a FVIIIXTEN expression cassette flanked by HBoV1 ITRs and / or recombinant BEV (AcBIVVBac.Polh.HBoV1.NS1Tn7) encoding the HBoV1 NS1 gene in the presence of the AcMNPV polyhedrin promoter. Figure 25B shows the schematic map of AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRsTn7 and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEVs. Figure 25C is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected with the illustrated AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRsTn7 and AcBIVVBac.Polh.HBoV1.NS1Tn7 BEVs at different MOIs or at different ratios at a fixed MOI. DNA bands corresponding to the sizes of the FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA), and Sf9 cell genomic DNA (genomic DNA) are indicated by arrows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Provided herein is a baculovirus expression vector system suitable for expressing two or more foreign sequences.Also provided is a method of using the baculovirus expression vector system described herein, for example, to generate ceDNA.

[0089] I. Definition Herein, a noun with the term "a" or "an" refers to one or more of that noun: 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" can be used interchangeably herein.

[0090] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical values ​​set forth. In general, the term "about" is used to modify numerical values ​​above and below the stated value by a variance of up to ten percent higher or lower.

[0091] As used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0092] "Nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide 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") in either single-stranded form or double-stranded helix, or any phosphoester analogues thereof, such as phosphorothioates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term encompasses double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. In describing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand with the homologous sequence to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids described herein.

[0093] As used herein, "inverted terminal repeat" (or "ITR") refers to a nucleic acid subsequence located at either the 5' or 3' end of a single stranded nucleic acid sequence that includes a set of nucleotides followed downstream by its reverse complement, i.e., a palindromic sequence. The intervening sequence of nucleotides between the initial sequence and the reverse complement can be of any length, including zero. In one embodiment, an ITR useful for the present disclosure includes 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 non-T-shaped hairpin structure, e.g., a U-shaped hairpin structure. In some embodiments, an ITR promotes long-term survival of a nucleic acid molecule in a cell nucleus. In some embodiments, an ITR promotes permanent survival of a nucleic acid molecule in a cell nucleus (e.g., for the entire lifespan of a cell). In some embodiments, an ITR promotes stability of a nucleic acid molecule in a cell nucleus. In some embodiments, the ITRs promote maintenance of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs promote persistence of the nucleic acid molecule in the cell nucleus. In some embodiments, the ITRs inhibit or prevent degradation of the nucleic acid molecule in the cell nucleus.

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

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

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

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

[0098] Exemplary autonomous parvoviruses include, but are not limited to, porcine parvovirus, minute virus of mice, canine parvovirus, mink entertitus virus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus.Other autonomous parvoviruses are known to those skilled in the art.See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers).

[0099] The term "non-AAV" as used herein encompasses nucleic acids, proteins, and viruses from the family Parvovirus, excluding all adeno-associated viruses (AAV) of the Parvovirus 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.

[0100] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, AAV serotypes thereof, and the 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 later discovered. See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

[0101] The term "derived from" as used herein means a component isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) 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 species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive a nucleotide or polypeptide can be deliberately directed or deliberately randomized, or a mixture of each. Mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from a first can be a fortuitous event (e.g., due to the infidelity of a polymerase), and identification of the derived nucleotide or polypeptide can be performed by suitable screening methods, for example, as described herein. Mutagenesis of a polypeptide typically involves manipulation of a polynucleotide that encodes the polypeptide.In some embodiments, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence is at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 201%, at least 202%, at least 203%, at least 204%, at least 205%, at least 206%, at least 207%, at least 208%, at least 209%, at least 300%, at least 301%, at least 302%, at least 303%, at least 304%, at least 305%, at least 306%, 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence. In other embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 90% identical to the non-AAV ITR (or, respectively, AAV ITR), where the non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, AAV ITR). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 80% identical to the non-AAV ITR (or, respectively, the AAV ITR), where the non-AAV (or AAV) ITRs retain the functional properties of the non-AAV ITR (or, respectively, the AAV ITR).In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 70% identical to the non-AAV ITR (or, respectively, AAV ITR), where the non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, AAV ITR). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 60% identical to the non-AAV ITR (or, respectively, AAV ITR), where the non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, AAV ITR). In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 50% identical to the non-AAV ITR (or, respectively, AAV ITR), where the non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, AAV ITR).

[0102] In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR. In some embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, where the fragment is at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 9 ...15 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 15 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 ITRs derived from a non-AAV (or AAV) ITR maintain the functional properties of the non-AAV ITR (or, respectively, the AAV ITR).In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, where the fragment comprises at least about 129 nucleotides and the ITR derived from a non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, the AAV ITR). In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, where the fragment comprises at least about 102 nucleotides and the ITR derived from a non-AAV (or AAV) ITR maintains the functional properties of the non-AAV ITR (or, respectively, the AAV ITR).

[0103] In some embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of the non-AAV (or AAV) ITR, 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 non-AAV (or AAV) ITR.

[0104] In certain embodiments, a nucleotide or amino acid sequence derived from a second nucleotide or amino acid sequence, when properly aligned, is at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 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%, or 100% sequence identity, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence. In other embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 90% identical to the homologous portion of the non-AAV ITR (or, respectively, AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITRs derived from a non-AAV (or AAV) ITR are at least 80% identical to the homologous portion of the non-AAV ITR (or, respectively, AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence.In some embodiments, the ITRs derived from the non-AAV (or AAV) ITRs are at least 70% identical to the homologous portion of the non-AAV ITR (or, respectively, AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITRs derived from the non-AAV (or AAV) ITRs are at least 60% identical to the homologous portion of the non-AAV ITR (or, respectively, AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, the ITRs derived from the non-AAV (or AAV) ITRs are at least 50% identical to the homologous portion of the non-AAV ITR (or, respectively, AAV ITR) when properly aligned, where the first nucleotide or amino acid sequence maintains the biological activity of the second nucleotide or amino acid sequence.

[0105] A "capsid-free" or "capsid-less" vector or nucleic acid molecule refers to a vector construct that lacks a capsid. In some embodiments, the capsid-free vector or nucleic acid molecule does not include sequences encoding, for example, AAV Rep proteins. In some embodiments, the capsid-free or capsid-less vector may include covalently closed ends, and is referred to herein as "closed-end DNA (ceDNA)". Generally, the ceDNA described herein can be made using the baculovirus expression system of the present invention, and the ceDNA generally includes at least one nucleic acid encoding a heterologous gene product flanked on either side by ITRs (e.g., AAV or non-AAV ITRs).

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

[0107] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein after the export of the growing protein chain across the rough endoplasmic reticulum is initiated. Those skilled in the art are aware that signal peptides are generally fused to the N-terminus of a polypeptide and cleaved from the complete or "full-length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of its sequence maintains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide can be used, for example, human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide or a functional derivative thereof.

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

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

[0110] As used herein, the term "gene cassette" or "expression cassette" refers to a DNA sequence that can direct the expression of a particular polynucleotide sequence in a suitable host cell, including a promoter operably linked to a polynucleotide sequence of interest. A gene cassette is located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region and includes nucleotides that affect the 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 and a transcription termination sequence will usually be located 3' of the coding sequence. In some embodiments, the gene cassette includes a polynucleotide that encodes a gene product. In some embodiments, the gene cassette includes a polynucleotide that encodes a miRNA. In some embodiments, the gene cassette includes a heterologous polynucleotide sequence.

[0111] A polynucleotide encoding a product, such as an miRNA, or a gene product (e.g., a polypeptide, such as a therapeutic protein) can include a promoter and / or other expression (e.g., transcription or translation) control sequence operably associated with one or more coding regions. In operably associated, a coding region for a gene product, such as 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. 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 if the nature of the link 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, such as enhancers, operators, repressors, and transcription termination signals, are also operably associated with a coding region to direct expression of the gene product.

[0112] "Expression control sequence" refers to a regulatory nucleotide sequence, such as a promoter, enhancer, terminator, etc., that provides for the expression of a coding sequence in a host cell. Expression control sequences generally encompass any regulatory nucleotide sequence that promotes efficient transcription and translation of 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, but are not limited to, expression control sequences that function in vertebrate cells, such as, but are not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter in conjunction with intron A), Simian Virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other expression control sequences include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences that can control gene expression in eukaryotic cells. Additional suitable expression control sequences include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Other expression control sequences include intron sequences, posttranscriptional regulatory elements, and polyadenylation signals. Additional exemplary expression control sequences are described elsewhere in this disclosure.

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

[0114] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. Such includes, but is not limited to, 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, as well as the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, such as 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, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage. The term "yield" as used herein refers to the amount of polypeptide produced by expression of a gene.

[0115] "Vector" refers to any vehicle for cloning and / or transferring nucleic acid to a host cell. A vector can be a replicon to which another nucleic acid segment can be attached to effect replication of the attached segment. "Replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an automatic unit of replication in vivo, i.e., capable of replicating under its own control. The term "vector" encompasses vehicles for introducing nucleic acid into cells in vitro, ex vivo, or in vivo. Many vectors are known and used in the art, e.g., plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment to complementary cohesive termini.

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

[0117] The term "host cell" as used herein refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of ssDNA or vectors. The term encompasses the progeny of an original cell that has been transduced. Thus, "host cell" as used herein generally refers to a cell that has been transduced with an exogenous DNA sequence. The progeny of a single parent cell may not necessarily be completely identical in morphology or genome 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.

[0118] The term "selectable marker" refers to a distinguishing factor (usually an antibiotic or chemical resistance gene, which can be selected based on the effect of the marker gene, i.e., resistance to antibiotics, resistance to herbicides), colorimetric marker, enzyme, fluorescent marker, 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 markers known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc.; as well as genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc.

[0119] The term "reporter gene" refers to a nucleic acid that encodes a distinguishing factor that can be distinguished based on the effect of the reporter gene, where the effect is used to trace the inheritance of the nucleic acid of interest, to distinguish cells or organisms that have inherited the nucleic acid of interest, and / or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable marker genes can be considered as reporter genes.

[0120] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located '3' to the promoter sequence. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even include synthetic DNA segments. Different promoters can direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental and physiological conditions. A promoter that causes a gene to be expressed in most cell types most of the time is commonly referred to as a "constitutive promoter". A promoter that causes a gene to be expressed in a particular cell type is commonly referred to as a "cell-specific promoter" or "tissue-specific promoter". A promoter that causes a gene to be expressed at a particular stage of development or cell differentiation is commonly referred to as a "development-specific promoter" or "cell differentiation-specific promoter". A promoter that causes or induces expression of a gene after exposure or treatment of cells with a drug, biological molecule, chemical, ligand, light, or the like that induces the promoter, is generally called an "inducible promoter" or "regulatable promoter." Moreover, in most cases, the exact boundaries of regulatory sequences are not completely defined, so that DNA fragments of different lengths can have the same promoter activity.

[0121] A promoter sequence is typically bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. 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, will be found within the promoter sequence.

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

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

[0124] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of a cell, usually in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genomic integration sequences, phages or nucleotide sequences, linear, circular or supercoiled, single-stranded or double-stranded DNA or RNA from any source, where many nucleotide sequences are joined or recombined into unique constructs that can introduce promoter fragments and DNA sequences for selected gene products with appropriate 3' non-translated sequences into cells.

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

[0126] "Cloning vector" means a "replicon", which is a unit length of continuously replicating nucleic acid, including an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be attached in order to effect replication of the attached segment. Certain cloning vectors allow for replication in one cell type, such as bacteria, and expression in another cell, such as a eukaryotic cell. Cloning vectors typically contain one or more sequences that can be used for the insertion of a nucleic acid sequence of interest into the vector and / or for selection of cells that contain one or more multiple cloning sites.

[0127] The term "expression vector" refers to a vehicle designed to allow 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.

[0128] Vectors are introduced into host cells by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or DNA vector transporter. "Culture", "to culture", and "culturing", as used herein, refer to the incubation of cells under in vitro conditions that allow the cells to grow or divide or to maintain the cells alive. "Cultured cells", as used herein, refer to cells that are propagated in vitro.

[0129] The term "percent identity" as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences, where possible. "Identity" can be readily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods for determining identity are codified in publicly available computer programs.Sequence alignment and percent identity calculations can be performed using sequence analysis software, such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA). 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 referenced program unless otherwise specified. As used herein, "default values" will mean any set of values ​​or parameters originally loaded by the software when first started.

[0130] As used herein, nucleotides corresponding to nucleotides of a particular sequence of the present disclosure are identified by alignment of the sequences of the present disclosure to maximize identity to the reference sequence. The numerical values ​​used to identify equivalent amino acids in the reference sequence are based on the numerical values ​​used to identify the corresponding amino acids in the sequences of the present disclosure.

[0131] As used herein, the term "heterologous" or "exogenous" refers to a molecule that is not normally found in a given context, e.g., a cell or polypeptide. For example, an exogenous or heterologous molecule can be introduced into a cell and is present only after manipulation of the cell, e.g., by transfection, or other forms of genetically engineered or heterologous amino acid sequences can be present in a protein such that it is not found in nature.

[0132] As used herein, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not naturally occur with a given polynucleotide sequence. In one embodiment, the heterologous nucleotide sequence encodes a polypeptide that can extend the half-life of a therapeutic protein, e.g., a clotting factor, e.g., FVIII. In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of a therapeutic protein, e.g., a clotting factor, e.g., FVIII. In other embodiments, the heterologous nucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of a therapeutic protein without significantly affecting its biological activity or function (e.g., procoagulant activity). In some embodiments, the therapeutic protein is linked or connected to the polypeptide encoded by the heterologous nucleotide sequence by a linker. Non-limiting examples of polypeptide moieties encoded by a heterologous nucleotide sequence include immunoglobulin constant regions or portions thereof, albumin or fragments thereof, albumin binding moieties, transferin, the PAS polypeptides of U.S. Patent Application Publication No. 20100292130, HAP sequences, transferin or fragments thereof, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, albumin binding small molecules, XTEN sequences, FcRn binding moieties (e.g., the complete constant region or a portion thereof that binds to FcRn), single chain Fc regions (e.g., the U.S. Pat. Appln. 20100292130A1), and the like. Examples of suitable heterologous nucleotide sequences include ScFc regions, such as those described in US Pat. No. 2008 / 0260738, WO 2008 / 012543, or WO 2008 / 1439545, polyglycine linkers, polyserine linkers, peptides and small polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) with varying degrees of secondary structure from less than 50% to more than 50%, or combinations of two or more thereof. In some embodiments, the polypeptide encoded by the heterologous nucleotide sequence is linked to a non-polypeptide moiety.Non-limiting examples of non-polypeptide moieties include polyethylene glycol (PEG), albumin-binding small molecules, polysialic acid, hydroxyethyl starch (HES), derivatives thereof, or any combination thereof.

[0133] As used herein, the term "optimized" refers to a polynucleotide sequence that encodes a polypeptide, where the polynucleotide sequence has been mutated to enhance the properties of the polynucleotide sequence. In some embodiments, optimization is performed to increase transcription levels, increase translation levels, increase steady-state mRNA levels, increase or decrease the binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase the yield of the polypeptide produced by the polynucleotide sequence. Examples of modifications that can be made to a polynucleotide sequence to optimize the polynucleotide sequence include codon optimization, G / C content optimization, removal of repetitive sequences, removal of AT-rich elements, removal of cryptic cleavage sites, removal of cis-acting elements that suppress transcription or translation, addition or removal of poly-T or poly-A sequences, addition of sequences around the transcription start site that enhance transcription function, such as Kozak consensus sequences, removal of sequences that can form stem-loop structures, removal of destabilizing sequences, and combinations of two or more thereof.

[0134] As used herein, the term "bacmid" refers to a shuttle vector capable of propagation in both E. coli and insect cells. A recombinant bacmid refers to a bacmid that includes a heterologous sequence (e.g., a heterologous sequence encoding a heterologous gene).

[0135] II. Baculovirus Expression Vector System Provided herein is a baculovirus expression vector system comprising a baculovirus shuttle vector (bacmid) and / or a stable cell line engineered to produce a therapeutic product. Provided herein is a baculovirus expression vector system comprising a recombinant bacmid comprising two or more foreign sequence insertion sites, and one or more donor vectors capable of mediating the insertion of a foreign sequence (e.g., a heterologous gene) into the foreign sequence insertion sites.

[0136] In some embodiments, the therapeutic product is a protein. In some embodiments, the therapeutic product is a nucleic acid. In some embodiments, the therapeutic product is a recombinant protein used in various applications, such as, for example, vaccines, protein replacement therapy (e.g., enzyme replacement therapy), and recombinant proteins for basic and applied research. In certain embodiments, the therapeutic product is a DNA therapeutic agent, which is a plasmid-like, non-capsid nucleic acid molecule that encodes a target sequence. The DNA therapeutic agent can be in the form of a covalently closed-end DNA (ceDNA). Generally, ceDNA comprises a therapeutic protein-encoding gene located between a first inverted terminal repeat (5'ITR) and a second ITR (3'ITR).

[0137] In certain embodiments, the 5'ITR and 3'ITR are adeno-associated virus (AAV) ITRs or non-AAV ITRs. In certain embodiments, the non-AAV ITRs are ITRs obtained from members of the virus family Parvovirus. Suitable ITR sequences include AAV ITRs of AAV serotypes known to those skilled in the art. Suitable AAV and non-AAV ITR sequences are described in International Publication No. WO2019032898(A1), International Publication No. WO2020033863(A1), and International Publication No. WO2017152149(A1), the disclosures of which are incorporated herein by reference in their entirety. For example, the non-AAV ITR sequence can be derived from goose parvovirus (GPV) or parvovirus B19 (also referred to herein as "B19").

[0138] A. Baculovirus Shuttle Vector (Bacmid) Baculoviruses are the most prominent viruses that infect insects. Over 500 baculovirus isolates have been identified, the majority of which originate from lepidopteran insects. The two most common isolates are Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV). When producing viral or non-viral vectors for gene therapy, it is often necessary to infect insect host cells with several baculovirus expression vectors. The generation of each baculovirus expression vector is time-consuming, raising the cost of production, which represents a major drawback of the baculovirus expression vector system prior to the present invention.

[0139] In certain embodiments, the baculovirus expression vector system provided herein comprises a recombinant bacmid, herein referred to as "BIVVBac". A representative schematic diagram of BIVVBac is shown in FIG. 1C. In certain embodiments, BIVVBac is a genetically modified AcMNPV that comprises at least two foreign sequence insertion sites. The baculovirus expression vector system that comprises a bacmid that comprises at least two foreign sequence insertion sites allows for a reduction in the total number of baculovirus expression vectors that need to be produced. In some embodiments, a single baculovirus expression vector of the present invention is required to produce a gene therapy vector.

[0140] In certain embodiments, BIVVBac comprises a first and a second foreign sequence insertion site. The first and second foreign sequence insertion sites may be different and utilize various mechanisms to drive the insertion of foreign sequences (e.g., heterologous sequences, heterologous genes). The insertion of foreign sequences is driven by any method known in the art. For example, foreign sequences are inserted by transposition or site-specific recombination. The foreign sequence insertion site is designed to be contained within a reporter gene such that the reporter gene is disrupted upon insertion of the foreign sequence. Disruption of the reporter gene can help identify bacmid clones that have foreign sequences inserted therein. 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.

[0141] In certain embodiments, the first and second foreign sequence insertion sites are located in different loci in AcMNPV.In certain embodiments, the first and second foreign sequence insertion sites are located in different non-essential loci in AcMNPV.Various non-essential loci of AcMNPV are known to those skilled in the art.For example, polyhedrin gene and EGT gene are non-essential AcMNPV genes for virus replication in insect cells.Therefore, in certain embodiments, the first foreign sequence insertion site is located in the polyhedrin locus of AcMNPV, and the second foreign sequence insertion site is located in the EGT locus of AcMNPV.

[0142] In certain embodiments, the first foreign sequence insertion site allows the insertion of the foreign sequence by transposition. In certain embodiments, the first foreign sequence insertion site comprises a selective target site for the insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a selective target site for the insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a selective target site that is an attachment site for a bacterial transposon. Suitable bacterial transposons and their corresponding attachment sites are known to those skilled in the art. For example, transposon Tn7 is known for its ability to transpose to a specific site of a bacterial chromosome (attTn7) at high frequency. Thus, in certain embodiments, the first foreign sequence insertion site is a selective target site that is an attachment site for a Tn7 transposon (e.g., attTn7). In some embodiments, the first foreign sequence insertion site is a selective target site that is an attachment site for a mini-Tn7 transposon (e.g., mini-attTn7, a minimal DNA sequence required for recognition by and binding of a Tn7 transposon).

[0143] In certain embodiments, the first foreign sequence insertion site is fused in frame with a reporter gene. The reporter gene can be any reporter gene known in the art, such as, for example, luciferase (Luc), green fluorescent protein (GFP), red fluorescent protein (RFP), a fluorescent protein of a particular color, chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. In certain embodiments, the first foreign sequence insertion site is fused in frame with a reporter gene encoding an enzyme capable of metabolizing a chromogenic substrate. The enzyme capable of metabolizing a chromogenic substrate can be an enzyme known in the art with the same properties, such as LacZ or a functional part thereof. The reporter gene encoding an enzyme capable of metabolizing a chromogenic substrate is used in color-based screening of positive insertion events. For example, LacZ is used in blue-white screening, where a functional LacZ gene product cleaves the chromogenic substrate X-gal or Blue-gal, resulting in the production of a blue pigment. In blue-white screening, the presence of functional LacZ by α-complementation means that the insertion event was unsuccessful, with blue colonies representing clones in which the foreign sequence was not inserted, whereas white colonies represent clones in which the foreign sequence was successfully inserted, thereby preventing the production of a functional LacZ gene product and representing cleavage of the chromogenic substrate (e.g., X-gal).

[0144] Thus, in certain embodiments, the first foreign sequence insertion site is a selective target site for a bacterial transposon that is fused in frame with the sequence encoding LacZα or a functional part thereof.In certain embodiments, the first foreign sequence insertion site is a selective target site that is an attachment site for a Tn7 transposon (e.g., attTn7) that is fused in frame with the sequence encoding LacZα or a functional part thereof.For successful transposition, the transposon disrupts the sequence encoding LacZα, resulting in disruption of the production of functional LacZ gene product.

[0145] In certain embodiments, the second foreign sequence insertion site allows the insertion of the foreign sequence by site-specific recombination. In certain embodiments, the second foreign sequence insertion site comprises a selective target site that can mediate site-specific recombination events. Various site-specific recombinase techniques are known to those skilled in the art. For example, the Cre-LoxP system mediates site-specific recombination by Cre recombinase that can recognize 34 base pair DNA sequences called loxP sites. Thus, the second foreign sequence insertion site is a selective target site for Cre-mediated recombination. In certain embodiments, the second foreign sequence insertion site is a selective target site that comprises a LoxP site or a variant thereof that can be recognized by Cre recombinase.

[0146] The recombinant bacmid of the present invention comprises other elements necessary for its ability to be propagated in both bacterial cells (e.g., E. coli) and insect cells. For example, the recombinant bacmid of the present invention comprises a bacterial replicon. In certain embodiments, the bacmid comprises a bacterial replicon. Various bacterial replicons are known to those skilled in the art, including, for example, replicons originating from the F plasmid. In certain embodiments, a suitable bacterial replicon is the mini-F replicon, which is a derivative of the F plasmid, composed of the DNA regions oriS and incC necessary for replication and regulation. In certain embodiments, the bacterial replicon is a low copy number replicon. In certain embodiments, the low copy number replicon is a mini-F replicon.

[0147] Other elements of the recombinant bacmid of the present invention include one or more selectable marker sequences, and other reporter genes. Examples of selectable markers known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, etc.; as well as genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase genes, etc. In certain embodiments, the recombinant bacmid includes a selectable marker sequence that includes an antibiotic resistance gene. In certain embodiments, the antibiotic resistance gene is a kanamycin resistance gene and confers resistance to kanamycin. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. In some cases, the selectable marker can be considered a reporter. In certain embodiments, the recombinant bacmid comprises a reporter gene that encodes a fluorescent protein. In certain embodiments, the fluorescent protein is a red fluorescent protein.

[0148] Those skilled in the art will recognize that the various elements of the recombinant bacmids described herein are in operable linkage with each other. Each of the various coding sequences in the bacmid may be in operable linkage with regulatory regions, including, for example, promoter sequences. Any promoter sequence known in the art may be suitable.

[0149] Thus, the recombinant bacmid of the present invention comprises a mini-F replicon, an antibiotic resistance gene, LacZα or a functional portion thereof containing an attachment site for a Tn7 transposon, a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter, and a LoxP site or a variant thereof.

[0150] In certain exemplary embodiments, the recombinant bacmid of the present invention is BIVVBac, shown in FIG. 1C. In certain embodiments, BIVVBac encodes the AcMNPV C6 genome and is engineered to encode two foreign sequence insertion sites: 1) a mini-attTn7 at the polyhedrin locus, and 2) a LoxP at the EGT locus. The mini-attTn7 insertion sequence is fused in frame with a Lac-promoter-driven E. coli LacZα fragment for X-gal-mediated blue / white screening of recombinant bacmids after Tn7-mediated transposition, and a LoxP site for Cre-mediated in vitro or in vivo recombination. BIVVBac also contains a red fluorescent protein gene under the control of the AcMNPV39K promoter, followed by an ets polyadenylation signal.

[0151] In certain exemplary embodiments, BIVVBac is introduced into a bacterial strain capable of Tn7-mediated transposition. In certain embodiments, the bacterial strain is an E. coli strain. In certain embodiments, the bacterial strain is an E. coli DH10B bacterium. In certain embodiments, a helper plasmid encoding the Tn7 transposase genes tnsA-E and tetracycline resistance is introduced into the DH10B E. coli carrying BIVVBac. The DH10B E. coli carrying BIVVBac and the helper plasmid is referred to herein as BIVVBac. DH10B It is called.

[0152] Any foreign sequence (e.g., heterologous sequence) can be introduced into BIVVBac via Tn7-mediated transfer. In certain embodiments, the foreign sequence is introduced into BIVVBac by inserting a Tn7 transfer vector containing the foreign sequence into BIVVBac. DH10BThe BIVVBac containing the exogenous sequence is introduced into the BIVVBac via Tn7-mediated transposition by introducing the exogenous sequence into the BIVVBac, resulting in a BIVVBac containing the exogenous sequence. In certain embodiments, the BIVVBac containing the exogenous sequence comprises a bacterial replicon, a first selectable marker sequence, an exogenous sequence (e.g., a heterologous sequence) inserted into a first reporter gene, where the inserted exogenous sequence disrupts the reading frame of the first reporter gene, a second reporter gene operably linked to a baculovirus-inducible promoter, and a selective target site capable of mediating a site-specific recombination event.

[0153] In certain exemplary embodiments, for purposes of generating gene therapy vectors, AAV or non-AAV Rep genes are introduced into BIVVBac via Tn7-mediated transfer. In certain embodiments, a Tn7 transfer vector containing an AAV or non-AAV Rep gene is introduced into BIVVBac. DH10B, resulting in BIVVBac containing the Rep gene. In certain embodiments, the recombinant bacmid comprises: a mini-F replicon; a first antibiotic resistance gene; a sequence encoding B19 Rep inserted into LacZα or a functional portion thereof, where the inserted B19 Rep disrupts the reading frame of LacZα or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof. In certain embodiments, the recombinant bacmid comprises: a mini-F replicon; a first antibiotic resistance gene; a sequence encoding GPV Rep inserted into LacZα or a functional portion thereof, where the inserted GPV Rep disrupts the reading frame of LacZα or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof. In certain embodiments, the recombinant bacmid comprises a mini-F replicon, a first antibiotic resistance gene; a sequence encoding AAV2 Rep inserted into LacZα or a functional portion thereof, where the inserted AAV2 Rep interrupts the reading frame of LacZα or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

[0154] Any foreign sequence (e.g., heterologous sequence) can be introduced into BIVVBac via Cre-mediated recombination.In certain embodiments, foreign sequence is introduced into BIVVBac via Cre-mediated recombination, in which case foreign sequence is present on the Cre-LoxP transfer vector described herein.The method of Cre-mediated recombination is well known to those skilled in the art.

[0155] In certain exemplary embodiments, for the purpose of generating gene therapy vector, therapeutic protein-encoding gene flanked by symmetric or asymmetric AAV or non-AAV inverted terminal repeat (ITR) is introduced into BIVVBac via Cre-mediated recombination.In certain embodiments, recombinant bacmid comprises: mini-F replicon; antibiotic resistance gene; LacZα or its functional part, which contains attachment site for T7 transposon; a first selective target site (e.g., a first LoxP site) that can mediate site-specific recombination events; a therapeutic protein-encoding gene flanked by symmetric or asymmetric AAV or non-AAV ITR; and a second selective target site (e.g., a second LoxP site) that can mediate site-specific recombination events.

[0156] Thus, for purposes of generating a gene therapy vector, the recombinant bacmid comprises: a sequence encoding B19 Rep inserted into LacZα or a functional portion thereof, where the inserted B19 Rep interrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site containing heterologous sequences, where the heterologous sequences comprise, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from parvovirus B19; a sequence encoding a protein; one or more expression control sequences operably linked to the sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat derived from parvovirus B19.

[0157] Thus, for purposes of generating a gene therapy vector, the recombinant bacmid comprises: a sequence encoding GPV Rep inserted into LacZα or a functional portion thereof, where the inserted GPV Rep interrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site containing heterologous sequences, where the heterologous sequences comprise, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from GPV; a sequence encoding a protein; one or more expression control sequences operably linked to the sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat derived from GPV.

[0158] Thus, for purposes of generating a gene therapy vector, the recombinant bacmid comprises: a sequence encoding AAV2 Rep inserted into LacZα or a functional portion thereof, where the inserted AAV2 Rep interrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site containing heterologous sequences, where the heterologous sequences comprise, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from AAV2; a sequence encoding a protein; one or more expression control sequences operably linked to the sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat derived from AAV2.

[0159] Thus, for purposes of generating a gene therapy vector, the recombinant bacmid comprises: a sequence encoding human bocavirus (HBoV1) Rep, LacZα, or a functional portion thereof, inserted into a mini-attTn7 site, where the inserted HBoV1 Rep interrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site containing a heterologous sequence, where the heterologous sequence comprises, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from HBoV1; a sequence encoding a protein; one or more expression control sequences operably linked to the sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat derived from HBoV1.

[0160] In certain embodiments, BIVVBac bacmid is used to generate closed-end DNA (ceDNA). In some embodiments, ceDNA is generated by using a single baculovirus expression vector. In this "OneBAC" approach, a single baculovirus expression vector (e.g., BIVVBac) encodes all essential elements required for ceDNA generation in the baculovirus system, and could potentially be used in any baculovirus-permissive cell line for ceDNA generation. This approach is depicted in FIG. 13A.

[0161] In certain embodiments, ceDNA is produced by using multiple baculovirus expression vectors. In this "TwoBAC" approach, all essential elements required for ceDNA production will be inserted into two different baculoviruses (e.g., two BIVVBac bacmids) and used for co-infection in any cell line that is permissive for baculovirus infection. This approach is depicted in Figure 13B.

[0162] In certain embodiments, ceDNA is produced by a stable cell line. In this approach, the essential elements required for ceDNA production are inserted into both components of the baculovirus system. This approach is depicted in Figure 13C. The stable cell line can be generated by stably integrating a protein coding sequence under the control of a baculovirus gene promoter (e.g., a baculovirus constitutive promoter). In certain embodiments, the stable cell line is a stable insect cell line.

[0163] B. Transfer Vector The recombinant bacmid described herein comprises at least two foreign sequence insertion sites. In certain embodiments, the at least two foreign sequence insertion sites are 1) mini-attTn7 for Tn7-mediated transfer, and 2) LoxP for Cre-mediated site-specific recombination. Thus, the baculovirus expression vector system of the present invention further comprises two or more transfer vectors comprising foreign sequences inserted into the foreign sequence insertion sites of the recombinant bacmid.

[0164] Tn7 transfer vector In certain embodiments, the transfer vector comprises a foreign sequence to be inserted into the mini-attTn7 of the recombinant bacmid. In certain embodiments, the transfer vector is a Tn7 transfer vector. In certain embodiments, the transfer vector is a mini-Tn7 transfer vector. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 (Tn7L and Tn7R) that can mediate the transfer of a foreign sequence into the mini-attTn7 of the recombinant bacmid.

[0165] In certain exemplary embodiments, for the purpose of making gene therapy vectors, Tn7 transfer vectors contain coding sequences for AAV or non-AAV Rep. In some embodiments, Rep expression constructs are provided, in which the rep gene is a non-AAV or AAV gene cloned under the control of a baculovirus gene promoter in pFastBac1 transfer vector (Invitrogen). In certain embodiments, the baculovirus gene promoter is the promoter for the immediate early (ie1). In certain embodiments, the baculovirus gene promoter is the promoter for the polyhedrin gene. Suitable baculovirus gene promoters are known to those skilled in the art. In certain embodiments, the suitable baculovirus gene promoter is an immediate early, early, late, or very late gene promoter.

[0166] In certain embodiments, the Rep coding sequence is modified such that the standard start codon of Rep78 is modified to a non-standard start codon. In certain embodiments, the other AUG codon before the start codon of Rep52 is mutated to tolerate either a silent mutation (if out of frame) or to encode a conservative amino acid substitution (if in frame) to allow expression of Rep78 and Rep52 polypeptides from a single mRNA transcript by a leaky ribosomal scanning mechanism.

[0167] In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 (Tn7L and Tn7R) adjacent to a baculovirus gene promoter operably linked to a Rep gene. In certain embodiments, the Rep gene is selected from the group consisting of B19 Rep, GPV Rep, HBoV1, and AAV2 Rep. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 adjacent to a polyhedrin promoter operably linked to B19 Rep as shown in Figures 3A and 3B. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 adjacent to an ie1 promoter operably linked to B19 Rep as shown in Figures 3A and 3B. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 adjacent to a polyhedrin promoter operably linked to GPV Rep as shown in Figures 3C and 3D. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 flanked by the polyhedrin promoter operably linked to AAV2 Rep, as shown in Figures 3E and 3F. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 flanked by the polyhedrin promoter operably linked to HBoV1 Rep (data not provided).

[0168] Cre-LoxP transfer donor vector In certain embodiments, the transfer vector is a Cre-LoxP transfer donor vector. In certain embodiments, the Cre-LoxP transfer donor vector is a nucleic acid vector that comprises a first origin of replication for propagating the nucleic acid vector in a first bacterial strain, where the first origin of replication is a conditional origin of replication; a second origin of replication for propagating the nucleic acid vector in a second bacterial strain; a multiple cloning site for inserting a foreign sequence (e.g., a heterologous sequence); a selectable marker sequence; a reporter gene; and / or a selective target site that can mediate a site-specific recombination event. In certain embodiments, the first origin of replication is conditional on the presence of a π-protein, such as an R6Kγ origin of replication. In certain embodiments, the first origin of replication is an R6Kγ origin of replication. Thus, in certain embodiments, the first bacterial strain comprises a π-protein. In certain embodiments, the second origin of replication is a pUC57 origin of replication. In certain embodiments, the selective target site that can mediate site-specific recombination events comprises LoxP sites or variants thereof, and the site-specific recombination events are mediated by Cre recombinase. The Cre-LoxP transfer vector described herein can have any other elements suitable for appropriate identification of the host cell that contains the vector. For example, any selectable marker sequence and / or reporter gene known to those skilled in the art can be incorporated into the Cre-LoxP transfer vector described herein.

[0169] In certain embodiments, the Cre-LoxP transfer vector is a pUC57 vector containing LoxP recombination sites, an enhanced GFP (eGFP) marker gene in the presence of the AcMNPV ie1 promoter preceded by the transcriptional enhancer hr5 element followed by the AcMNPV p10 polyadenylation signal, an ampicillin resistance marker, a conditional R6Kγ origin of replication, and a multiple cloning site. Features of the Cre-LoxP transfer vector include: 1) a multiple cloning site (MCS) for inserting transgenes; 2) LoxP sites for Cre-mediated in vitro or in vivo recombination; 3) two origins of replication including: i) ColE1 Ori for propagating the vector in E. coli strains such as DH5α, NEB Stable, PMC103, and DH10B; and ii) a conditional R6KγOri for propagating the vector in E. coli strains expressing π (pi) proteins such as pir+ and pir116; 4) an ampicillin antibiotic resistance gene for selecting recombinant bacmids with kanamycin and ampicillin after inserting the Cre-LoxP donor vector into the LoxP sites of BIVVBac by Cre-mediated recombination; and 5) an enhanced GFP reporter gene for identifying the stability of the transgene in the recombinant BEV while replicating in insect cells in serial passages. It will be understood by one of skill in the art that a multiple cloning site may or may not be maintained in a Cre-LoxP transfer vector containing an exogenous sequence (e.g., a transgene) depending on how the exogenous sequence was inserted therein (e.g., by using terminal or non-terminal restriction sites, or the nature of the restriction enzyme used to clone the exogenous sequence therein).

[0170] In certain exemplary embodiments, for the purpose of making gene therapy vector, Cre-LoxP transfer vector comprises foreign sequence (e.g., heterologous sequence).For example, in certain embodiments, Cre-LoxP transfer vector comprises therapeutic protein-coding gene flanked by symmetric or asymmetric AAV or non-AAV inverted terminal repeat (ITR).In certain embodiments, therapeutic protein-coding gene flanked by symmetric or asymmetric AAV or non-AAVITR is present at the multiple cloning site position of Cre-LoxP transfer vector.

[0171] It will be recognized by those skilled in the art that Tn7 transfer vector and Cre-LoxP transfer donor vector are examples of transfer vectors that can be used for inserting foreign sequences to generate recombinant bacmids of the present invention. In certain embodiments, the foreign sequences inserted using the transfer vectors described herein are interchangeable between transfer vectors. For example, for the purpose of generating a gene therapy vector, the Rep gene is introduced into the recombinant bacmid by a Tn7 transfer vector, and a therapeutic protein-encoding gene is introduced by a Cre-LoxP transfer vector. In another example, for the purpose of generating a gene therapy vector, the Rep gene is introduced into the recombinant bacmid by a Cre-LoxP transfer vector, and a therapeutic protein-encoding gene is introduced by a Tn7 transfer vector.

[0172] In certain embodiments, the Cre-LoxP transfer vector of the invention comprises a LoxP sequence and an enhanced green fluorescent protein (eGFP)-encoding gene and is used to clone a therapeutic protein-encoding gene (e.g., hFVIIIco6XTEN with flanking symmetric or asymmetric ITRs, where the ITRs are non-AAV or AAV ITRs).

[0173] In certain embodiments, a recombinant bacmid is provided in which a Rep-encoding gene is inserted into the mini-attTn7 site of the polyhedrin locus by transposition, and a therapeutic protein-encoding gene with symmetric or asymmetric ITRs is inserted into the LoxP site of the EGT locus by Cre-mediated recombination. In certain embodiments, the Rep gene and ITRs are non-AAV or AAV.

[0174] Thus, in certain embodiments, a recombinant bacmid is provided that comprises: a sequence encoding an AAV Rep (e.g., AAV2 Rep) inserted into LacZα or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα), where the inserted Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site that comprises heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated AAV 5' inverted terminal repeat from which it is derived; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated AAV 3' inverted terminal repeat.

[0175] In certain embodiments, a recombinant bacmid is provided that comprises: a sequence encoding a non-AAV Rep inserted into LacZα or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα), where the inserted Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site that comprises heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated non-AAV 5' inverted terminal repeat from a first parvovirus non-AAV genome; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated non-AAV 3' inverted terminal repeat from a second parvovirus non-AAV genome. In certain embodiments, the first and second parvovirus non-AAV genomes are different.

[0176] In certain embodiments, provided herein is a recombinant bacmid that comprises the following: a sequence encoding B19 Rep inserted into LacZα, or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα, where the inserted B19 Rep disrupts the reading frame of LacZα, or a functional portion thereof; and a multiple cloning site that comprises heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from parvovirus B19; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from parvovirus B19.

[0177] In certain embodiments, provided herein is a recombinant bacmid that comprises: a sequence encoding GPV Rep inserted into LacZα or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα), where the inserted GPV Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from GPV; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from GPV.

[0178] In certain embodiments, provided herein is a recombinant bacmid comprising: a sequence encoding AAV2 Rep inserted into LacZα or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα), where the inserted AAV2 Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from AAV2; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from AAV2.

[0179] In certain embodiments, provided herein is a recombinant bacmid comprising: a sequence encoding HBoV1 Rep inserted into LacZα or a functional portion thereof (e.g., inserted into a mini-attTn7 site within LacZα), where the inserted HBoV1 Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising heterologous sequences, the heterologous sequences comprising, from 5' to 3': a wild-type or truncated 5' inverted terminal repeat derived from HBoV1; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated 3' inverted terminal repeat derived from HBoV1.

[0180] In certain embodiments, a recombinant bacmid is provided that includes: a sequence encoding a non-AAV Rep inserted into LacZα or a functional part thereof (e.g., inserted into a mini-attTn7 site in LacZα), where the inserted Rep disrupts the reading frame of LacZα or a functional part thereof; and a multiple cloning site that includes a heterologous sequence, the heterologous sequence including, from 5' to 3': a wild-type or truncated non-AAV 5' inverted terminal repeat; a protein-encoding sequence; one or more expression control sequences operably linked to the protein-encoding sequence; and a wild-type or truncated non-AAV 3' inverted terminal repeat. In certain embodiments, the non-AAV Rep and the non-AAV inverted terminal repeat are selected from the ITRs of a non-AAV genome of the virus family Parvovirus. In certain embodiments, the 5'ITR and the 3'ITR are ITRs of the same parvovirus non-AAV genome. In certain embodiments, the 5'ITR and the 3'ITR are ITRs of different parvovirus non-AAV genomes. In such embodiments, the non-AAV Rep is at least one Rep of a parvovirus non-AAV genome. In such embodiments, one skilled in the art will be able to determine the most suitable Rep to be used in embodiments in which the 5'ITR and 3'ITR are ITRs of different parvovirus non-AAV genomes.

[0181] In certain embodiments, the recombinant bacmid comprises a wild-type or truncated AAV 5'ITR and a wild-type or truncated parvoviral non-AAV 3'ITR. In certain embodiments, the recombinant bacmid comprises a wild-type or truncated parvoviral non-AAV 5'ITR and a wild-type or truncated AAV 3'ITR. In such embodiments, one skilled in the art will be able to determine the most suitable Rep to be used in embodiments where the 5'ITR and 3'ITR are ITRs of different parvoviral genomes.

[0182] III. Foreign Sequences It will be understood by those skilled in the art that the baculovirus expression vector system described herein can be used to produce any desired product. For example, the baculovirus expression vector system described herein can be used to produce recombinant proteins or viral or non-viral vectors for gene therapy. Therefore, the following description of the production of nucleic acid molecules is not limiting in any way.

[0183] nucleic acid molecule In certain embodiments, the baculovirus expression vector system described herein can be used for the production of nucleic acid molecules. For example, the production of nucleic acid molecules for gene therapy. Thus, certain embodiments described herein relate to the production of plasmid-like capsid-free nucleic acid molecules that code for target sequences. Capsid, the protein shell of a virus, surrounds the genetic material of the virus. It is known that capsid protects the viral genome, delivers the genome to the host, and assists the function of virion by interacting with the host. However, viral capsid may limit the packaging capacity of vectors and / or cause immune reactions, especially when used in gene therapy.

[0184] AAV vector has emerged as one of the more common types of gene therapy vector.However, the existence of capsid limits the usefulness of AAV vector by gene therapy.In particular, capsid itself can limit the size of the transgene contained in vector to less than 4.5kb.Various therapeutic proteins that can be useful in gene therapy can easily exceed this size even before expression control sequence is added.

[0185] Furthermore, the proteins that compose the capsid can serve as antigens that can be targeted by the subject's immune system. AAV is very common in the general population, and most people are exposed to AAV throughout their lives. As a result, most potential gene therapy recipients will likely already have developed an immune response to AAV, and therefore are likely to reject this therapy.

[0186] In certain embodiments, the present invention is directed to the production of a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette, for example, a gene cassette encoding a therapeutic protein and / or a miRNA. In some embodiments, the first ITR and the second ITR are adjacent to a gene cassette comprising a heterologous polynucleotide sequence. In some embodiments, the nucleic acid molecule does not comprise a gene encoding a capsid protein, a replication protein, and / or an assembly protein. In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a clotting factor. In some embodiments, the gene cassette encodes a miRNA. In certain embodiments, the gene cassette is located between the first ITR and the second ITR. In some embodiments, the nucleic acid molecule further comprises one or more non-coding regions. In certain embodiments, the one or more non-coding regions comprise a promoter sequence, an intron, a post-transcriptional regulatory element, a 3'UTR poly(A) sequence, or any combination thereof.

[0187] In one embodiment, the gene cassette is a single-stranded nucleic acid. In another embodiment, the gene cassette is a double-stranded nucleic acid.

[0188] Some aspects of the present disclosure are directed to nucleic acid molecules comprising gene cassettes, e.g., gene cassettes encoding therapeutic proteins and / or miRNAs. In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a clotting factor. In some embodiments, the gene cassette encodes a miRNA. In some embodiments, the nucleic acid molecule further comprises at least one non-coding region. In certain embodiments, the at least one non-coding region comprises a promoter sequence, an intron, a post-transcriptional regulatory element, a 3'UTR poly(A) sequence, or any combination thereof.

[0189] 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 β-actin / rabbit β-globin intron, and has been modified to eliminate five existing ATG sequences to reduce false translation initiation. In certain embodiments, the intron sequence includes an SV40 small T intron. In some embodiments, the intron sequence is located 5' to 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. In some embodiments, the chimeric intron includes the nucleic acid sequence of SEQ ID NO: 23.

[0190] 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 double stranded nucleic acid (ceDNA).

[0191] 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 herein by reference in its entirety. In some embodiments, the gene cassette is a "hFVIIIco6XTEN" gene cassette described in International Application No. PCT / US2017 / 015879. In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding a B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII) fused with an XTEN 144 peptide.

[0192] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII driven by an mTTR promoter. In some embodiments, the mTTR promoter comprises the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the gene cassette further comprises an A1MB2 enhancer element. In some embodiments, the A1MB2 enhancer element comprises the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the gene cassette further comprises a chimeric or synthetic intron. In some embodiments, the chimeric intron consists of a chicken β-actin / rabbit β-globin intron, as well as modified to eliminate five existing ATG sequences to reduce false translation initiation. 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. In some embodiments, the chimeric intron comprises the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the gene cassette further comprises a woodchuck post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO:24. In some embodiments, the gene cassette further comprises a bovine growth hormone polyadenylation (bGHpA) signal. In some embodiments, the bGHpA signal comprises the nucleic acid sequence of SEQ ID NO:25. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:19. In some embodiments, the gene cassette comprises the nucleic acid sequence of SEQ ID NO:19.

[0193] In some embodiments, the gene cassette comprises a nucleotide sequence encoding a codon-optimized FVIII and XTEN peptide driven by a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) containing an A1MB2 enhancer element, a hybrid synthetic intron (chimeric intron), a woodchuck posttranscriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal. In some embodiments, the gene cassette comprises the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the gene cassette comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 19.

[0194] Inverted terminal repeats In certain embodiments, the 5'ITR and 3'ITR are adeno-associated virus (AAV) ITRs or non-AAV ITRs. In certain embodiments, the non-AAV ITRs are ITRs obtained from members of the virus family parvovirus. Suitable ITR sequences include AAV ITRs of AAV serotypes known to those skilled in the art. Suitable AAV and non-AAV ITR sequences are described in International Publication Nos. WO2019032898(A1), WO2020033863(A1), and WO2017152149(A1), the disclosures of which are incorporated herein by reference in their entirety. For example, the non-AAV ITR sequence may be derived from goose parvovirus (GPV) or parvovirus B19 (also referred to herein as "B19"). In some embodiments, the ITRs are not derived from the AAV genome. In some embodiments, the ITRs are non-AAV ITRs. In some embodiments, the ITRs are ITRs of a non-AAV genome of the virus family Parvovirus selected from the group consisting of, but not limited to, 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 erythrovirus parvovirus B19 (a human virus; also referred to herein as "B19"). In another embodiment, the ITRs are derived from a Muscovy duck parvovirus (MDPV) strain. In certain embodiments, the MDPV strain is attenuated, e.g., MDPV strain FZ91-30. In other embodiments, the MDPV strain is pathogenic, e.g., MDPV strain YY. In some embodiments, the ITRs are derived from a porcine parvovirus, such as porcine parvovirus U44978. In some embodiments, the ITRs are derived from a minute virus of mice, such as minute virus of mice U34256.In some embodiments, the ITRs are derived from a canine parvovirus, such as canine parvovirus M19296. In some embodiments, the ITRs are derived from a mink enteritis virus, such as mink enteritis virus D00765. In some embodiments, the ITRs are derived from a Dependoparvovirus. In one embodiment, the Dependoparvovirus is a Dependovirus goose parvovirus (GPV) strain. In a particular embodiment, the GPV strain is attenuated, such as GPV strain 82-0321V. In another particular embodiment, the GPV strain is pathogenic, such as GPV strain B. Examples of suitable parvovirus ITR sequences are set forth in Table 1.

[0195] [Table 1-1] [Table 1-2]

[0196] Therapeutic Proteins In some embodiments, the present invention provides a nucleic acid molecule comprising a gene cassette that encodes a first ITR, a second ITR, and a target sequence, the gene cassette encoding the target sequence encoding a therapeutic protein. In some embodiments, the gene cassette encodes one therapeutic protein. In some embodiments, the gene cassette encodes two or more therapeutic proteins. 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.

[0197] 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. Any therapeutic protein can be produced by the baculovirus expression vector system of the present disclosure, including but not limited to the production of coagulation factors. In some embodiments, the clotting 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 clotting 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 coagulation factor comprises VWF or a variant or fragment thereof.

[0198] Growth factors In some embodiments, the present disclosure provides a nucleic acid molecule that comprises a gene cassette that encodes a first ITR, a second ITR, and a target sequence, the gene cassette being a target sequence that encodes a therapeutic protein, and the therapeutic protein comprises a growth factor.The growth factor can be selected from any growth factor known in the art.In some embodiments, the growth factor is a hormone.In other embodiments, the growth factor is a cytokine.In some embodiments, the growth factor is a chemokine.

[0199] 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 ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, and ephrin B3. In some embodiments, the growth factor is erythropoietin (EPO). In some embodiments, the growth factor is fibroblast growth factor (FGF). In some embodiments, the FGF is selected from FGF1, FGF2, 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 growth hormone (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 (GDF-9). 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 neurotrophin. 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 neurotrophin 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 a 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).

[0200] MicroRNA (miRNA) MicroRNAs (miRNAs) are small non-coding RNA molecules (approximately 18-22 nucleotides) that negatively regulate gene expression by either inhibiting translation or inducing messenger RNA (mRNA) degradation. Since their discovery, miRNAs have been implicated in various cellular processes, such as apoptosis, differentiation, and cell proliferation, as well as they have been found to play important roles in carcinogenesis. The ability of miRNAs to regulate gene expression makes miRNA expression a valuable tool in gene therapy in vivo.

[0201] In some embodiments, the present disclosure provides a nucleic acid molecule that comprises a gene cassette that encodes a first ITR, a second ITR, and a target sequence, wherein the target sequence encodes miRNA, and the first ITR and / or the second ITR are non-adeno-associated virus ITRs (e.g., the first ITR and / or the second ITR are non-AAV derived).The miRNA can be any miRNA known in the art.In some embodiments, the miRNA downregulates the expression of target gene.In certain embodiments, the target gene is selected from SOD1, HTT, RHO, or any combination thereof.

[0202] In some embodiments, the gene cassette encodes one miRNA. In some embodiments, the gene cassette encodes two or more miRNAs. In some embodiments, the gene cassette encodes two or more different miRNAs. In some embodiments, the gene cassette encodes two or more copies of the same miRNA. In some embodiments, the gene cassette encodes two or more variants of the same therapeutic protein. In certain embodiments, the gene cassette encodes one or more miRNAs and one or more therapeutic proteins.

[0203] In some embodiments, the miRNA is a naturally occurring miRNA. In some embodiments, the miRNA is an engineered miRNA. In some embodiments, the miRNA is an artificial miRNA. In certain embodiments, the miRNA comprises the miHTT engineered miRNA disclosed by Evers et al., Molecular Therapy 26(9):1-15 (epub ahead of print June 2018). In certain embodiments, the miRNA comprises the miR SOD1 artificial miRNA disclosed by Dirren et al., Annals of Clinical and Translational Neurology 2(2):167-84 (February 2015). In certain embodiments, the miRNA comprises miR-708, which targets RHO (Behrman et al., JCB 192(6):919-27 (2011).

[0204] In some embodiments, the miRNA upregulates the expression of a gene by downregulating the expression of an inhibitor of the gene. In some embodiments, the inhibitor is a natural, e.g., wild-type, inhibitor. In some embodiments, the inhibitor is the result of a mutated, heterologous, and / or misexpressed gene.

[0205] Expression control elements In some embodiments, the nucleic acid molecule or vector produced by the baculovirus expression vector system described herein further comprises at least one expression control sequence.As used herein, an expression control sequence is any regulatory nucleotide sequence that promotes the efficient transcription and translation of the coding nucleic acid to which the sequence is operably linked, such as a promoter sequence or a promoter-enhancer combination.For example, the isolated nucleic acid molecule produced by the method of the present disclosure can be operably linked to at least one transcription control sequence.

[0206] The gene expression control sequence may be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, as well as other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters derived from cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus.

[0207] Other constitutive promoters are known to those skilled in the art. The promoters useful as gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of inducers. For example, metallothionein promoters are induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0208] 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 liver cells. Examples of liver-specific promoters include, but are not limited to, the mouse transthyretin promoter (mTTR), the native human factor VIII promoter, the native human factor IX promoter, the human alpha-1-antitrypsin promoter (hAAT), the human albumin minimal promoter, and the mouse albumin promoter. In certain embodiments, the promoter comprises the mTTR promoter. The mTTR promoter is described in RH Costa et al., 1986, Mol. Cell. Biol. 6:4697. The F8 promoter is described in Figueiredo and Brownlee, 1995, J. Biol. Chem. 270:11828-11838. In certain embodiments, the promoter comprises any of the mTTR promoters (e.g., mTTR202 promoter, mTTR202opt promoter, mTTR482 promoter) as disclosed in U.S. Patent Application Publication No. 2019 / 0048362, which is incorporated by reference in its entirety.

[0209] In some embodiments, the nucleic acid molecule comprises a tissue-specific promoter. In certain embodiments, the tissue-specific promoter drives the expression of a therapeutic protein, such as a clotting factor, in the liver, for example, in hepatocytes and / or endothelial cells. In certain embodiments, the promoter is selected from the group consisting of mouse transthyretin promoter (mTTR), native human factor VIII promoter, human alpha-1-antitrypsin promoter (hAAT), human albumin minimal promoter, mouse albumin promoter, tristetraprolin (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, and any combination thereof. In some embodiments, the promoter is selected from a liver-specific promoter (e.g., alpha 1-antitrypsin (AAT)), a muscle-specific promoter (e.g., muscle creatine kinase (MCK), myosin heavy chain alpha (αMHC), myoglobin (MB), and desmin (DES)), a synthetic promoter (e.g., SPc5-12, 2R5Sc5-12, dMCK, and tMCK), and any combination thereof.

[0210] The expression level can be further increased to achieve therapeutic efficiency using one or more enhancers. One or more enhancers can be provided either alone or together with one or more promoter elements. Typically, the expression control sequence includes multiple enhancer elements and tissue-specific promoters. In one embodiment, the enhancer includes one or more copies of the alpha-1-microglobulin / bikunin enhancer (Rouet et al., 1992, J. Biol. Chem. 267:20765-20773; Rouet et al., 1995, Nucleic Acids Res. 23:395-404; Rouet et al., 1998, Biochem. J. 334:577-584; Ill et al., 1997, Blood Coagulation Fibrinolysis 8:S23-S30). In another embodiment, the enhancer is derived from a liver-specific transcription factor binding site, e.g., EBP, DBP, HNF1, HNF3, HNF4, HNF6, etc., with Enh1 including HNF1, (sense)-HNF3, (sense)-HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4 (antisense).

[0211] In a particular embodiment, a promoter useful for the present disclosure is the ET promoter, also known as GenBank number AY661265. See also Vigna et al., Molecular Therapy 11(5):763 (2005). Other examples of suitable vectors and expression control sequences are described in WO 02 / 092134, EP 1395293, or U.S. Patent Nos. 6,808,905, 7,745,179, or 7,179,903, which are incorporated herein by reference in their entirety.

[0212] Generally, the expression control sequence shell optionally includes 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed sequences will include a promoter region that includes a promoter sequence for transcriptional control of an operably linked coding nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream activator sequences, as desired.

[0213] In certain embodiments, a nucleic acid molecule generated by the baculovirus expression vector system described herein comprises one or more miRNA target sequences, for example, operably linked to a transgene.

[0214] In some embodiments, the target sequence is the miR-223 target, which has been reported to block expression most effectively in myeloid progenitor cells and at least partially in more primitive HSPCs.The miR-223 target can block expression in differentiated myeloid cells, such as granulocytes, monocytes, macrophages, myeloid dendritic cells, etc.The miR-223 target can also be suitable for gene therapy applications that rely on robust transgene expression in lymphoid or erythroid lineages.The miR-223 target can also block expression very effectively in human HSCs.

[0215] In some embodiments, the target sequence is a miR-142 target. In some embodiments, a complementary sequence of a hematopoietic-specific microRNA, such as miR-142 (142T), is incorporated into a nucleic acid molecule containing a transgene, rendering the transcript encoding the transgene susceptible to miRNA-mediated downregulation. By this method, expression of the transgene can be prevented in hematopoietic antigen-presenting cells (APCs) while maintaining it in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose stringent post-transcriptional control on transgene expression, resulting in stable delivery and long-term expression of the transgene. In some embodiments, miR-142 regulation prevents immune-mediated clearance of transduced cells and / or induces antigen-specific regulatory T cells (T regs), mediating robust immune tolerance to the transgene-encoded antigen.

[0216] In some embodiments, the target sequence is a miR181 target. Chen CZ and Lodish H, Seminars in Immunology (2005) 17(2):155-165, discloses miR-181, a miRNA that is specifically expressed in B cells in mouse bone marrow (Chen and Lodish, 2005). It has also been disclosed that some human miRNAs are associated with leukemia.

[0217] The target sequence may be fully or partially complementary to the miRNA. The term "fully complementary" means that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes the target sequence. The term "partially complementary" means that the target sequence is only partially complementary to the sequence of the miRNA that recognizes the target sequence, so that the partially complementary sequence is still recognized by the miRNA. In other words, a partially bilateral target sequence in the context of the present disclosure is effective in performing the recognition of the corresponding miRNA and preventing or reducing the expression of a transgene in cells that express the miRNA. Examples of miRNA target sequences are described in WO2007 / 000668, WO2004 / 094642, WO2010 / 055413, or WO2010 / 125471, which provisional patent applications are incorporated herein by reference in their entirety.

[0218] different parts In some embodiments, the transgene encodes a heterologous amino acid sequence. The heterologous amino acid sequence can be linked to the transgene product. In some embodiments, the heterologous amino acid sequence can be linked to the N-terminus or C-terminus of the transgene product. For example, for a transgene encoding FVIII, the heterologous amino acid sequence can be linked to the N-terminus or C-terminus of the FVIII amino acid, or can be inserted between two amino acids in the FVIII amino acid sequence. In some embodiments, the heterologous amino acid sequence can be inserted into the FVIII polypeptide at any site disclosed in WO 2013 / 123457 (A1) and WO 2015 / 106052 (A1) or US Patent Publication No. 2015 / 0158929 (A1), which are incorporated herein by reference in their entirety.

[0219] In some embodiments, the heterologous amino acid sequence is inserted within the B domain of FVIII or a fragment thereof.

[0220] In some embodiments, the heterologous moiety comprises one or more XTEN sequences, fragments, variants, or derivatives thereof. As used herein, "XTEN sequence" refers to a polypeptide that is extended in length by a non-naturally occurring, substantially non-repetitive sequence that is primarily composed of small hydrophilic amino acids, where the sequence has no or only a low degree of secondary or tertiary structure under physiological conditions. As a heterologous moiety, XTEN can function as a half-life extension moiety. In addition, XTEN can provide desirable properties, such as, but not limited to, enhanced pharmacokinetic parameters and solubility properties.

[0221] In some embodiments, XTEN sequences useful for this disclosure are peptides or polypeptides having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or polypeptide having from about 20 to more than about 3000 amino acid residues, from 30 to more than about 2500 residues, from 40 to more than about 2000 residues, from 50 to more than about 1500 residues, from 60 to more than about 1000 residues, from 70 to more than about 900 residues, from 80 to more than about 800 residues, from 90 to more than about 700 residues, from 100 to more than about 600 residues, from 110 to more than about 500 residues, or from 120 to more than about 400 residues. In a particular embodiment, XTEN is a peptide or polypeptide having an amino acid sequence of more than 42 and less than 144 amino acids in length. Includes.

[0222] XTEN sequences of the present disclosure can include one or more sequence motifs of 5-14 (e.g., 9-14) amino acid residues, or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence motif, where the motif comprises, consists essentially of, or consists of 4-6 types of amino acids (e.g., 5 amino acids) selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). Examples of XTEN sequences that can be used as heterologous moieties in the chimeric proteins of the present disclosure are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0239554(A1), 2010 / 0323956(A1), 2011 / 0046060(A1), 2011 / 0046061(A1), 2011 / 0077199(A1), or 2011 / 0172146(A1). ), or in International Publication Nos. WO 2010 / 091122(A1), 2010 / 144502(A2), 2010 / 144508(A1), 2011 / 028228(A1), 2011 / 028229(A1), or 2011 / 028344(A2), each of which is incorporated by reference in its entirety.

[0223] IV.Host cells Suitable host cells are known to those skilled in the art. By "host cell" is meant any cell that carries or is capable of carrying any substance of interest.

[0224] In some embodiments, the host cells suitable for use in the present invention are cells of insect origin. In some embodiments, suitable insect host cells include, for example, cell lines isolated from Spodoptera frugiperda (Sf) or cell lines isolated from Trichoplusia ni (Tni). A person skilled in the art would be able to easily identify the suitability of any Sf or Tni cell line. Examples of insect host cells include, but are not limited to, Sf9 cells, Sf21 cells, High Five™ cells. Examples of insect host cells include, but are not limited to, any Sf or Tni cell line free of adventitious virus contamination, such as Sf-rhadovirus negative (Sf-RVN) and Tn-nodavirus negative (Tn-NVN) cells. Other suitable host insect cells are known to those skilled in the art. In one particular embodiment, the insect host cell is an Sf9 cell.

[0225] In some embodiments, the host cell suitable for use in the present invention is a cell of bacterial origin. In some embodiments, the suitable bacterial host cell can be any bacterial host cell known to those skilled in the art. In certain embodiments, the suitable bacterial host cell cannot unravel cruciform DNA structures. In certain embodiments, the suitable bacterial host strain comprises a disruption in the SbcCD complex. In some embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene and / or the SbcD gene. In certain embodiments, the disruption in the SbcCD complex comprises a gene disruption in the SbcC gene. Various bacterial host strains comprising a gene disruption in the SbcC gene are known in the art. For example, but not limited to, bacterial host strain PMC103 comprises the genotypes sbcC, recD, mcrA, ΔmcrBCF; bacterial host strain PMC107 comprises the genotypes recBC, recJ, sbcBC, mcrA, ΔmcrBCF; and bacterial host strain SURE comprises the genotypes recB, recJ, sbcC, mcrA, ΔmcrBCF, umuC, uvrC.

[0226] In some embodiments, suitable host cells for use in the present invention are cells of mammalian origin, such as human origin. Those skilled in the art are recognized for their ability to preferentially determine the particular host cell line most suitable for their purposes. Exemplary host cell lines include, but are not limited to, CHO, DG44, and DUXB11 (Chinese hamster ovary line, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with the SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), PER.C6®, NS0, CAP, BHK21, and HEK293 (human kidney).

[0227] Introduction of the vector of the present disclosure into a host cell can be accomplished by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (e.g., 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, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988).

[0228] The host cells containing the vector of the present disclosure are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium that contains nutrients necessary for cell growth. The nutrients necessary for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the medium may contain one or more selection factors. Optionally, the medium may contain calf serum or fetal calf serum (FCS). Generally, the growth medium will select for cells containing the vector, for example, by drug selection or deprivation of essential nutrients that are supplemented by the selectable marker of the vector.

[0229] V. How to use The baculovirus expression vector system provided herein is used in the production of products encoded by foreign sequences inserted into the recombinant bacmids described herein. Scalable production of products can be achieved by several approaches known in the art.

[0230] One approach involves the infection of suitable insect host cells that support the growth of baculovirus. In certain embodiments, the recombinant bacmid containing the foreign sequence described herein is first propagated in a suitable bacterial host cell (e.g., E. coli). The recombinant bacmid is then isolated from the bacterial host cell and transfected into a suitable insect host cell using a suitable transfection reagent (e.g., CELLFECTIN). The insect host cell produces recombinant baculovirus particles, which can then infect host insect cells for viral amplification of the foreign sequence.

[0231] In certain embodiments, the present invention provides a method for producing a product encoded by a foreign sequence, comprising transfecting the recombinant bacmid described herein into a suitable insect cell under suitable conditions to produce a recombinant baculovirus; and infecting a second suitable insect cell with the recombinant baculovirus under suitable conditions to produce a product encoded by a foreign sequence.In certain embodiments, for the purpose of producing gene therapy, the recombinant bacmid comprises a Rep coding sequence and a protein coding sequence flanked on both sides by ITRs.

[0232] In certain embodiments, provided herein is a method for producing a nucleic acid molecule, comprising transfecting a recombinant bacmid as described herein into a suitable insect cell under suitable conditions to produce a recombinant baculovirus; and infecting a second suitable insect cell with the recombinant baculovirus under suitable conditions to produce a nucleic acid molecule.In certain embodiments, provided herein is a method for producing a ceDNA, comprising transfecting a recombinant bacmid as described herein into a suitable insect cell under suitable conditions to produce a recombinant baculovirus; and infecting a second suitable insect cell with the recombinant baculovirus under suitable conditions to produce a ceDNA.

[0233] In another approach, a stable cell line can be generated by stably integrating a protein encoding sequence under the control of a baculovirus gene promoter (e.g., a baculovirus constitutive gene promoter). In certain embodiments, the stable cell line is a stable insect cell line. Stable integration of the sequence can be performed by any method known to those skilled in the art. Methods for stable integration of nucleic acids into various host cell lines are known in the art (see the Examples below for a more detailed description of exemplary producer cell lines created by stable integration of nucleic acids). For example, recursive selection (e.g., by using a selectable marker) can be used to select cells that have integrated a nucleic acid containing a selectable marker (and an AAV cap and rep gene and / or rAAV genome). In other embodiments, the nucleic acid is integrated into the cell line in a site-specific manner to generate a producer cell line. Several site-specific recombination systems are known in the art, such as, for example, 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).

[0234] In the stable cell line approach, in one embodiment, the BEV encoding the complement protein required for proper expression of the protein encoding sequence is introduced into the stable cell line. In certain embodiments, for the purpose of producing gene therapy, the stable cell line contains a therapeutic protein-encoding gene adjacent to symmetric or asymmetric AAV or non-AAVITR stably integrated therein. The BEV containing the encoding suitable Rep is then introduced into the stable cell line under the necessary conditions for the production of gene therapy.

[0235] Methods for generating specific stable cell lines are described herein (see Example 8). Examples of specific stable cell lines for making ceDNA include cell lines with the plasmid shown in FIG. 8B stably integrated therein. These stable cell lines are listed in Table 5. In one particular method, to make ceDNA, a BEV encoding a suitable Rep is introduced into the stable cell line under the conditions required for making ceDNA.

[0236] In yet another approach, the production of the product encoded by the foreign sequence can be achieved using a stable cell line in a baculovirus-free manner. In certain embodiments, for the purpose of producing gene therapy, the stable cell line contains a therapeutic protein-encoding gene flanked by symmetric or asymmetric AAV or non-AAV ITRs stably integrated therein. In certain embodiments, the production of baculovirus-free in a stable cell line includes transient expression of Rep protein in the stable cell line under the control of a baculovirus promoter. Suitable baculovirus gene promoters are known to those of skill in the art. In certain embodiments, the baculovirus gene promoter is the immediate early (ie) gene promoter of Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). In certain embodiments, the baculovirus gene promoter is the OpIE2 promoter of OpMNPV. Various methods of mediating transient gene expression are known to those of skill in the art. In certain embodiments, transient gene expression can be achieved by polyethyleneimine (PEI)-mediated transfection.

[0237] The downstream purification of the product involves any method known to those skilled in the art.For example, for viral or non-viral vectors for gene therapy, the target is purified by a plasmid DNA isolation kit that includes a silica-based column that separates low molecular weight DNA from RNA, high molecular weight DNA, proteins, and other impurities by ion exchange chromatography.

[0238] All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0239] 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.

[0240] Having generally described the present disclosure, a further understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0241] BIVVBac Precursor Bacmid Among expression vectors, baculoviruses stand out due to their extra-large gene-carrying capacity of up to several tens of kb, with some reports of 100 kb. This transgene capacity has been exploited for the generation of recombinant AAV vectors (up to 38 kb expression cassettes). It was speculated that the large transgene-carrying capacity of baculoviruses could also be exploited for the generation of DNA therapeutic agents for non-viral gene therapy. Therefore, a novel versatile baculovirus shuttle vector (bacmid) was created, specifically designed to accommodate a larger number of transgenes than can be achieved by existing bacmid tools. This versatile bacmid (called "BIVVBac") could be used for the generation of rAAV vectors for in vivo gene therapy, as well as for the production of any desired protein, e.g., recombinant protein.

[0242] The BIVVBac bacmid was derived from bMON14272 (Invitrogen) encoding the AcMNPV C6 genome (Figure 1A) and engineered to encode two insertion sites: 1) a mini-attTn7 at the polyhedrin locus, and 2) a LoxP at the EGT locus. The mini-attTn7 insertion sequence is fused in frame with a Lac-promoter-driven E. coli LacZα fragment for X-gal-mediated blue / white screening of recombinant bacmids after Tn7-mediated transposition, as well as a LoxP site for Cre-mediated in vitro or in vivo recombination. The LoxP recombination site was inserted into bMON14272, which contains the mini-attTn insertion sequence at the polyhedrin locus, in two steps. First, a plasmid consisting of the Rudolph red fluorescent protein (RFP) gene in the presence of the AcMNPV 39K promoter followed by the ets polyadenylation signal, LoxP recombination sites, and sequences flanking the AcMNPV EGT locus was synthesized by GenScript® (Piscataway, NJ) (FIG. 1B) (SEQ ID NO: 9). The synthetic plasmid DNA was then co-transfected with AvrII-linearized bMON14272 in Sf9 cells (ATCC® CRL-1711) using Cellfectin (Invitrogen) transfection reagent according to the manufacturer's instructions. The RFP expression cassette and LoxP sequences were recombined at the EGT locus by homologous recombination in Sf9 cells. Progeny viruses generated in cell-free supernatants were plaque purified, and RFP+ plaques were amplified in Sf9 cells to generate P1 (passage 1) viruses. Baculovirus DNA was then isolated from the P1 virus and used as a template for PCR, which used internal and external primers from the transfer plasmid to amplify the region spanning the predicted recombination junction, and the resulting amplimers were sequenced. The recombinant virus was further amplified and used for viral DNA isolation, then transformed into E. coli DH10B bacteria (O'Reilly et al., 1992), followed by selection with kanamycin, X-Gal, and IPTG.Bacmid DNA isolated from the kanamycin-resistant blue E. coli clone was analyzed by restriction enzyme mapping and named "BIVVBac" (Figure 1C). A helper plasmid from pMON7124 (Invitrogen), encoding the Tn7 transposase genes tnsA-E and tetracycline resistance, was transformed into DH10B E. coli carrying BIVVBac, followed by selection with kanamycin and tetracycline. One of the double-resistant clones was used to generate electro-competent cells, as previously described (Sharma and Schimke, 1996), and the resulting new E. coli strain was transformed with BIVVBac. DH10B (FIG. 1D). This strain was then used to insert AAV2, B19, or GPV replication (Rep) protein genes into the polyhedrin locus by Tn7 transposition, as described below. EXAMPLES

[0243] Cre-LoxP donor vector A DNA construct consisting of LoxP recombination sites, an enhanced GFP (eGFP) marker gene in the presence of the AcMNPV ie1 promoter preceded by a transcription enhancer hr5 element and followed by an AcMNPV p10 polyadenylation signal, an ampicillin resistance marker, a conditional R6Kγ origin of replication, and a multiple cloning site was synthesized by GenScript® (Piscataway, NJ) (SEQ ID NO: 10) (FIG. 2A). This synthetic DNA was cloned into a pUC57 vector, and the resulting construct was named "Cre-LoxP donor vector" (FIG. 2B). The key features of the Cre-LoxP donor vector are: (1) a multiple cloning site (MCS) for inserting transgenes; (2) a LoxP site for Cre-mediated in vitro or in vivo recombination; (3) two origins of replication: (3a) ColE1 Ori for propagating the vector into E. coli strains, such as DH5α, NEB Stable, PMC103, and DH10B; and (3b) a conditional R6KγOri for propagating the vector into E. coli strains expressing π protein, such as pir+ and pir116; (4) an ampicillin antibiotic resistance gene for selecting recombinant bacmids with kanamycin and ampicillin after inserting the Cre-LoxP donor vector into the LoxP site of “BIVVBac” by Cre-LoxP recombination; and (5) an enhanced GFP reporter gene for identifying the stability of the transformed gene in the recombinant BEV while replicating in Sf9 cells in serial passages. EXAMPLES

[0244] Replication (Rep) protein expression construct B19.Rep: The coding sequence for B19 Rep was obtained from B19 strain HV (GenBank accession number AF162273) and the wild-type sequence was synthesized by GenScript® (Piscataway, NJ) (SEQ ID NO: 11) (FIG. 3A). The synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector in the presence of the AcMNPV polyhedrin promoter and a gentamicin-resistant clone was used to generate the pFastBac.Polh.B19.Rep transfer vector. This vector was transformed with BIVVBac DH10BRecombinant BEV, AcBIVVBac.Polh.B19.RepTn7, was generated by transforming it into E. coli. The titered BEV was then used for infection in a polyclonal Sf cell line encoding hFVIIIco6XTEN with symmetric B19 ITRs, and the ceDNA vector was purified from the infected cell pellet as described below. Preliminary results revealed that the polyhedrin promoter-driven B19.Rep was unable to "rescue" ceDNA. We hypothesized that the high level of unspliced ​​B19.Rep expression at the very late phase of the baculovirus infection cycle is inefficient and / or toxic to cells. To this hypothesis, the pFastBac.IE1.B19.Rep transfer vector was generated by replacing the polyhedrin promoter with the AcMNPV-constitutive immediate early (ie1) promoter (Figure 3B). Using this vector, recombinant BEV, AcBIVVBac.IE1.B19.Rep Tn7 were generated and then tested in a polyclonal Sf cell line encoding hFVIIIco6XTEN with symmetric B19 ITRs. The results showed that AcMNPV-constitutive ie1-driven B19.Rep was able to rescue ceDNA vectors from both cell lines tested compared to polyhedrin-driven Rep. This data suggests that stoichiometric expression of Rep protein is important for ceDNA production in Sf9 cells, as previously observed for rAAV production.

[0245] GPV.Rep: The coding sequence of GPV Rep was obtained from GPV strain B (GenBank accession number GPU25749). Unlike B19, GPV.Rep produces spliced ​​proteins (Rep78 and Rep52) from a single mRNA transcript, and the Rep78 coding sequence was genetically modified to allow expression of Rep78 and Rep52 polypeptides from a single mRNA species by a leaky ribosomal scanning mechanism to achieve stoichiometric expression of both proteins. The AUG start codon of the Rep78 open reading frame (orf), the adjacent proline codon, and the 12 downstream AUG triplets occurring before the start codon of the Rep52orf were altered by gene synthesis. The Rep78 start codon and adjacent nucleotides were mutated to an inefficient translation initiation signal consisting of a CUG triplet shown in the context of the Kozak consensus sequence. The AUG triplet occurring between the start codon of Rep78 orf and the AUG start codon of Rep52 orf was modified to tolerate either silent mutations (in the case of out-of-frame AUG codons) or to code for conservative amino acid substitutions (in the case of in-frame AUG codons). Finally, the modified GPV.Rep coding sequence was synthesized by GenScript® (SEQ ID NO: 12) (Figure 3C) and the synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector in the presence of the AcMNPV polyhedrin promoter to generate the pFastBac.Polh.GPV.Rep transfer vector (Figure 3C). This vector was then transformed into the BIVVBac DH10B The recombinant BEV, AcBIVVBac.Polh.GPV.Rep was transformed into E. coli. Tn7 was prepared.

[0246] AAV2.Rep: The coding sequence of AAV2 Rep was obtained from the AAV2 genome (GenBank accession number NC_001401). Unlike B19.Rep, AAV2.Rep also produces spliced ​​proteins (Rep78 and Rep52) from a single mRNA transcript, and the Rep78 coding sequence was genetically modified to allow expression of Rep78 and Rep52 polypeptides from a single mRNA transcript by a leaky ribosomal scanning mechanism to achieve stoichiometric expression of both proteins, as previously described (Smith et al., 2009) (Figure 3E). The modified AAV2.Rep coding sequence was synthesized by GenScript® (SEQ ID NO: 13) (Figure 3E), and the synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector in the presence of the AcMNPV polyhedrin promoter to generate the pFastBac.Polh.AAV2.Rep transfer vector (Figure 3F). This vector was then transformed into BIVVBac DH10B The recombinant BEV, AcBIVVBac.Polh.AAV2.RepTn7, was generated by transformation into E. coli. EXAMPLES

[0247] Human FVIIIco6XTEN expression construct Human FVIIIco6XTEN expression constructs with non-AAV or AAV ITRs: Gene constructs containing codon-optimized human FVIII with XTEN 144 peptide (FVIIIco6XTEN) in the presence of liver-specific TTPp promoter, woodchuck posttranscriptional regulatory element (WPRE), bovine growth hormone polyadenylation (bGHpA) signal and non-AAV or AAV truncated flanking ITRs were described in WO2019032898A1. These constructs, created here by replacing either the 5' or 3' ITRs with non-AAV or AAV wild-type sequences, were tested in parallel with new constructs (Table 2). These new constructs were cultured and maintained in E. coli strain PMC103, which contains a deleted sbcC gene that encodes an exonuclease that recognizes and eliminates cruciform DNA structures. PMC103 E. coli was able to support the growth of hFVIIIco6XTEN constructs with non-AAV or AAV symmetric or asymmetric ITRs after optimizing temperature and bacterial culture growth conditions. All new constructs were selected on ampicillin resistance plates and screened by restriction enzyme mapping to determine the correct genetic structure. The hFVIIIco6XTEN expression cassette constructs were then used to generate Cre-LoxP donor vectors.

[0248] [Table 2]

[0249] Human FVIIIco6XTEN Cre-LoxP donor vector with non-AAV or AAV ITRs The hFVIIIco6XTEN expression cassettes from constructs-1, -3, and -7 (Table 2) were excised with PstI enzyme and cloned into the same site of Cre-LoxP donor vector. The resulting constructs were transformed into NEB® stable competent E. coli strain (New England Biolabs), and ampicillin-resistant clones were screened by restriction enzyme mapping. Correct clones were named with the prefix "pCL" for "plasmid Cre-LoxP" in pCLDV-1, -3, and -7 (Table 2) (Figure 4B). Similarly, to generate other donor vectors, the hFVIIIco6XTEN expression cassettes from constructs-2, -4, -5, -6, -8, and -9 were excised with PstI and / or PvuII enzyme and cloned into the same site of Cre-LoxP donor vector. These constructs were transformed into PMC103 E. coli strain, due to the long linear repeats of the wild-type ITR sequences, and ampicillin-resistant clones were screened by restriction enzyme mapping.The resulting Cre-LoxP donor vectors, pCLDV-2, -4, -5, -6, -8, and -9, were then inserted into the "BIVVBac" bacmids encoding either B19, GPV, or AAV2 Rep at the Tn7 site, respectively, at the LoxP recombination sites (Table 3).The sequences of the ITRs in Table 3 are listed in Table 1.

[0250] [Table 3] EXAMPLES

[0251] Replicating (Rep) Baculovirus Expression Vector (BEV) AAV replication depends on nonstructural (replication) proteins; Rep78, Rep68, Rep52, and Rep40. There are several activities of AAV Rep78 and Rep68 that have been characterized to participate in viral DNA replication, such as binding to AAV ITRs, sequence- and strand-specific endonuclease activity, and ATP-dependent DNA helicase activity. Either Rep68 or Rep78 binds to a specific region (Rep binding site) within the ITRs and nicks one strand of the double-stranded replication intermediate at a unique site called the Terminal Resolution Site (TRS). The endonuclease reaction results in the covalent attachment of Rep to the newly generated 5' end. The 3' end of the nick serves as a primer for the extension of the ITRs. The helicase activity of the covalently attached Rep molecule can unravel the secondary structure of the ITRs. The process of terminal resolution provides a means for the recovery of the ends and the generation of progeny viral genomes. Therefore, Rep is essential for ITR-mediated vector production in eukaryotic cells, and to "rescue" the ITR-flanked hFVIIIco6XTEN vector genome from Sf9 cells, we generated recombinant BEVs encoding non-AAV or AAV Rep. To generate these BEVs, we first transformed BIVVBac DH10BE. coli (Figure 1D) was supertransformed with the transfer vectors pFastBac.IE1.B19.Rep (Figure 3B), pFastBac.Polh.GPV.Rep (Figure 3D), and pFastBac.Polh.AAV2.Rep (Figure 3F), and then transformants were selected in kanamycin, gentamicin, X-Gal, and IPTG. Site-specific transfer of the Rep expression cassette and gentamicin resistance gene at the mini-attTn7 insertion site of BIVVBac disrupted LacZα (fused in frame with mini-attTn7), resulting in white colonies of E. coli in X-Gal-mediated double antibiotic selection. The resulting recombinant bacmid DNA was isolated from E. coli white colonies by alkaline lysis miniprep and digested with restriction enzymes to determine the correct genetic structure. Restriction mapping results showed the expected fragments for each recombinant bacmid, suggesting site-specific transfer of Rep at the polyhedrin locus of BIVVBac (Figure 5A). Further confirmation was obtained by PCR amplifying the region spanning the expected insertion site using internal and external primers of the transfer plasmid and sequencing the resulting amplicons.

[0252] The correct recombinant bacmids encoding B19.Rep, GPV.Rep, or AAV2.Rep were transfected in Sf9 cells 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 described (Jarvis, 2014). Tn7 (Figure 5B), AcBIVVBac.Polh.GPV.Rep Tn7 (Figure 5C), and AcBIVVBac.Polh.AAV2.Rep Tn7Six plaque-purified RFP+ clones from (Figure 5D) were cultured at 0.5 × 10 6 AcBIVVBac.IE1.B19.Rep was amplified to P1 (passage 1) in Sf9 cells seeded at 100 / mL. Four to five days post-infection, P1 viruses were harvested by low-speed centrifugation, and infected cell pellets were tested for B19.REP, GPV.REP, or AAV2.REP detection by immunoblotting using anti-B19 NS1 (MyBioSource, Inc.), anti-GPV.REP (GenScript®), and anti-AAV2.REP303.9 (American Research Products Inc.) monoclonal or polyclonal antibodies, respectively. Tn7 Immunoblot analysis of plaque-purified clones (Figure 5E) showed a single species of B19.REP with the predicted mass of 74 kDa, suggesting B19.REP expressed from an unspliced ​​mRNA in Sf9 cells. Interestingly, comparable levels of B19.REP expression were observed in all viral clones tested, suggesting stoichiometric levels of B19.REP expression in the presence of the AcMNPV IE1 promoter in Sf9 cells. Unlike B19.Rep, AcBIVVBac.Polh.GPV.Rep Tn7 (Figure 5F), or AcBIVVBac.Polh.AAV2.Rep Tn7(FIG. 5G) Immunoblot analysis of plaque-purified clones showed that two species of REP with predicted masses of 73 kDa and 49 kDa corresponded to Rep78 and Rep52 polypeptides, suggesting that GPV.REP and AAV2.REP were expressed from spliced ​​mRNA transcripts in Sf9 cells. Comparable levels of Rep78 or Rep52 were observed in all viral clones tested, suggesting stable GPV.REP and AAV2.REP expression in the presence of the AcMNPV polyhedrin promoter in Sf9 cells. The level of REP78 expression was approximately half that of REP52, further suggesting that the correct stoichiometric expression of both polypeptides was achieved from a single mRNA transcript by a leaky ribosomal scanning mechanism. The highest REP-expressing clone of each BEV was selected for further amplification in Sf9 cells and then used for infection in stable cell lines encoding hFVIIIco6XTEN flanked by non-AAV and AAV symmetric or asymmetric ITRs for ceDNA vector generation. EXAMPLES

[0253] Replicating (Rep) and human FVIIIco6XTEN baculovirus expression vector (BEV) To test whether BIVVBac can be used to accommodate multiple transgenes, a family of derivative vectors was generated that encode two transgene expression cassettes: 1) Rep and 2) hFVIIIco6XTEN flanked by non-AAV and AAV symmetric or asymmetric ITRs. These BEVs were generated in two steps. First, the Rep expression cassette was inserted into the mini-attTn7 site of the polyhedrin locus by Tn7 transposition as described above. The resulting recombinant bacmid was then used to insert the hFVIIIco6XTEN expression cassette into the LoxP site of the EGT locus by in vitro Cre-LoxP recombination using Cre recombinase (New England Biolabs). In the process, Cre-LoxP donor vectors (pCLDV-1 to -9) (Table 2) encoding hFVIIIco6XTEN with B19, GPV, and AAV ITRs were inserted into AcBIVVBac.IE1.B19.Rep7 (Figure 5B), AcBIVVBac.Polh.GPV.Rep (Figure 5C), or AcBIVVBac.Polh.AAV2.Rep (Figure 5D) bacmids, respectively. Recombination reactions were transformed into DH10B E. coli and transformants were selected on kanamycin, gentamicin, and ampicillin. Triple antibiotic resistant colonies were screened by restriction enzyme mapping and / or by PCR by amplifying the region spanning the predicted insertion site using primers inside and outside the transfer plasmid and sequencing the resulting amplimers.

[0254] Correct recombinant bacmids encoding both transgene cassettes were maxi prep purified and transfected in Sf9 cells using Cellfectin® (Invitrogen) transfection reagent. Four to five days after transfection, progeny baculoviruses were collected and plaque purified in Sf9 cells. Six plaque-purified RFP+ and GFP+ clones (Figures 6A, 6B, and 6C) of each recombinant BEV (BEV-1 to -9) (Table 4) were cultured at 0.5 × 10 in a T25 flask in ESF921 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). 6 The BEV clones were amplified to P1 (passage 1) in Sf9 cells seeded at 1000 / mL. During 4-5 days post-infection, all clones showed a progression of infection, identified by the number of GFP+ and RFP+ cells for each recombinant BEV, suggesting that the virus could replicate normally and that the insertion of multiple transgenes in the same baculovirus genome had no adverse effect on progeny virus production. P1 viruses were collected by low-speed centrifugation, and infected cell pellets were processed for either REP detection by immunoblotting or ceDNA isolation using the PureLink Maxi Prep DNA isolation kit (Invitrogen). Immunoblotting was performed to detect B19.REP, GPV.REP, or AAV2.REP as described above. Immunoblotting results showed that all six clones were positive for each REP tested, suggesting that the insertion of the hFVIIIco6XTEN cassette at the EGT locus had no adverse effect on immediate-early or polyhedrin-driven REP expression. Finally, the highest REP-expressing clones of each BEV were further amplified to generate BEV working stocks (P2), followed by titration in Sf9 cells. The titrated BEV was used for infection in Sf9 cells to generate hFVIIIco6XTEN ceDNA vectors, as described below.

[0255] [Table 4] EXAMPLES

[0256] Human FVIIIco6XTEN ceDNA vector generation from baculovirus Recombinant BEVs encoding both hFVIIIco6XTEN and Rep genes (Table 4) were tested for ceDNA production. Sf9 cells were infected with titered working stocks (P2) of each BEV at a multiplicity of infection (MOI) of 3 pfu / cell. Cells were gently tumbled for 1.5 hours at room temperature, pelleted at 500xg for 5 minutes, the supernatant was aspirated, and cells were washed once with 10 mL of fresh ESF-921 medium. Cells were suspended in 50 mL of ESF-921 medium and then incubated for 72 hours at 28°C in a shaker incubator. 72 hours after infection, infected cells were harvested and pellets were washed once with 1x PBS to remove residual baculovirus particles and / or culture medium. ceDNA vectors were then isolated by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions. To identify the yield and purity of each ceDNA vector, the elution fractions were analyzed by 0.8-1.2% agarose gel. As a first round of analysis, ceDNA vectors isolated from Sf9 cells infected with BEV-5 (Table 4, Figure 7A) were tested. All six clones of BEV-5 were tested for ceDNA production. One of the clones showed a DNA band corresponding to the size of the hFVIIIco6XTEN transgene (approximately 7.0 kb), indicating that the polyhedrin-driven GPV REP is able to rescue the asymmetric ITR-flanked hFVIIIco6XTEN vector DNA (Figure 7B). To visualize the banding pattern and confirm the size of the ceDNA, the samples were tested again in different amounts. Multiple bands of high molecular weight were observed (Figure 7C). This data supports the concept of ceDNA production from a single recombinant BEV encoding a non-AAV Rep, as well as an ITR-flanked hFVIIIco6XTEN transgene. EXAMPLES

[0257] Human FVIIIco6XTEN stable insect cell line It was speculated that the insect cell genome could also be modified to produce a DNA therapeutic agent following baculovirus infection. To achieve this goal, a plasmid encoding a neomycin resistance marker in the presence of the AcMNPV immediate early 1 (ie1) promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal was synthesized using GenScript® (Piscataway, NJ) (SEQ ID NO: 14) (Figure 8A). This synthetic DNA was transfected with Cellfectin® (Invitrogen TM) was used to co-transfect Sf9 cells with plasmids encoding hFVIIIco6XTEN expression cassettes (Table 3) flanked by non-AAV and AAV symmetric and asymmetric ITRs. 24 hours after transfection, cells were visualized under a fluorescent microscope to determine transfection efficiency, and the results showed >80% GFP+ cells, suggesting a higher transfection efficiency. 72 hours after transfection, cells were selected with G418 antibiotic (Sigma Aldrich) suspended in complete TNMFH medium (Grace's Insect Medium supplemented with 10% FBS + 0.1% Pluronic F68) at a final concentration of 1.0 mg / mL. After about one week of selection, about 50% of transformed cells were recovered, suggesting that the neomycin resistance marker was stably integrated into this cell population. Surviving cells were removed from the selection medium and fed with fresh complete TNMFH medium until confluence growth. As adherent cultures, confluent cells were gradually expanded into larger culture vessels where they could continue to divide. Each cell line was then adapted to suspension culture in shake flasks for one passage in complete TNMFH and one passage in ESF-921 medium supplemented with 10% FBS. Finally, each cell line was 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 by passage every 4 days and cell growth was monitored. Each cell line was named with the prefix "Sf" according to the inserted hFVIIIco6XTEN construct (Figure 8B) (Table 5).

[0258] [Table 5] EXAMPLES

[0259] Generation of human FVIIIco6XTEN ceDNA vector from stable cell lines To test the stable cell line approach, polyclonal populations of each cell line (Table 4) encoding hFVIIIco6XTEN expression cassettes with non-AAV and AAV symmetric or asymmetric ITRs were used. Cells were infected with titered working stocks (P2) of each recombinant BEV encoding Rep (FIG. 5B-D) at a multiplicity of infection (MOI) of 3 pfu / cell. Cells were gently tumbled for 1.5 h at room temperature, pelleted at 500xg for 5 min, the supernatant was aspirated, and cells were washed once with 10 mL of fresh ESF-921 medium. Finally, cells were suspended in 50 mL of ESF-921 medium and then incubated for 72 h at 28 °C in a shaker incubator. After 72 h of infection, infected cells were harvested and pellets were washed once with 1x PBS to remove residual baculovirus particles and / or culture medium. The ceDNA vectors were then isolated by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions. To identify the yield and purity of each ceDNA vector, the eluted fractions were analyzed by 0.8-1.2% agarose gel electrophoresis. The results showed that, as previously observed, the DNA bands corresponded to the size of hFVIIIco6XTEN (approximately 7.0 kb) with different degrees of intensity as well as a high molecular weight band (Figure 9A-C). Interestingly, all cell lines tested showed that the DNA bands corresponded to the size of the hFVIIIco6XTEN expression cassette, regardless of the adjacent symmetric and / or asymmetric ITRs. Compared to B19 or AAV2 lines, GPV cell lines-3, -4, and -5 showed multiple bands of various sizes. EXAMPLES

[0260] Human FVIIIco6XTEN expression from ceDNA To determine whether ceDNA can be used as a non-viral gene therapy vector to treat hemophilia A patients, an in vitro analysis was performed to test human FVIII expression from ceDNA in hepatocellular carcinoma human Huh7 cells. First, Huh7 cells were cultured in 24-well plates at 5×10 in modified IMEM medium (Gibco™) supplemented with 10% FBS and 0.1% antibiotic mixture (Invitrogen™). 5The cells were seeded at 1000 x 1000 cells / mL and then incubated for 48 hours in a 37°C incubator with CO2 (5%) to achieve a monolayer with quiescent cell growth. This growth condition was achieved to mimic hFVIIIco6XTEN expression in the nuclei of non-dividing liver cells in vivo. The monolayer was transfected with approximately 1 μg of gel-purified ceDNA corresponding to the size of the hFVIIIco6XTEN expression cassette using Lipofectamine 3000 (Invitrogen™) according to the manufacturer's instructions. Plasmid DNA encoding hFVIIIco6XTEN flanked by symmetrically truncated ITRs was used as a control (pCLDV-1, -3, and -7, Table 3). 18 hours after transfection, the transfection mixture was aspirated and the cells were fed with fresh IMEM medium supplemented with 10% FBS and 0.1% antibiotic mixture. After 48 and 72 hours of feeding, cell-free supernatants were removed from each treatment and secreted hFVIIIco6XTEN was measured by Chromogenix Coatest® SP Factor VIII chromogenic assay according to the manufacturer's instructions. FVIII activity was normalized by standards and plotted in U / mL in FIG. 10. The assay results showed increasing levels of hFVIII expression over time in all samples tested except hFVIII.B19Δ135.ceDNA. This data suggests that FVIII was expressed from the latently transfected ceDNA vector in the nuclei of non-dividing monolayers of Huh7 cells. ceDNA obtained from GPV cell lines-3, -4, and -5 showed lower levels of FVIII expression compared to AAV strains-6 and -8, suggesting that the GPV ITRs are likely cleaved during integration into the cellular genome, as explained above. Surprisingly, ceDNA obtained from B19 cell lines-1 and -2 showed no detectable hFVIIIco6XTEN expression, whereas the plasmid DNA used as a control (pCLDV-1, Table 3) did.Overall, GPV and AAV ceDNA vectors demonstrated sustained hFVIII expression at physiological levels in Huh7 cells, suggesting that ceDNA can be used as a non-viral gene therapy vector (Figure 10). Individual cell lines are listed in Table 5. EXAMPLES

[0261] Generation of human FVIIIco6XTEN ceDNA vector by transient transfection The transient gene expression system is one of the most important techniques for performing functional analysis of the baculovirus-insect cell system. This system was developed to express foreign genes under the restriction of baculovirus promoters in transient expression plasmids. Furthermore, this system offers a shorter turnaround in vector construction and protein synthesis, avoids the difficulties associated with viral infection and cell lysis, and provides a robust means for observing cellular trafficking of proteins. Baculovirus gene promoters are generally divided into immediate-early, early, late, and very-late promoters according to the initiation of transcription in the infection cycle. Among these, only the immediate-early (ie) gene promoters are recognized by host RNA polymerase II as well as independent of viral transcription factors, which makes them suitable for baculovirus-free heterologous protein expression in insect cells. The most widely commercially available transient gene expression system is developed based on the immediate-early (ie) gene promoter of Orgia pseudotsugata multiple nuclear large nuclear polyhedrosis virus (OpMNPV). Compared with other immediate early (ie) gene promoters, the OpIE2 promoter of OpMNPV was found to provide strong activity for heterologous protein expression in Sf cells (Bleckmann et al., 2016). More recently, a baculovirus-free system based on polyethylenimine (PEI)-mediated transient gene expression under the control of the OpIE2 promoter was also described (Puente-Massaguer et al., 2020).

[0262] Therefore, we hypothesized that transient expression of Rep protein could "rescue" human FVIIIco6XTEN ceDNA vector from stable cell lines. To test this hypothesis, we utilized PEI-mediated transient Rep protein expression in the presence of OpMNPV immediate early (OpIE2) promoter for the generation of baculovirus-free in stable cell lines. The OpIE2 promoter sequence from the OpMNPV genome (GenBank accession number NC_001875.2) was synthesized by GenScript® (Piscataway, NJ) (SEQ ID NO: 15). The synthetic promoter sequence was cloned to replace the polyhedrin or immediate early promoter in the Rep protein expression construct (Figure 11A, 11C, 11E). The resulting transient expression plasmids, pFastBac.OpIE2.B19.Rep (Figure 11B), pFastBac.OpIE2.GPV.Rep (Figure 11D), and pFastBac.OpIE2.AAV2.Rep (Figure 11F), will be used to transfect stable cell lines (Table 5) encoding human FVIIIco6XTEN expression cassettes with non-AAV and AAV symmetric or asymmetric ITRs.

[0263] The stable cell lines encoding human FVIIIco6XTEN expression cassettes with non-AAV and AAV symmetric or asymmetric ITRs (Table 5) will be supertransformed with transient expression plasmids encoding B19.Rep, GPV.Rep, or AAV2.Rep in the presence of the OpMNPV immediate early (OpIE2) promoter (Figure 11), and a plamid encoding a puromycin resistance marker in the presence of the AcMNPV immediate early 1 (ie1) promoter preceded by the transcription enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal, to generate a new set of stable cell lines. The transformed cells will be selected by puromycin and expanded into shake flask cultures as described in Example 8. These cell lines will be used as producer cell lines for baculovirus-free human FVIIIco6XTEN ceDNA vector generation. EXAMPLES

[0264] Modified FVIIIXTEN expression cassette We hypothesized that the level of transgene expression cassette could be increased by codon-optimizing the cDNA for the targeted host. Physiological levels of FVIII expression from the V1.0 FVIIIco6XTEN expression cassette have been demonstrated in previous studies, as described in US Patent Publication No. 20190185543. However, to further improve target specificity and reduce immunogenicity, the FVIIIXTEN cDNA was codon-optimized with depleted CpG repeats to avoid the innate immune response generated against the DNA vector encoding the FVIIIXTEN expression cassette with parvovirus ITRs. A modified V2.0 FVIIIXTEN expression cassette was generated that contains B-domain deleted (BDD) codon optimized human factor VIII (BDDcoFVIII) fused with XTEN 144 peptide (FVIIIXTEN) under the control of a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) with an enhancer element (A1MB2), a hybrid synthetic intron (chimeric intron), a woodchuck post-transcriptional regulatory element (WPRE), and a bovine growth hormone polyadenylation (bGHpA) signal. The V2.0 FVIIIXTEN expression cassette comprises the nucleic acid sequence of SEQ ID NO:19.

[0265] Initial in vivo efficacy studies showed a considerable improvement in FVIII activity compared to the V1.0 FVIIIXTEN expression cassette (data not shown). Therefore, the V2.0 FVIIIXTEN expression cassette was used to generate recombinant BIVVBac bacmid as described above (Example 6). Engineered parvovirus ITRs, including AAV2 WT (Figure 12A), B19 WT or minimal (SEQ ID NO: 16) (Figure 12B), and GPVΔ120 (SEQ ID NO: 17) or GPVΔ186 (SEQ ID NO: 18) (Figure 12C), were then inserted into the V2.0 FVIIIXTEN expression cassette (SEQ ID NO: 19) and tested for the generation of FVIIIXTEN ceDNA vectors in the baculovirus system using three different approaches as described below. EXAMPLES

[0266] Approach to creating FVIIIXTEN ceDNA In the baculovirus insect cell system, recombinant BEV delivers the gene of interest under the presence of a strong promoter, and provides the transcription complex that is essential for viral replication in insect cells. This system provides the flexibility of inserting the transgene of interest in the baculovirus genome and / or the insect cell genome in the form of a stable cell line. These advantages of the baculovirus system were exploited in the design of three different approaches to ceDNA production to provide flexibility in platform selection due to ease of scalability.

[0267] OneBAC: To investigate the use of the OneBac approach for transgene expression, the BIVVBac bacmid was used. As described in Example 1, BIVVBac was designed to accept the insertion of multiple transgenes by two different mechanisms at two different sites in the baculovirus genome. An optimized FVIIIXTEN expression cassette (SEQ ID NO: 19) was inserted into the mini-attTn7 site of the polyhedrin locus in BIVVBac with parvovirus ITRs in the same backbone by Tn7 transposition, and an ITR-specific replication (Rep) gene expression cassette was inserted into the LoxP site of the EGT locus by Cre-LoxP recombination as described above (Example 6). Recombinant BEV was then generated and used to infect Sf9 cells to generate FVIIIXTEN ceDNA, as shown in Figure 13A. As described below, different promoters were used to control Rep expression levels to prove the concept of the OneBac approach for ceDNA production.

[0268] TwoBAC: To explore the use of the TwoBac approach for transgene expression, an optimized FVIIIXTEN expression cassette was inserted with the parvovirus ITRs and / or an ITR-specific replication (Rep) gene expression cassette was inserted into the mini-attTn7 site in the polyhedrin locus of two different BIVVBac bacmids by Tn7 transposition, as described in Example 5 above. Recombinant BEVs were then generated and used for co-infection in Sf9 cells to generate FVIIIXTEN ceDNA, as shown in Figure 13B. The difficulties associated with the TwoBAC approach were explored by using different ratios of multiplicities of infection (MOI) of the two baculoviruses, as well as fine-tuning the Rep expression level to obtain reproducible ceDNA productivity, as described in the following experiments.

[0269] Stable cell lines: To investigate the use of a stable cell line approach to transgene expression, a stable cell line was generated using an optimized FVIIIXTEN expression cassette with parvovirus ITRs, as described in Example 8. A recombinant bacmid was also generated by inserting an ITR-specific replication (Rep) gene expression cassette into a mini-attTn7 site in the polyhedrin locus of a BIVVBac bacmid by Tn7 transposition, as described in Example 5 above. Recombinant Rep.BEV was then generated and used to infect a FVIIIXTEN stable cell line to generate FVIIIXTEN ceDNA, as shown in FIG. 13C. The difficulties associated with the stable cell line approach were investigated by enriching FVIIIXTEN transformants by FACS cell sorting using GFP as a proxy to facilitate the process of generating stable cell lines, as described in the following experiment. EXAMPLES

[0270] FVIIIXTEN ceDNA (ceFVIIIXTEN) vector construction from OneBAC Recombinant BEV encoding V2.0 FVIIIXTEN with AAV2 WT or B19 WT ITRs and their respective Rep genes in the presence of the AcMNPV polyhedrin promoter was tested for FVIIIXTEN ceDNA (ceFVIIIXTEN) production in Sf9 cells using the OneBAC approach as described above. 6 1000 / mL cells were infected with OneBAC BEV at an MOI of 0.1, 0.5, 1.0, 2.0, or 3.0 plaque-forming units (pfu) / cell (Figures 14A, 15A). Cells were suspended in 50 mL of serum-free ESF-921 medium and then incubated in a 28 °C shaker incubator for 72–96 h or until viability reached 60–70%. Approximately 96 h after infection, infected cells were harvested and pellets were processed using a Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions for ceFVIIIXTEN isolation. To identify the productivity of ceFVIIIXTEN, the final elution fractions were analyzed in 0.8–1.2% agarose gel electrophoresis.

[0271] ceFVIIIXTEN AAV2 ITR Agarose gel analysis of AAV2 OneBAC (Figure 14B) showed a DNA band corresponding to the size of ceFVIIIXTEN AAV2 ITR (approximately 8.5 kb) at all MOIs tested with the highest productivity obtained at an MOI of 1.0 pfu / cell compared to other treatments (Figure 14C). This result was contrary to earlier data showing that ceFVIIIXTEN derived from HBoV1 ITR constructs showed increased productivity with increasing viral load (data not shown). Without being bound by theory, this may suggest that AAV2 Rep has a different mechanism of binding and endonuclease activity at the terminal release site of AAV2 WT ITR for DNA replication compared to HBoV1-NS1 protein, which may be due to the different hairpin structure (T-shaped vs. U-shaped) in each ITR.

[0272] ceFVIIIXTEN B19 ITR Agarose gel analysis of B19 OneBAC (FIG. 15B) showed a DNA band corresponding to the size of ceFVIIIXTEN B19 ITR (approximately 8.5 kb) at all MOIs tested with low levels of productivity (FIG. 15C). Unlike AAV2 or HBoV1, higher viral load of B19 OneBAC did not improve ceFVIIIXTEN productivity, potentially due to higher levels of B19-NS1 expressed late in infection in the presence of a strong (polyhedrin) baculovirus promoter. These results suggest that the combination of full-length (WT) parvovirus ITR and stoichiometric REP expression may be important for obtaining higher ceDNA productivity in the baculovirus system.

[0273] In conclusion, these experiments demonstrated that the OneBAC approach provides a proof of concept for ceDNA production from a single recombinant BEV encoding FVIIIXTEN with parvovirus ITR and NS1 transgenes. It demonstrates the viability and functionality of multiple transgenes inserted into different loci of a baculovirus shuttle vector (BIVVBac) and its potential use for recombinant AAV vector generation in the baculovirus-insect cell system. EXAMPLES

[0274] FVIIIXTEN ceDNA (ceFVIIIXTEN) vector construction from TwoBAC To investigate the TwoBAC approach to ceDNA transgene expression, several different conditions of coinfection were tested by either coinfecting Sf9 with TwoBAC at different MOIs while keeping the ratio constant, or coinfecting Sf9 with TwoBAC at different ratios while keeping the MOI constant. In each case, the virus inoculum was not removed and the cells were incubated in a shaker incubator at 28 °C until viability reached 60-70%. Approximately 96 h after infection, the infected cells were harvested and the pellet was processed using the Maxi Prep DNA isolation kit (Invitrogen) for ceFVIIIXTEN isolation according to the manufacturer's instructions. To identify the productivity of ceFVIIIXTEN, the final elution fractions were analyzed in 0.8-1.2% agarose gel electrophoresis.

[0275] ceFVIIIXTEN AAV2 ITR Approximately 2.0 × 10 cells were seeded in 50 mL of serum-free ESF-921 medium. 6 / mL cells, respectively, at an MOI of 0.01, 0.1, 0.3, 1.0, or 3.0 pfu / cell to keep the ratio constant at 1:10. Tn7 BEV and AcBIVVBac.Polh.AAV2.RepΔVP80 at an MOI of 0.001, 0.01, 0.03, 0.1, or 0.3 pfu / cell Tn7 A titered working stock (P2) of BEV (previously described in U.S. Patent Application No. 63 / 069,115) was co-infected (Figures 16A-16B). Agarose gel analysis of AAV2 TwoBAC showed different degrees of ceFVIIIXTEN (ceDNA) production capacity compared to contaminating baculovirus DNA (vDNA) at different MOIs of coinfection. MOIs of 0.1-0.01 and 3.0-0.3 pfu / cell showed similar degrees of ceFVIIIXTEN (ceDNA) production capacity, but the later coinfection MOI showed lower levels of contaminating vDNA (Figure 16C).

[0276] These results suggest that the AAV2 TwoBAC approach shows comparable levels of ceFVIIIXTEN productivity relative to OneBAC, but there are significant differences in the levels of other impurities co-purified by this method of purification. Without being bound by theory, this may further highlight the importance of stoichiometric AAV2 REP78 / REP52 expression to achieve higher ceFVIIIXTEN productivity in Sf9 cells.

[0277] ceFVIIIXTEN B19 ITR Approximately 2.0 × 10 cells were seeded in 50 mL of serum-free ESF-921 medium. 6 / mL cells, AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITRs at an MOI of 0.1, 0.3, 0.5, 1.0, 3.0, or 5.0 pfu / cell. Tn7 BEV and AcBIVVBac.Polh.B19-NS1 at an MOI of 0.01, 0.03, 0.05, 0.1, 0.3, or 0.5 pfu / cell to maintain a 1:10 ratio or at an MOI of 0.02, 0.06, 0.1, 0.2, or 0.6 pfu / cell to maintain a constant 1:5 ratio, respectively. Tn7 A titered working stock (P2) of BEV was co-infected (Figures 17A-17B). Unlike AAV2 TwoBAC, agarose gel analysis of B19 TwoBAC showed comparable levels of ceFVIIIXTEN (ceDNA) and contaminating baculovirus DNA (vDNA) productivity at different MOIs of co-infection (Figure 17C). Interestingly, none of the conditions tested showed an improvement in ceFVIIIXTEN B19 ITR productivity, as seen with B19 OneBAC.

[0278] In conclusion, both the B19 OneBAC and B19 TwoBAC approaches prove the concept of ceDNA production from non-AAV parvoviral ITRs in the baculovirus system.

[0279] ceFVIIIXTEN GPV ITR Approximately 2.0 × 10 cells were seeded in 50 mL of serum-free ESF-921 medium. 6 / mL cells, AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITRs at an MOI of 0.1, 0.3, 0.5, 1.0, 3.0, or 5.0 pfu / cell. Tn7 BEV and AcBIVVBac.Polh.GPV-NS1 at MOIs of 0.01, 0.03, 0.05, 0.1, 0.3, or 0.5 pfu / cell to maintain a 1:10 ratio or at MOIs of 0.02, 0.06, 0.1, or 0.2 pfu / cell to maintain a constant 1:5 ratio, respectively. Tn7 BEV, titered working stock (P2) was co-infected (Figure 18A-18B). Unlike AAV2 or B19 TwoBAC, agarose gel analysis of GPV TwoBAC showed no detectable DNA bands corresponding to the size of ceFVIIIXTEN (approximately 8.5 kb) at all MOIs tested, except for a faint band observed at MOIs of 5.0 to 0.5 pfu / cell co-infection (Figure 18C). The ceFVIIIXTEN productivity obtained by GPV TwoBAC correlated with that of GPV OneBAC, where V1.0 FVIIIco6XTEN was used with the GPV asymmetric ITR, as described above (Figure 7B, see Example 7).

[0280] In both studies, truncated GPV ITRs were tested due to the cloning complexities in obtaining full-length WT GPV ITRs on both ends of the FVIIIXTEN transgene cassette. Nevertheless, both GPV OneBAC and GPV TwoBAC approaches provide proof of concept for ceDNA production, as well as demonstrate the significance of optimal MOI ratios and promoter selection to achieve higher productivity of FVIIIXTEN ceDNA in Sf9 cells. EXAMPLES

[0281] FVIIIXTEN ceDNA (ceFVIIIXTEN) vector generation from stable cell lines To test the stable cell line approach, polyclonal populations of each cell line generated with AAV2 WT, B19 minimal, or GPVΔ120 ITR flanked V2.0 FVIIIXTEN were incubated with approximately 2.0 × 10 cells in 50 mL of serum-free ESF921 medium. 6 Cells were seeded at 1000 / mL and infected with titered working stocks (P2) of ITR-specific REP.BEV at different MOIs as indicated. In each case, the virus inoculum was not removed and the cells were incubated for 72–96 h or until viability reached 60–70% in a shaker incubator at 28 °C. Approximately 96 h after infection, infected cells were harvested and pellets were processed for ceFVIIIXTEN isolation using the PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. To determine the productivity of ceFVIIIXTEN, the final elution fractions were analyzed in 0.8–1.2% agarose gel electrophoresis.

[0282] ceFVIIIXTEN AAV2 ITR Cell lines encoding V2.0 FVIIIXTEN with AAV2 WT ITR were developed as previously described (Example 8). The polyclonal Sf.FVIIIXTEN.AAV2.WT.ITR cell population was then inoculated with AcBIVVBac.Polh.AAV2.RepΔVP80 at an MOI of 0.001, 0.01, 0.03, 0.1, 0.3, or 0.5 pfu / cell. Tn7AAV2 stable cell lines were infected with a titered working stock (P2) of BEV (described in US Patent Application Serial No. 63 / 069,115) (Figures 19A, 19B). Agarose gel analysis of ceFVIIIXTEN isolated from AAV2 stable cell lines showed comparable levels of productivity at all MOIs tested, except for the lowest MOI of 0.001 pfu / cell, which showed no detectable DNA band corresponding to the size of ceFVIIIXTEN (approximately 8.5 kb). Interestingly, cells infected with REP.BEV at MOIs of 0.01 and 0.03 pfu / cell showed higher productivity with ceFVIIIXTEN, in conjunction with contaminating baculovirus DNA (vDNA), compared to higher MOIs (Figure 19C). These results suggest that, unlike the AAV2 OneBAC or AAV2 TwoBAC approaches, the stable cell line approach requires a lower viral inoculum to “rescue” the stably integrated FVIIIXTEN expression cassette flanked by AAV2 WT ITRs from the Sf9 cell genome.

[0283] In conclusion, this study proves the concept of ceDNA production by cell lines stably integrated with a FVIIIXTEN expression cassette flanked by parvoviral ITRs.

[0284] ceFVIIIXTEN B19 ITRs Cell lines encoding V2.0 FVIIIXTEN with B19min.ITR were developed as previously described (Example 8). The polyclonal Sf.FVIIIXTEN.B19.min.ITR cell population was then inoculated with AcBIVVBac.Polh.B19-NS1 at an MOI of 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, or 5.0 pfu / cell. Tn7The titered working stock (P2) of BEV was infected (Figures 20A, 20B). Agarose gel analysis of ceFVIIIXTEN isolated from B19 stable cell line showed increasing levels of DNA band intensity with increasing MOI of REP.BEV. However, the DNA band was slightly lower than the expected size of ceFVIIIXTEN (about 8.5 kb) (Figure 20C). Interestingly, a similar banding pattern was observed for V1.0 ceFVIIIXTEN obtained from B19 stable cell line (Example 9, Figure 9A), albeit with different ITRs. In contrast, ceFVIIIXTEN obtained from Sf9 cells infected with B19 OneBAC or TwoBAC encoding FVIIIXTEN with B19 WT ITRs showed the expected size of DNA band for V2.0 ceFVIIIXTEN (about 8.5 kb) (Figures 15C and 17C).

[0285] These results suggest that although terminal release sites and REP binding elements are present in truncated or minimal variants of the B19 ITRs, the full-length (WT) sequence is required for B19-NS1 to bind and cleave DNA for efficient replication.

[0286] ceFVIIIXTEN GPV ITR Cell lines encoding V2.0 FVIIIXTEN with GPVΔ120 ITR were developed as previously described (Example 8). The polyclonal Sf.FVIIIXTEN.GPVΔ120.ITR cell population was then inoculated with AcBIVVBac.Polh.GPV-NS1 at an MOI of 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, or 5.0 pfu / cell. Tn7 A titered working stock (P2) of BEV was infected (Figures 21A, 21B). Unlike the B19 cell line, agarose gel analysis of ceFVIIIXTEN isolated from the GPV stable cell line showed that the level of productivity decreased with increasing MOI of infection.

[0287] Interestingly, in the case of ceFVIIIXTEN GPV ITR, an opposite trend in ceFVIIIXTEN productivity was observed compared to the other parvovirus ITR. The lowest MOI (0.01 pfu / cell) used for infection in GPV cell lines showed the highest band intensity for ceFVIIIXTEN (ceDNA) compared to other MOIs tested (Figure 21C). These results suggest that, at least in the case of stable cell lines, very low levels of GPV-NS1 are required for GPV-ITR-mediated ceDNA production. Furthermore, the data suggest that the low productivity of ceFVIIIXTEN observed in GPV OneBAC or TwoBAC (Figures 7C, 18C) is probably due to high virus load, which indirectly corresponds to a higher level of GPV-NS1 expression than originally required for ceFVIIIXTEN production.

[0288] Nonetheless, the stable cell line approach proves the concept of ceDNA production from cell lines in which a non-AAV parvovirus has stably integrated with a flanking FVIIIXTEN expression cassette. EXAMPLES

[0289] FVIIIXTEN ceDNA (ceFVIIIXTEN) vector construction In the baculovirus-insect cell system, recombinant BEV delivers the gene of interest in the presence of a strong promoter, as well as providing the transcription complex that is essential for viral replication in insect cells. Typically, the baculovirus DNA genome replicates in the nucleus to produce tens of millions of progeny virus particles, each containing a full-length DNA genome. It has been demonstrated that baculovirus genomic DNA is co-purified with ceDNA while isolating DNA from insect cells using a plasmid DNA-based purification method, such as a silica gel column. The columns of commercially available plasmid DNA kits are generally not designed to separate DNA based on the molecular weight of the DNA, and therefore typically all forms of DNA present in a sample can bind to these columns. Moreover, the binding capacity of high molecular weight DNA may be different from low molecular weight DNA, and the columns of anion exchange-based kits are not optimized based on the binding efficiency of different sizes of DNA.

[0290] We hypothesized that the high molecular weight DNA (>20 kb) observed in the ceDNA preps was likely baculovirus and / or Sf9 cell genomic DNA co-purified with the low molecular weight ceFVIIIXTEN (approximately 8.5 kb) (see, e.g., Figures 14C-21C). Previously, we used an indirect approach to reduce baculovirus DNA by knocking out baculovirus capsid genes, such as VP80, that are necessary for infectious progeny virus production. 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 is efficient in reducing baculovirus DNA contamination, it fails to reduce cellular genomic DNA, which is present in a significant amount (approximately 60%) of the total DNA obtained from the infected cell pellet.

[0291] In this study, we used a direct approach to separate FVIIIXTEN ceDNA from remaining undesired DNA and demonstrated the efficient yield of purified FVIIIXTEN (>95% purity) from total DNA preps from infected cell pellets. This novel approach takes advantage of preparative electrophoresis, which is widely used to separate different protein molecules according to size and charge. See, for example, Michov, B. (2020) Electrophoresis. Berlin, Boston: De Gruyter, pp. 405-424. For example, by using Bio-Rad Model 491 prep cells or other such units, complex molecules can be separated based on their size.

[0292] In this study, we used preparative electrophoresis techniques to separate FVIIIXTEN ceDNA from high molecular weight DNA and successfully obtained purified ceDNA that was used for in vivo studies, as described below.

[0293] The entire workflow for ceDNA purification is shown in Figure 22, where the process begins with the scale-up of Sf9 cell culture from 0.5 L to 1.5 L or larger volumes in serum-free insect cell culture medium (Figure 22A). 6 Typically, approximately 1.3 x 10 cells / mL are required to reach a desired cell density. 6After 2 days of incubation with a seeding density of 1000 / mL, cells were infected with OneBAC or TwoBAC BEV at an optimized MOI (depending on the approach used for ceDNA production) and incubated in a shaker incubator at 28 °C until viability reached approximately 60-70%, which typically takes about 4 days (Figure 22B). After viability reached approximately 70%, cells were harvested and processed for total DNA purification by anion exchange chromatography kit column, such as PureLink HiPure Expi Plasmid Gigaprep Purification Kit (Invitrogen), according to the manufacturer's instructions. To identify DNA production capacity and integrity, an aliquot of purified DNA material was checked in 0.8-1.2% agarose gel electrophoresis (Figure 22C). The purified material was then loaded into a preparative agarose gel electrophoresis apparatus containing a 0.5% preparative agarose gel and a 0.25% stacking agarose gel, assembled according to the manufacturer's instructions. Samples are electrophoresed at low voltage (constant about 40 volts), a buffer recirculation flow rate of about 50 mL / min, and an elution buffer flow rate of 50 μL / min for 6-7 days at 4° C. to collect each fraction in the fraction collection chamber for 70-80 minutes. After the continuous elution electrophoresis, 20 μL of each fraction was checked in a 0.8-1.2% agarose gel electrophoresis to identify the purity of the FVIIIXTEN ceDNA (FIG. 22D). The desired fractions were collected and precipitated with 3M NaOAc, pH 5.5, and 100% EtOH at −200° C. for 1-2 hours. Finally, the precipitated FVIIIXTEN ceDNA was pelleted at high speed and washed once with 70% EtOH before being resuspended in TE, pH 8.0 buffer. The purified FVIIIXTEN ceDNA was checked again on 0.8-1.2% agarose gel electrophoresis to confirm purity and integrity before being injected into animals for in vivo efficacy studies (Figure 22E). EXAMPLES

[0294] In vivo effects of FVIIIXTEN ceDNA (ceFVIIIXTEN) Systemic administration of ceFVIIIXTEN in HemA mice To verify the functionality of ceDNA in vivo, purified ceFVIIIXTEN with AAV2 or HBoV1 WT ITRs was transfected with hFVIIIR593C at 0.3 μg, 1.0 μg, or 2.0 μg / mouse (corresponding to 12 μg, 40 μg, and 80 μg / kg, respectively). + / + / HemA mice were systemically injected by tail vein hydrodynamic injection. Plasma samples were taken from the injected mice at 7 day intervals and FVIII activity was measured by chromogenic assay as described above.

[0295] Plasma FVIII activity normalized to percent normal for ceFVIIIXTEN-injected cohorts is shown in Figure 23. Results showed that a dose-dependent response in HemA mice with supraphysiological levels of FVIII expression (>500% of normal) was observed at the highest dose of AAV2 or HBoV1 ceDNA tested. However, a gradual decrease in FVIII expression levels was observed up to 140 days after injection, after which the levels stabilized in the ceFVIIIXTEN AAV2 ITR-injected cohort. Interestingly, the FVIII expression levels of ceFVIIIXTEN HBoV1 ITR showed a similar expression trend to mice injected with eFVIIIXTEN AAV2 ITR (data not shown).

[0296] In conclusion, these in vivo efficacy studies confirm the functionality of ceFVIIIXTEN and demonstrate that parvoviral ITRs can be used to generate functional ceDNA encoding a transgene of interest in the baculovirus-insect cell system. EXAMPLES

[0297] FVIIIXTEN HBoV1 ITRs ceDNA vector generation from OneBAC OneBAC BEV encoding both the FVIIIXTEN HBoV1 ITR and HBoV1 NS1 genes was tested for FVIIIXTEN ceDNA production in Sf9 cells. 6 100 / mL of cells were infected with titered 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) / cell (Figure 24A). Cells were suspended in 50 mL of serum-free ESF-921 medium and then incubated for 72-96 h or until viability reached 60-70% in a 28°C shaker incubator. Approximately 96 h after infection, infected cells were harvested and pellets were processed for FVIIIXTEN ceDNA vector isolation by PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. To identify FVIIIXTEN ceDNA vector productivity, the final elution fractions were analyzed in 0.8-1.2% agarose gel electrophoresis.

[0298] AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS1 encoding FVIIIXTEN with HBoV1 ITRs and polyhedrin-driven HBoV1-NS1 LoxP Agarose gel analysis of BEV (Figure 24B) is shown in Figure 24C. The results showed that DNA bands corresponded to the size of FVIIIXTEN HBoV1 ITR (approximately 8.5 kb) ceDNA at all doses tested, with increased productivity with increasing MOI.

[0299] This result was contrary to the productivity of ceDNA obtained by AAV2 ITRs OneBAC, where a decrease in productivity was observed before the increase in viral load.Without being bound by theory, HBoV1-NS1 protein may have a unique binding mechanism for DNA replication and endonuclease activity at the terminal release site of HBoV1 ITR, which may be due to the unique structure of REH and LEH ITRs.

[0300] In conclusion, these experiments demonstrated that the OneBAC approach provides a proof of concept for ceDNA production from a single recombinant BEV encoding FVIIIXTEN with HBoV1 ITR and NS1 transgenes. It also demonstrates the feasibility and functionality of multiple transgenes inserted into different loci of a baculovirus shuttle vector (BIVVBac) and its potential use for recombinant AAV vector generation in the baculovirus-insect cell system. EXAMPLES

[0301] FVIIIXTEN HBoV1 ITRs ceDNA vector construction from TwoBAC To explore the TwoBAC approach to transgene expression, a clonal recombinant BEV encoding FVIIIXTEN HBoV1 ITR with polyhedrin-driven HBoV1-NS1 BEV was tested for co-infection at different MOIs of 1:10 and 1:5 ratios or at different ratios of 0.3, 1.0, 3.0, and 5.0 pfu / cell for the production of FVIIIXTEN ceDNA vectors in Sf9 cells (Figure 25A). 6 Cells were seeded at 1000pfu / mL in 50 mL of serum-free ESF-921 medium and transfected with AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRs at MOIs of 0.1, 0.3, 0.5, 1.0, 3.0, and 5.0 pfu / cell. Tn7 BEV and AcBIVVBac.Polh.HBoV1-NS1 at MOIs of 0.01, 0.03, 0.05, 0.1, 0.3, and 0.5 pfu / cell to maintain a constant 1:10 ratio, or at MOIs of 0.02, 0.06, 0.1, 0.2, 0.6, and 1.0 pfu / cell to maintain a constant 1:5 ratio, respectively. Tn7Cells were coinfected with a titered working stock (P2) of BEV. Similarly, cells were coinfected at ratios of 1:1, 1:2, 1:5, or 1:10 at MOIs of 0.3, 1.0, 3.0, or 5.0 pfu / cell (Figure 25B). In each case, the virus inoculum was not removed and cells were incubated in a shaker incubator at 28 °C until viability reached 60-70%. Approximately 96 h after infection, infected cells were harvested and pellets were processed for FVIIIXTEN ceDNA vector isolation by PureLink Maxi Prep DNA isolation kit (Invitrogen) according to the manufacturer's instructions. To identify ceDNA production capacity, the final elution fractions were analyzed in 0.8-1.2% agarose gel electrophoresis.

[0302] As expected, agarose gel analysis showed different degrees of FVIIIXTEN ceDNA production ability under different conditions.However, compared with other conditions tested, TwoBAC co-infected with MOI of 3.0 pfu / cell showed increased FVIIIXTEN ceDNA production ability with the highest viral load ratio of 1:10 (Figure 25C).Higher viral load appears to improve FVIIIXTEN HBoV1 ITRs ceDNA production ability, which is consistent with the observation in OneBAC BEV (see Example 6).This further suggests the requirement of higher levels of HBoV1-NS1 for HBoV1-ITR-dependent FVIIIXTEN ceDNA replication in Sf9 cells.

[0303] The results with OneBAC or TwoBAC indicate that the level of HBoV1-NS1 replication has a profound effect on the productivity of FVIIIXTEN ceDNA in the baculovirus system.

[0304] In conclusion, these experiments demonstrated that the TwoBAC approach provides a proof of concept for ceDNA production from two recombinant BEVs encoding FVIIIXTEN with HBoV1 ITR and / or NS1 transgenes. These experiments also demonstrate the importance of the promoter to achieve optimal MOI ratios and / or higher productivity of FVIIIXTEN ceDNA in Sf9 cells.

[0305] array

[0306] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

Claims

1. Bacterial replicons; a first selectable marker sequence; a heterologous sequence inserted into the first reporter gene, wherein the inserted heterologous sequence disrupts the reading frame of the first reporter gene; a second reporter gene operably linked to a baculovirus-inducible promoter; and Selective target sites capable of mediating site-specific recombination events Including, The heterologous sequence comprises a 5' inverted terminal repeat (ITR) and / or a heterologous gene encoding a parvoviral Rep protein in a recombinant bacmid.

2. (i) the heterologous sequence comprises a second selectable marker sequence, optionally wherein the second selectable marker sequence comprises a gentamicin resistance gene; (ii) the bacterial replicon is a mini-F replicon; (iii) the first selectable marker sequence comprises a kanamycin resistance gene; (iv) the first reporter gene encodes LacZα or a functional portion thereof; (v) the second reporter gene encodes a red fluorescent protein; (vi) the baculovirus-inducible promoter is the 39K promoter; (vii) the selective target site comprises a LoxP site or a variant thereof; and / or (viii) The recombinant bacmid of claim 1, wherein the site-specific recombination event is mediated by Cre recombinase.

3. a first heterologous sequence inserted into the first reporter gene, wherein the inserted heterologous sequence disrupts the reading frame of the first reporter gene; a first selective target site capable of mediating a site-specific recombination event; a multiple cloning site containing a second heterologous sequence; and a second selective target site capable of mediating a site-specific recombination event Including, The second heterologous sequence is a recombinant bacmid that includes a 5' inverted terminal repeat (ITR) and / or a 3' inverted terminal repeat (ITR).

4. The recombinant bacmid of claim 3, wherein the first heterologous sequence comprises a first heterologous gene; optionally, the first heterologous sequence comprises an expression control sequence operably linked to the protein-encoding sequence; further optionally, the expression control sequence comprises a baculovirus promoter; even further optionally, the baculovirus promoter is an immediate early, early, late, or very late promoter; still further optionally, the baculovirus promoter is selected from the group consisting of a polyhedrin promoter, an immediate early 1 promoter, and an immediate early 2 promoter.

5. 5. The recombinant bacmid of claim 4, wherein the first heterologous sequence encodes a Rep protein from the genome of a member of the viral family Parvoviridae; optionally, the Rep protein is selected from the group consisting of B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

6. The recombinant bacmid of claim 3 , wherein the second heterologous sequence comprises a second heterologous gene.

7. 7. The recombinant bacmid of claim 6, wherein the second heterologous sequence comprises an expression control sequence operably linked to the protein-encoding sequence; optionally, the expression control sequence comprises a tissue-specific promoter, a polyadenylation signal, and / or a post-transcriptional regulatory element; further optionally, the tissue-specific promoter is a tristetraprolin (TTP) or mouse transthyretin (mTTR) promoter; even further optionally, the polyadenylation signal is a bovine growth hormone polyadenylation signal; or the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

8. 4. The recombinant bacmid of claim 3, wherein the 5' ITR is derived from the genome of a first member of the viral family Parvoviridae, and the 3' ITR is derived from the genome of a second member of the viral family Parvoviridae; optionally, the first and second members are the same; or the first and second members are different.

9. the 5' ITR and 3' ITR are derived from a parvovirus selected from the group consisting of B19, GPV, HBoV1, and AAV2, and optionally (i) the 5′ ITR is a wild-type or truncated 5′ ITR derived from B19; (ii) the 5′ ITR is a wild-type or truncated 5′ ITR derived from GPV; (iii) the 5′ ITR is a wild-type or truncated 5′ ITR derived from AAV2; (iv) the 5′ ITR is the wild-type 5′ ITR from HBoV1; (v) the 3′ ITR is a wild-type or truncated 3′ ITR derived from B19; (vi) the 3′ ITR is a wild-type or truncated 3′ ITR derived from GPV; (vii) the 3′ ITR is a wild-type or truncated 3′ ITR derived from AAV2; or (viii) The recombinant bacmid of claim 3, wherein the 3' ITR is a wild-type 3' ITR derived from HBoV1.

10. (A) The recombinant bacmid further comprises: (i) a bacterial replicon; optionally, the bacterial replicon is a mini-F replicon; and / or (ii) selectable marker sequences; optionally, the one or more selectable marker sequences comprise one or more antibiotic resistance genes; further optionally, the one or more antibiotic resistance genes are selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, and a gentamicin resistance gene; Includes; (B) the first reporter gene encodes LacZα or a functional portion thereof; (C) the recombinant bacmid further comprises at least a second and a third reporter gene; optionally, the second and third reporter genes each encode a fluorescent protein; and further optionally, the fluorescent protein is a green fluorescent protein or a red fluorescent protein; (D) the first and second selective target sites comprise LoxP sites or variants thereof; and / or (E) The recombinant bacmid of claim 3, wherein the site-specific recombination event is mediated by Cre recombinase.

11. The recombinant bacmid of claim 3 , wherein the second heterologous sequence encodes a therapeutic protein.

12. 12. The recombinant bacmid of claim 11, wherein the therapeutic protein is a clotting factor; optionally, the clotting factor is Factor VIII (FVIII) or FVIII-XTEN.

13. 13. The recombinant bacmid of claim 12, wherein the second heterologous sequence comprises the nucleic acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO:

29.

14. A set of recombinant bacmids comprising a first bacmid and a second bacmid, The first bacmid contains a sequence encoding Rep inserted into the mini-attTn7 site, wherein the inserted Rep disrupts the reading frame of a reporter gene or a functional portion thereof; and The second bacmid contains the heterologous sequence, the heterologous sequence being, from 5' to 3': a wild-type or truncated 5′ inverted terminal repeat (ITR) derived from the first genome of a member of the viral family Parvoviridae; protein-coding sequences; one or more expression control sequences operably linked to the protein-encoding sequence; and Wild-type or truncated 3' inverted terminal repeats (ITRs) derived from a second genome of a member of the virus family Parvoviridae A set of recombinant bacmids comprising:

15. the 5' ITR and 3' ITR are derived from a parvovirus selected from the group consisting of B19, GPV, HBoV1, and AAV2; and / or 15. The set of recombinant bacmids of claim 14, wherein the heterologous sequence comprises the nucleic acid sequence of SEQ ID NO:20, SEQ ID NO:19, or SEQ ID NO:29.

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