TW202328455A - Baculovirus expression system - Google Patents

Abstract

Provided herein is a baculovirus expression vector system for the production of a desired protein. In one aspect, the desired protein is a closed-ended DNA (ceDNA) molecule comprising wild-type and/or truncated inverted terminal repeats derived from a genome of a member of the viral family Parvoviridae.

Description

Baculovirus Expression System

Related applications

This application is a continuation of International Application No. PCT/US2021/047218 (filed August 23, 2021), which claims priority to U.S. Provisional Application No. 63/069,073 (filed August 23, 2020), which The entire text is hereby incorporated by reference in its entirety. References to Electronically Submitted Sequence Listings

The contents of the sequence listing (name: 725669_SA9-474PCCON.txt; size: 67.8KB; creation date: January 14, 2022) submitted electronically as an ASCII text file are incorporated herein by reference in their entirety.

Gene therapy offers the possibility of a durable means of treating a variety of diseases. Recent developments in gene therapy treatments employ the use of viral or non-viral vectors. Several techniques are currently used to generate gene therapy vectors.

For example, the Baculovirus Expression Vector System (BEVS) is an established system for generating gene therapy vectors. In this system, insect host cells are infected with recombinant baculovirus. Insect host cells provide the necessary protein processing machinery to produce products encoded by recombinant baculoviruses. BEVS have been used to generate products for many different applications, including vaccines, therapeutic proteins, protein crystallography, and products for basic and applied research. When producing viral vectors for gene therapy, several baculovirus expression vectors are often used and infected into insect host cells. The production of each baculovirus expression vector is time consuming and drives up production costs.

Accordingly, there is a need in the art for improved baculovirus expression vector systems that avoid the limitations of existing systems.

Provided herein is a baculovirus expression vector system comprising a baculovirus shuttle vector (bacmid, bacmid) and/or a stable cell line engineered to produce a therapeutic drug product. The baculovirus expression vector system provided herein comprises a recombinant baculovirus shuttle vector containing two or more sites for insertion of foreign sequences, and one or more sites capable of mediating the insertion of foreign sequences (eg, heterologous genes) Donor vector in the foreign sequence insertion site.

In one aspect, provided herein is a baculovirus shuttle vector comprising: a bacterial replicon; a selectable marker sequence; a first reporter comprising a first preferential target site for insertion of a transposon gene; a second reporter gene operably linked to a baculovirus-inducible promoter; and a second preferential target site capable of mediating site-specific recombination events.

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.

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

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 portion thereof. In certain exemplary embodiments, the chromogenic substrate is blue-gal or X-gal.

In certain exemplary embodiments, said first preferential target site for insertion of a transposon does not disrupt the reading frame of said first reporter gene. In certain exemplary embodiments, the first preferential target site is an attachment site for a bacterial transposon. In certain exemplary embodiments, the first preferential target site is an attachment site for a T7 transposon. In certain exemplary embodiments, the transposon is a T7 transposon.

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

In certain exemplary embodiments, the baculovirus-inducible promoter is a 39K promoter.

In certain exemplary embodiments, the second preferential target site comprises a LoxP site or a variant thereof.

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

In another aspect, provided herein is a baculovirus shuttle vector comprising: a miniature F replicon; an antibiotic resistance gene; a LacZα gene or a functional portion thereof comprising a T7 transposon attachment site; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site or a variant thereof.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a bacterial replicon; a first selectable marker sequence; a heterologous sequence inserted into a 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 a preferential target site capable of mediating site-specific recombination events point.

In certain exemplary embodiments, the heterologous sequence comprises a heterologous gene. In certain exemplary embodiments, the heterologous gene comprises a sequence encoding a protein.

In certain exemplary embodiments, the heterologous sequences further comprise expression control sequences. In certain exemplary embodiments, said expression control sequence is operably linked to said sequence encoding a protein.

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-early1 promoter and immediate-early2 promoter ).

In certain exemplary embodiments, the expression control sequence comprises a polyadenylation signal.

In certain exemplary embodiments, the protein is a Rep protein isolated from the genome of a member of the virus family Parvoviridae. In certain exemplary embodiments, the protein is a parvovirus Rep protein. In certain exemplary embodiments, the parvovirus Rep protein is selected from B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

In certain exemplary embodiments, the heterologous sequence comprises a second selectable marker sequence. In certain exemplary embodiments, the second selectable marker sequence comprises a gentamycin resistance gene.

In certain exemplary embodiments, the bacterial replicon is a miniature 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 red fluorescent protein. In certain exemplary embodiments, wherein the baculovirus-inducible promoter is a 39K promoter. In certain exemplary embodiments, the second preferential target site comprises a LoxP site or a variant thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a heterologous sequence inserted into a miniature attTn7 site, wherein the heterologous sequence encodes a Rep, and wherein The inserted Rep disrupts the reading frame of the LacZα gene or its functional part; and the LoxP site or its variants.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a miniature attTn7 site, wherein the heterologous sequence encodes a Rep, and wherein the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and a LoxP site point or its variants.

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

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

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a bacterial replicon; a first antibiotic resistance gene; a heterologous sequence inserted into a miniature attTn7 site, wherein the heterologous sequence encodes an AAV2 Rep, and wherein the inserted AAV2 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof.

In another aspect, provided herein is a nucleic acid vector comprising: a first origin of replication for propagating the nucleic acid vector in a first bacterial strain, wherein 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; Multiple cloning sites for inserting heterologous sequences; Selectable marker sequence; Reporter gene; And capable of mediating site-specific recombination Priority target sites for events.

In certain exemplary embodiments, the first origin of replication is conditional on the presence of the pi protein. In certain exemplary embodiments, the first origin of replication is R6Kγ.

In certain exemplary embodiments, the first bacterial strain comprises a pi protein.

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

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

In certain exemplary embodiments, the reporter gene encodes a fluorescent protein. In certain exemplary embodiments, the fluorescent protein is green fluorescent protein.

In certain exemplary embodiments, the preferential target site comprises a LoxP site or a variant thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

In another aspect, provided herein is a nucleic acid vector comprising: a first origin of replication for propagating the nucleic acid vector in a first bacterial strain, wherein 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; multiple cloning sites comprising heterologous sequences; selectable marker sequences; reporter genes; and capable of mediating site-specific recombination events Priority target sites.

In certain exemplary embodiments, the heterologous sequence comprises a heterologous gene. In certain exemplary embodiments, the heterologous gene comprises a sequence encoding a protein.

In certain exemplary embodiments, the heterologous sequences further comprise expression control sequences. In certain exemplary embodiments, said expression control sequence is operably linked to said sequence encoding a protein. 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 murine 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 sequences comprise post-transcriptional regulatory elements. In certain exemplary embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a coagulation factor. In certain exemplary embodiments, the coagulation factor is Factor VIII (FVIII). In certain exemplary embodiments, the coagulation factor is FVIII-XTEN.

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, said 5' ITR and said 3' ITR are derived from parvoviruses. In certain exemplary embodiments, the parvovirus is selected from 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.

In certain exemplary embodiments, the first origin of replication is conditional on the presence of the pi protein. In certain exemplary embodiments, the first origin of replication is R6Kγ.

In certain exemplary embodiments, the first bacterial strain comprises a pi protein.

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

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

In certain exemplary embodiments, the reporter gene encodes a fluorescent protein. In certain exemplary embodiments, the fluorescent protein is green fluorescent protein.

In certain exemplary embodiments, the preferential target site comprises a LoxP site or a variant thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a first heterologous sequence inserted into a first reporter gene, wherein the inserted heterologous sequence disrupts the reading frame of the first reporter gene; a first preferential target site capable of mediating a site-specific recombination event; a multiplex cloning site comprising a second heterologous sequence; and a site-specific recombination event capable of mediating the second priority target site.

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 the sequence encoding the protein.

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 a polyhedrin promoter, an immediate early 1 promoter, and an immediate early 2 promoter.

In certain exemplary embodiments, the protein is a Rep protein isolated from the genome of a member of the virus family Parvoviridae. In certain exemplary embodiments, the protein is a parvovirus Rep protein. In certain exemplary embodiments, the parvovirus Rep protein is selected from B19 Rep, AAV2 Rep, HBoV1 Rep, and GPV Rep.

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 sequence encoding the protein. In certain exemplary embodiments, the expression control sequences comprise tissue-specific promoters, polyadenylation signals, and/or post-transcriptional regulatory elements. In certain exemplary embodiments, the tissue-specific promoter is a tristetraprolin (TTP) or murine 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).

In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a coagulation factor. In certain exemplary embodiments, the coagulation factor is Factor VIII (FVIII). In certain exemplary embodiments, the coagulation factor is FVIII-XTEN.

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 member and the second member are the same. In certain exemplary embodiments, the first member and the second member 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 derived from AAV2. In certain exemplary embodiments, the 5' ITR is a wild-type or truncated 5' ITR derived from HBoV1. In certain exemplary embodiments, the 3'ITR is a wild-type or truncated 3'ITR derived from B19. In certain exemplary embodiments, the 3'ITR is a wild-type or truncated 3'ITR derived from GPV. In certain exemplary embodiments, the 3' ITR is a wild-type or truncated 3' ITR derived from AAV2. In certain exemplary embodiments, the 5' ITR is a wild-type 3' ITR derived from HBoV1.

In certain exemplary embodiments, the recombinant baculovirus shuttle vector further comprises a bacterial replicon. In certain exemplary embodiments, the bacterial replicon is a miniature F replicon.

In certain exemplary embodiments, the recombinant baculovirus shuttle vector 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 ampicillin resistance genes, kanamycin resistance genes, and gitamycin resistance genes.

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

In some exemplary embodiments, the recombinant baculovirus shuttle vector further comprises at least a second reporter gene and a third reporter gene. In certain exemplary embodiments, the second reporter gene 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.

In certain exemplary embodiments, the first preferential target site and the second preferential target site each comprise a LoxP site or a variant thereof. In certain exemplary embodiments, the site-specific recombination event is mediated by Cre recombinase.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a sequence encoding Rep inserted into a miniature attTn7 site, wherein the inserted Rep disrupts the LacZα gene or a reading frame of a functional portion thereof; and a multiplex cloning site comprising a heterologous sequence, wherein said heterologous sequence comprises, from 5' to 3': a wild-type or truncated genome derived from a first genome of a member of the family Parvoviridae a short 5' inverted terminal repeat; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild Type or truncated 3' inverted terminal repeat.

In certain exemplary embodiments, said first genome and said second genome of the family Parvoviridae are identical. In certain exemplary embodiments, said first genome and said second genome of the family Parvoviridae are different.

In certain exemplary embodiments, said Rep is derived from said first genome or said second genome of a member of said family of viruses, Parvoviridae.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a sequence encoding B19 Rep inserted into a miniature attTn7 site, wherein the inserted B19 Rep disrupts LacZα the reading frame of the gene or functional portion thereof; and a multiplex cloning site comprising a heterologous sequence comprising, from 5' to 3': a wild-type or truncated 5' reverse end derived from B19 a repeat sequence; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from B19.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a sequence encoding a GPV Rep inserted into a miniature attTn7 site, wherein the inserted GPV Rep disrupts LacZα the reading frame of the gene or functional portion thereof; and a multiplex cloning site comprising a heterologous sequence comprising, from 5' to 3': a wild-type or truncated 5' reverse end derived from GPV a repeat sequence; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from GPV.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a sequence encoding AAV2 Rep inserted into a miniature attTn7 site, wherein the inserted AAV2 Rep disrupts LacZα the reading frame of the gene or functional portion thereof; and a multiplex cloning site comprising a heterologous sequence comprising, from 5' to 3': a wild-type or truncated 5' reverse end derived from AAV2 a repeat sequence; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from AAV2.

In another aspect, provided herein is a recombinant baculovirus shuttle vector comprising: a sequence encoding AAV2 HBoV1 Rep inserted into a miniature 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, wherein the heterologous sequence comprises from 5' to 3': a wild-type 5' inverted terminal repeat sequence derived from HBoV1; a protein-encoding sequence; one or more expression control sequences operably linked to said protein-encoding sequence; and a wild-type 3' inverted terminal repeat sequence derived from HBoV1.

In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a coagulation factor. In certain exemplary embodiments, the coagulation factor is Factor VIII (FVIII). In certain exemplary embodiments, the coagulation factor is FVIII-XTEN.

In another aspect, there is provided a host cell comprising a baculovirus shuttle vector as described herein. In another aspect, there is provided a host cell comprising a recombinant baculovirus shuttle vector as described herein. In another aspect, there is provided a host cell comprising a vector as described herein.

In another aspect, provided herein is a method of producing a recombinant baculovirus shuttle vector as described herein, the method comprising: introducing a baculovirus shuttle vector as described herein and a donor nucleic acid molecule into a bacterial cell , said donor nucleic acid molecule comprising a bacterial replicon operably linked to a transposon comprising a heterologous sequence as described herein; growing said bacterial cell under conditions in which translocation occurs; and from The recombinant baculovirus shuttle vector is isolated from the bacterial cells.

In another aspect, this paper provides a method for producing the recombinant baculovirus shuttle vector as described herein, the method comprising: combining the recombinant baculovirus shuttle vector as described herein, the vector as described herein, and introducing Cre recombinase into bacterial cells; cultivating the bacterial cells under conditions where site-specific recombination occurs; and isolating the recombinant baculovirus shuttle vector from the bacterial cells.

In another aspect, provided herein is a method of producing a recombinant baculovirus, the method comprising transfecting the recombinant baculovirus shuttle vector described herein into insect cells under appropriate conditions.

In another aspect, provided herein is a method of producing a closed-end DNA (ceDNA) molecule, the method comprising: transfecting a recombinant baculovirus shuttle vector as described herein into an insect cell under appropriate conditions to produce a recombinant baculovirus; and infecting a second insect cell with the recombinant baculovirus under appropriate conditions to produce a ceDNA molecule.

In another aspect, provided herein is a method of producing a closed-ended DNA (ceDNA) molecule comprising a wild-type or truncated B19 inverted terminal repeat, the method comprising: combining the wild-type or truncated A recombinant baculovirus shuttle vector as described herein of the short form of the B19 inverted terminal repeat is transfected into an insect cell to produce a recombinant baculovirus; and a second insect cell is infected with the recombinant baculovirus under appropriate conditions to produce ceDNA molecules.

In another aspect, provided herein is a method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated GPV inverted terminal repeat, the method comprising: A recombinant baculovirus shuttle vector as described herein of the short GPV inverted terminal repeat is transfected into an insect cell to produce a recombinant baculovirus; and a second insect cell is infected with the recombinant baculovirus under appropriate conditions to produce ceDNA molecules.

In another aspect, provided herein is a method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated AAV2 inverted terminal repeat, the method comprising: combining the wild-type or truncated A recombinant baculovirus shuttle vector as described herein for the short form of the AAV2 inverted terminal repeat is transfected into an insect cell to produce a recombinant baculovirus; and a second insect cell is infected with the recombinant baculovirus under appropriate conditions to produce ceDNA molecules.

In another aspect, provided herein is a method of producing a closed-ended DNA (ceDNA) molecule comprising a wild-type HBoV1 inverted terminal repeat, the method comprising: adding a DNA molecule comprising a wild-type HBoV1 inverted terminal repeat under appropriate conditions A recombinant baculovirus shuttle vector as described herein is transfected into an insect cell to produce a recombinant baculovirus; and a second insect cell is infected with the recombinant baculovirus under appropriate conditions to produce a ceDNA molecule.

In another aspect, provided herein is a plasmid comprising a nucleic acid sequence comprising, from 5' to 3', a wild-type or truncated genome derived from a first member of the family Parvoviridae type 5' inverted terminal repeat; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild Type or truncated 3' inverted terminal repeat.

In some embodiments, disclosed herein is a set of recombinant baculovirus shuttle vectors comprising a first baculovirus shuttle vector and a second baculovirus shuttle vector, wherein the first baculovirus shuttle vector The viral shuttle vector comprises a Rep-encoding sequence inserted into the miniature attTn7 site, wherein the inserted Rep disrupts the reading frame of the reporter gene or a functional portion thereof; and wherein the second baculoviral shuttle vector comprises a heterologous sequence, wherein said heterologous sequence comprises from 5' to 3': a wild-type or truncated 5' inverted terminal repeat (ITR) derived from the first genome of a member of the family Parvoviridae; a sequence encoding a protein; a or a plurality of expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated 3' inverted terminal repeat (ITR) derived from a second genome of a member of the family Parvoviridae of Viridae. In some embodiments, the 5' ITR and the 3' ITR are derived from a parvovirus selected from B19, GPV, HBoV1 and AAV2.

In certain exemplary embodiments, the one or more expression control sequences comprise tissue-specific promoters, polyadenylation signals, and/or post-transcriptional regulatory elements. In certain exemplary embodiments, the tissue-specific promoter is a tristetraprolin (TTP) or murine 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).

In certain exemplary embodiments, the protein is a therapeutic protein. In certain exemplary embodiments, the therapeutic protein is a coagulation factor. In certain exemplary embodiments, the coagulation factor is Factor VIII (FVIII). In certain exemplary embodiments, the coagulation factor is FVIII-XTEN.

In certain exemplary embodiments, said first member and/or said second member are selected from B19, GPV, HBoV1 and AAV2. In certain exemplary embodiments, the first member and/or the second member are the same. In certain exemplary embodiments, the first member and/or the second member are different.

In another aspect, the present invention provides a stable cell strain comprising the nucleic acid sequence as described herein, wherein the nucleic acid sequence is stably integrated into the genome of the stable cell strain.

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.

In another aspect, provided herein is a method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated inverted terminal repeat, the method comprising introducing a recombinant baculovirus shuttle vector as described herein into Introduced into stable cell lines as described herein.

In another aspect, provided herein is a method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated inverted terminal repeat, the method comprising introducing into a host cell: a plasmid as described herein ; and a recombinant baculovirus shuttle vector as described herein.

In certain exemplary embodiments, the host cell is an insect cell. In certain exemplary embodiments, the insect cell is Sf9.

Provided herein is a baculovirus expression vector system suitable for expressing two or more foreign sequences. Also provided are methods of, for example, producing ceDNA using the baculovirus expression vector systems described herein. I.Definition _

It should be noted that the term "a/a (a)" or "an/an (an)" entity refers to one/one or more/multiple of said entities: for example, "a nucleotide sequence" is understood to represent a or multiple nucleotide sequences. Similarly, "a therapeutic protein" and "a miRNA" are understood to represent one or more therapeutic proteins and one or more miRNAs, respectively. Accordingly, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The term "about" is used herein to mean approximately, roughly, approximately, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Generally, the term "about" is used herein to modify values above and below the stated value by a difference of 10% upwards or downwards (higher or lower).

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations where construed in an alternative ("or").

"Nucleic acid," "nucleic acid molecule," "nucleotide," "sequence of one or more nucleotides," and "polynucleotide" are used interchangeably and refer to ribonucleosides in single-stranded form or in a double-stranded helix (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") The phosphate polymerized form of or any of its phosphate analogs, such as phosphorothioates and thioesters. 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, especially 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, this term includes double-stranded DNA found inter alia in linear or circular DNA molecules (eg, restriction fragments), plasmids, supercoiled DNA, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, sequences may be described in this text according to conventional convention, given in the 5' to 3' direction only along the non-transcribed strand of DNA (i.e., the strand with sequence homology to mRNA) sequence. A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein.

As used herein, "inverted terminal repeat" (or "ITR") refers to a nucleic acid subsequence located at the 5' or 3' end of a single-stranded nucleic acid sequence, comprising a set of nucleotides (the initial sequence), followed downstream by Its reverse complement is a palindromic sequence. The intervening nucleotide sequence between the original sequence and the reverse complement can be of any length, including zero. In one embodiment, the ITRs useful in the present disclosure comprise one or more "palindromic sequences". An ITR can have any number of functions. In some embodiments, the ITRs described herein form a hairpin structure. In some embodiments, the ITR forms a T-shaped hairpin structure. In some embodiments, the ITRs form non-T-shaped hairpin structures, such as U-shaped hairpin structures. In some embodiments, the ITR promotes long-term survival of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITR promotes permanent survival (eg, for the entire lifespan of the cell) of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITR promotes the stability of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITR promotes retention of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITR promotes the persistence of the nucleic acid molecule in the nucleus of the cell. In some embodiments, the ITR inhibits or prevents degradation of the nucleic acid molecule in the nucleus of the cell.

Thus, an "ITR" as used herein can fold back on itself and form a double-stranded segment. For example, the sequence GATCXXXXGATC contains the initial sequence of GATC and its complement (3'CTAG5') when folded to form a double helix. In some embodiments, the ITR comprises a continuous palindromic sequence (eg, GATCGATC) between the original sequence and the reverse complement. In some embodiments, the ITR comprises an interrupted palindromic sequence (eg, GATCXXXXGATC) between the initial sequence and the reverse complement. In some embodiments, complementary portions of continuous or interrupted palindromic sequences 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 portion. In some embodiments, only a portion of the ITR forms a hairpin loop. In other embodiments, the entire ITR forms a hairpin loop.

In the present disclosure, at least one ITR is an ITR of a non-adeno-associated virus (non-AAV). In certain embodiments, the ITR is an ITR of a non-AAV member of the family Parvoviridae. In some embodiments, the ITR is an ITR of a non-AAV member of the genus Dependovirus or Erythrovirus . In specific embodiments, the ITR is the ITR of a goose parvovirus (GPV), muscovy duck parvovirus (MDPV), or rhodovirus parvovirus B19 (also referred to as parvovirus B19 - also referred to herein as "B19") , primate red parvovirus 1, B19 virus and rhodovirus). In certain embodiments, one of the two ITRs is an AAV ITR. In other embodiments, one of the two ITRs in the construct is an ITR of an AAV serotype selected from: serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and any combination thereof. In a particular embodiment, the ITR is derived from AAV serotype 2, eg, the ITR of AAV serotype 2.

In certain aspects of the present disclosure, a nucleic acid molecule comprises two ITRs, a 5' ITR and a 3' ITR, wherein the 5' ITR is located at the 5' end of the nucleic acid molecule and the 3' ITR is located at the 3' end of the nucleic acid molecule. The 5'ITR and 3'ITR can be derived from the same virus or different viruses. In certain embodiments, the 5' ITR is derived from AAV and the 3' ITR is not derived from an AAV virus (eg, non-AAV). In some embodiments, the 3' ITR is derived from AAV and the 5' ITR is not derived from an AAV virus (eg, non-AAV). In other embodiments, the 5' ITR is not derived from an AAV virus (eg, non-AAV), and the 3' ITR is derived from the same or a different non-AAV virus.

The term "parvovirus" as used herein encompasses the family Parvoviridae, including but not limited to autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include, for example, members of the following genera: Bocavirus , Dependovirus, Rhodovirus, Amdovirus , Parvovirus , Densovirus , Repeat Iteravirus , Contravirus , Aveparvovirus , Copiparvovirus , Protoparvovirus , Tetraparvovirus , Double Ambidensovirus , Brevidensovirus , Hepandensovirus and Penstyldensovirus .

Exemplary autonomous parvoviruses include, but are not limited to, porcine parvovirus, mouse parvovirus, canine parvovirus, mink enteritis virus, bovine parvovirus, chicken parvovirus, feline distemper 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 eg FIELDS et al. VIROLOGY, Volume 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers).

The term "non-AAV" as used herein encompasses nucleic acids, proteins and viruses from the Parvoviridae family, excluding any adeno-associated virus (AAV) of the Parvoviridae family. "Non-AAV" includes, but is not limited to, autonomously replicating members of the following genera: Bocavirus, Dependovirus, Rhodovirus, Aleutianovirus, Parvovirus, Densovirus, Repeatovirus, Contravirus genus, avian parvovirus, ruminant parvovirus, proparvovirus, type 4 parvovirus, ambisense densovirus, brevedensovirus, hepatopancreatic densovirus, and prawn densovirus.

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, Sheep AAV, Goat AAV, Shrimp AAV , those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)) and any others currently known or discovered in the future AAV. See eg FIELDS et al. VIROLOGY, Volume 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers).

As used herein, the term "derived from" means that a component is isolated from or prepared using a specified molecule or organism, or that information (eg, an amino acid or nucleic acid sequence) is derived from a specified molecule or organism. For example, a nucleic acid sequence (eg, ITR) derived from a second nucleic acid sequence (eg, ITR) may include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of nucleotides or polypeptides, derived species can be obtained, for example, by naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis. Mutagenesis for derivatizing nucleotides or polypeptides can be intentionally directed or intentionally random, or a mixture of each. Mutagenesis of a nucleotide or polypeptide to produce a different nucleotide or polypeptide derived from a first nucleotide or polypeptide may be a random event (e.g., due to distortion of the polymerase), and the derived nucleotide or polypeptide Identification can be by appropriate screening methods (eg, as discussed herein). Mutagenesis of a polypeptide generally requires manipulation of the polynucleotide encoding 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% identical to the second nucleotide or amino acid sequence, respectively. %, 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%, or 100% sequence identity, wherein the first A nucleotide or amino acid sequence retains the biological activity of a second nucleotide or amino acid sequence. In other embodiments, the ITRs derived from the non-AAV (or AAV) ITRs are at least 90% identical to the non-AAV ITRs (or respectively, AAV ITRs), wherein the non-AAV (or AAV) ITRs retain the non-AAV ITRs (or respectively ground, AAV ITR) functional properties. In some embodiments, ITRs derived from non-AAV (or AAV) ITRs are at least 80% identical to non-AAV ITRs (or respectively, AAV ITRs), wherein the non-AAV (or AAV) ITRs retain the non-AAV ITRs (or respectively ground, AAV ITR) functional properties. In some embodiments, ITRs derived from non-AAV (or AAV) ITRs are at least 70% identical to non-AAV ITRs (or respectively, AAV ITRs), wherein the non-AAV (or AAV) ITRs retain the non-AAV ITRs (or respectively ground, AAV ITR) functional properties. In some embodiments, ITRs derived from non-AAV (or AAV) ITRs are at least 60% identical to non-AAV ITRs (or respectively, AAV ITRs), wherein the non-AAV (or AAV) ITRs retain the non-AAV ITRs (or respectively ground, AAV ITR) functional properties. In some embodiments, ITRs derived from non-AAV (or AAV) ITRs are at least 50% identical to non-AAV ITRs (or respectively, AAV ITRs), wherein the non-AAV (or AAV) ITRs retain the non-AAV ITRs (or respectively ground, AAV ITR) functional properties.

In certain embodiments, an ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of a 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 a non-AAV (or AAV) ITR, wherein the fragment comprises 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 Nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides , at least about 125 nucleotides, at least about 150 nucleotides, at least about 175 nucleotides, at least about 200 nucleotides, at least about 225 nucleotides, at least about 250 nucleotides, at least About 275 nucleotides, at least about 300 nucleotides, at least about 325 nucleotides, at least about 350 nucleotides, at least about 375 nucleotides, at least about 400 nucleotides, at least about 425 nucleotides, at least about 450 nucleotides, at least about 475 nucleotides, at least about 500 nucleotides, at least about 525 nucleotides, at least about 550 nucleotides, at least about 575 nucleotides nucleotides or at least about 600 nucleotides; wherein the ITR derived from an ITR of a non-AAV (or AAV) retains the functional properties of a non-AAV ITR (or, respectively, an AAV ITR). In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of a non-AAV (or AAV) ITR, wherein the fragment comprises at least about 129 nucleotides, And wherein ITRs derived from non-AAV (or AAV) ITRs retain the functional properties of non-AAV ITRs (or, respectively, AAV ITRs). In certain embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of a non-AAV (or AAV) ITR, wherein the fragment comprises at least about 102 nucleotides, And wherein ITRs derived from non-AAV (or AAV) ITRs retain the functional properties of non-AAV ITRs (or, respectively, AAV ITRs).

In some embodiments, the ITR derived from a non-AAV (or AAV) ITR comprises or consists of a fragment of a non-AAV (or AAV) ITR, wherein the fragment comprises a non-AAV (or AAV) ITR 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% of the length %, 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%.

In certain embodiments, when correctly aligned, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence is homologous to the homologous portion of the second nucleotide or amino acid sequence, respectively. have 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% Or 100% sequence identity, wherein a first nucleotide or amino acid sequence retains the biological activity of a second nucleotide or amino acid sequence. In other embodiments, an ITR derived from an ITR of a non-AAV (or AAV) is at least 90% identical to a homologous portion of a non-AAV ITR (or, respectively, an AAV ITR) when properly aligned, wherein the first nucleotide The acid or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, an ITR derived from an ITR of a non-AAV (or AAV) is at least 80% identical to a homologous portion of a non-AAV ITR (or, respectively, an AAV ITR) when properly aligned, wherein the first nucleotide The acid or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, an ITR derived from an ITR of a non-AAV (or AAV) is at least 70% identical to a homologous portion of a non-AAV ITR (or, respectively, an AAV ITR) when properly aligned, wherein the first nucleotide The acid or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, an ITR derived from an ITR of a non-AAV (or AAV) is at least 60% identical to a homologous portion of a non-AAV ITR (or, respectively, an AAV ITR) when properly aligned, wherein the first nucleotide The acid or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence. In some embodiments, an ITR derived from an ITR of a non-AAV (or AAV) is at least 50% identical to a homologous portion of a non-AAV ITR (or, respectively, an AAV ITR) when properly aligned, wherein the first nucleotide The acid or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

A "capsid-free" or "capsid-less" vector or nucleic acid molecule refers to a vector construct that does not have a capsid. In some embodiments, the capsid-free vector or nucleic acid molecule is free of sequences encoding, for example, an AAV Rep protein. In some embodiments, a capsid-free or capsid-less vector may comprise covalently closed ends, referred to herein as "closed-end DNA (ceDNA)." Typically, the ceDNA described herein can be produced using the baculovirus expression system of the invention, and the ceDNA typically comprises at least one nucleic acid encoding a heterologous gene product flanked on either side by ITRs (e.g., AAV or non-AAV ITR).

As used herein, a "coding region" or "coding sequence" is the portion of a polynucleotide that consists of codons that can be translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not normally translated into an amino acid, it can be considered part of the coding region, but any flanking sequences (such as promoters, ribosomal binding sites, transcription terminators , introns, etc.) are not part of the coding region. The boundaries of the coding region are usually determined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxy terminus of the resulting polypeptide). Two or more coding regions can be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors ). As a result, then, a single vector may contain only a single coding region, or two or more coding regions.

Certain proteins secreted by mammalian cells are associated with a secretory signal peptide that is cleaved from the mature protein once the export of the growing protein chain across the rough endoplasmic reticulum has initiated. As is generally known to those skilled in the art, signal peptides are usually fused to the N-terminus of a polypeptide and are cleaved from the intact 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 this sequence that retains the ability to direct secretion of the polypeptide is operably associated with said polypeptide. Alternatively, a heterologous mammalian signal peptide (eg, human tissue plasminogen promoter (TPA) or mouse β-glucuronidase signal peptide) or a functional derivative thereof can be used.

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

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

As used herein, the term "gene cassette" or "expression cassette" means a DNA sequence capable of directing the expression of a particular polynucleotide sequence in a suitable host cell, comprising a promoter operably linked to the polynucleotide sequence of interest . A gene cassette can encompass nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of the coding region and which affect the transcription, RNA processing, stability or translation of the associated coding region. If the coding region is intended to be expressed in eukaryotic cells, a polyadenylation signal and transcription termination sequence will generally be located 3' to the coding sequence. In some embodiments, a gene cassette comprises a polynucleotide encoding a gene product. In some embodiments, the gene cassette comprises a polynucleotide encoding a miRNA. In some embodiments, the gene cassette comprises a heterologous polynucleotide sequence.

A polynucleotide encoding a product (e.g., miRNA or gene product (e.g., a polypeptide, such as a therapeutic protein)) may include a promoter and/or other expression (e.g., transcription or translation) control sequences. In operable association, a coding region for a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in a manner that places the expression of the gene product under the influence or control of the one or more regulatory regions . For example, if induction of promoter function results in the transcription of mRNA encoding a gene product (encoded by the coding region), and if the nature of the linkage between the promoter and coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with A coding region and a promoter are "operably associated" if the DNA template is capable of transcription. Expression control sequences other than promoters (eg, enhancers, operators, repressors, and transcription termination signals) may also be operably associated with the coding region to direct gene product expression.

"Expression control sequence" refers to a regulatory nucleotide sequence that provides for the expression of a coding sequence in a host cell, such as a promoter, an enhancer, a terminator, and the like. Expression control sequences generally encompass any regulatory nucleotide sequence that facilitates the efficient transcription and translation of an encoding nucleic acid to which it is operably linked. Non-limiting examples of expression control sequences include promoters, enhancers, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. Various presentation control sequences are known to those skilled in the art. These include, but are not limited to, expression control sequences functional in vertebrate cells, such as, but not limited to, promoter and enhancer fragments from cytomegalovirus (immediate early promoter, combined 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 protein, bovine growth hormone, and rabbit beta globin, and others capable of controlling gene expression in eukaryotic cells. Additional suitable expression control sequences include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters inducible by interferons or interleukins). Other expression control sequences include intronic sequences, post-transcriptional regulatory elements, and polyadenylation signals. Additional exemplary presentation control sequences are discussed elsewhere in this disclosure.

Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (notably the internal ribosome entry site, or IRES, also known as the CITE sequence).

The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product (eg, RNA or polypeptide). It includes without limitation transcription of polynucleotides into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product and translation of mRNA into polypeptides. Manifestation produces a "gene product". As used herein, a gene product may be a nucleic acid, such as messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids that have been post-transcriptionally modified, such as polyadenylation or splicing, or that have been post-translationally modified, such as methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage) of peptides. As used herein, the term "yield" refers to the amount of polypeptide produced by gene expression.

"Vector" refers to any vehicle used for the selection and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be ligated to effect replication of the ligated segment. "Replicon" refers to any genetic element (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous replicating unit in vivo (ie, capable of replicating under its own control). The term "vector" includes vehicles for introducing nucleic acid into cells in vitro, ex vivo or in vivo. Many vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of polynucleotides into suitable vectors can be accomplished by ligating appropriate polynucleotide fragments into selection vectors with complementary cohesive ends.

Vectors can be engineered to encode selectable markers or reporters that provide for selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express additional coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: conferring resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc. and genes used as phenotypic markers, i.e. anthocyanin regulatory genes, isopentyltransferase genes, etc. Examples of reporters known and used in the industry include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β - Glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

The term "host cell" as used herein refers to, for example, microorganisms, yeast cells, insect cells and mammalian cells that can or have been used as recipients of ssDNA or vectors. The term includes progeny of a primary cell that has been transduced. Thus, a "host cell" as used herein generally refers to a cell that has been transduced with an exogenous DNA sequence. It is understood that the morphology or genome or total DNA complement of progeny of a single parent cell may not necessarily be identical to the original parent due to natural, accidental or deliberate mutation. In some embodiments, the host cell can be an in vitro host cell.

The term "selectable marker" refers to an identifier (usually an antibiotic or chemoresistance genes), wherein the effect is used to track the inheritance of the nucleic acid of interest and/or to identify cells or organisms that have inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: conferring resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc. and genes used as phenotypic markers, i.e. anthocyanin regulatory genes, isopentyltransferase genes, etc.

The term "reporter gene" refers to a nucleic acid encoding an identification factor that can be identified based on the effect of the reporter gene for tracking the heritability of a nucleic acid of interest, identifying cells or organisms that have inherited a nucleic acid of interest, and/or Measure gene expression induction or transcription. Examples of reporter genes known and used in the industry include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β- - Glucuronidase (Gus), etc. A selectable marker gene can also be considered a reporter gene.

"Promoter" is used interchangeably with "promoter sequence" and refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. The promoter may be derived in its entirety from a native gene, or consist of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most of the time are generally referred to as "constitutive promoters". Promoters that cause a gene to be expressed in a particular cell type are generally referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause genes to be expressed at specific stages of development or cell differentiation are generally referred to as "development-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause gene expression after exposure or treatment of cells with promoter-inducing agents, biomolecules, chemicals, ligands, light, etc. are generally called "inducible promoters" or "regulatory promoters" ". It is also recognized that since in most cases the exact boundaries of the regulatory sequences have not been fully defined, DNA fragments of different lengths can have the same promoter activity.

A promoter sequence is usually 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 levels detectable above background . Within the promoter sequence will be found a transcription initiation site (conveniently defined eg by mapping with nuclease S1 ) as well as a protein binding domain (consensus sequence) responsible for the binding of RNA polymerase.

In some embodiments, the nucleic acid molecule comprises a tissue-specific promoter. In certain embodiments, a tissue-specific promoter drives expression of a therapeutic protein (eg, a coagulation factor) in the liver (eg, in hepatocytes and/or endothelial cells). In a particular embodiment, 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, triple tetraproline (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, and any combination. In some embodiments, the promoter is selected from liver-specific promoters (e.g., α1-antitrypsin (AAT)), muscle-specific promoters (e.g., muscle creatine kinase (MCK), myosin heavy chain α (αMHC), myoglobin (MB), and desmin (DES)), synthetic promoters (eg, SPc5-12, 2R5Sc5-12, dMCK, and tMCK), and any combination thereof. In a specific embodiment, the promoter comprises a TTP promoter.

The term "restriction endonuclease" is used interchangeably with "restriction enzyme" and refers to an enzyme that binds and cleaves within a specific sequence of nucleotides within double-stranded DNA.

The term "plasmid" refers to an extrachromosomal element, usually carrying genes that are not part of the central metabolism of the cell, and usually in the form of a circular double-stranded DNA molecule. Such elements may be linear, circular or supercoiled autonomously replicating sequences, genomic integrating sequences, bacteriophage or nucleotide sequences derived from single- or double-stranded DNA or RNA from any source, wherein multiple nucleotide sequences have been spliced or recombined into a unique construct capable of introducing the promoter fragment and DNA sequence for the selected gene product and the appropriate 3' non-translated sequence into the cell.

Eukaryotic viral vectors that may be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, poxviruses (eg, vaccinia virus vectors), baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cell transfection agents), DNA-protein complexes, and biopolymers.

"Cloning vector" refers to a "replicon", which is a unit length of continuously replicating nucleic acid and which contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment can be ligated to achieve the desired Replication of connected segments. Certain cloning vectors are capable of replicating in one cell type (eg, bacteria) and expressing it in another cell type (eg, eukaryotic cells). Colonization vectors typically contain one or more sequences that can be used to select cells comprising the vector and/or one or more multiplex cloning sites for insertion of a nucleic acid sequence of interest.

The term "expression vector" refers to a vehicle designed to enable expression of an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with the regulatory region as described above.

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 ), using gene guns or DNA vector transporters. As used herein, "culture", "to culture" and "culturing" means to grow cells in vitro under conditions that allow the cells to grow or divide or maintain the cells in a viable state. As used herein, "cultured cells" refers to cells propagated in vitro.

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, as the case may be, the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by a match between strings of such sequences. "Identity" can be readily calculated by known methods including, but not limited to: 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 ed.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G. ed. ) Academic Press (1987); and Sequence Analysis Primer (eds. Gribskov, M. and Devereux, J.) Stockton Press, New York (1991). Preferred methods of determining identity are designed to give the best match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Sequence alignments 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, Wisconsin), the GCG program suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215 :403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wisconsin 53715 USA). It will be understood in the context of this application that if sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the referenced program unless otherwise specified. "Default values" as used herein shall mean any set of values or parameters initially loaded with software upon first initialization.

As used herein, nucleotides corresponding to nucleotides in a particular sequence of the disclosure are identified by aligning the sequences of the disclosure to maximize identity to a reference sequence. The numbering used to identify equivalent amino acids in the reference sequence is based on the numbering used to identify the corresponding amino acid in the sequences of the disclosure.

As used herein, the terms "heterologous" or "exogenous" mean that such molecules are not normally found in a given context (eg, in a cell or in a polypeptide). For example, an exogenous or heterologous molecule may be introduced into a cell and be present only after the cell has been manipulated, eg, by transfection or other form of genetic engineering, or a heterologous amino acid sequence may be present in a protein where it does not occur naturally.

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 capable of extending the half-life of a therapeutic protein (eg, a blood clotting factor such as FVIII). In another embodiment, the heterologous nucleotide sequence encodes a polypeptide that increases the hydrodynamic radius of a therapeutic protein (eg, a coagulation factor such as FVIII). In other embodiments, the heterologous nucleotide sequence encodes a polypeptide that improves one or more pharmacokinetic properties of the Therapeutic protein without significantly affecting its biological activity or function (eg, procoagulant activity). In some embodiments, the Therapeutic protein is linked or connected to the polypeptide encoded by the heterologous nucleotide sequence via a linker. Non-limiting examples of polypeptide portions encoded by heterologous nucleotide sequences include, inter alia, immunoglobulin constant regions or portions thereof, albumin or fragments thereof, albumin binding portions, transferrin, the PAS polypeptides of US Patent Application No. 20100292130, HAP sequence, transferrin or fragments thereof, C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, albumin-binding small molecule, XTEN sequence, FcRn-binding portion (e.g., the entire Fc region or its binding to Part of FcRn), single chain Fc region (ScFc region, e.g. as described in US 2008/0260738, WO 2008/012543 or WO 2008/1439545), polyglycine linker, polyserine linker, selected from The 6- Peptides and short polypeptides of 40 amino acids (with degrees of secondary structure varying from less than 50% to greater 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.

As used herein, the term "optimized" in reference to a nucleotide sequence refers to a polynucleotide sequence encoding a polypeptide, wherein 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 binding of regulatory proteins such as general transcription factors, increase or decrease splicing, or increase polypeptides produced by a polynucleotide sequence output. Examples of changes that can be made to a polynucleotide sequence to optimize it include codon optimization, G/C content optimization, removal of repetitive sequences, removal of AT-rich elements, removal of cryptic splice sites, removal of cis elements, adding or removing poly-T or poly-A sequences, adding transcription-enhancing sequences (such as Kozak consensus sequences) around the transcription start site, removing sequences that can form stem-loop structures, removing destabilizing sequences and their A combination of two or more.

As used herein, the term "baculovirus shuttle vector" refers to a shuttle vector that can propagate in both E. coli and insect cells. A recombinant baculovirus shuttle vector refers to a baculovirus shuttle vector comprising a heterologous sequence (eg, a heterologous sequence encoding a heterologous gene). II. Baculovirus expression vector system

Provided herein is a baculovirus expression vector system comprising a baculovirus shuttle vector (baculovirus shuttle vector) and/or a stable cell line engineered to produce a therapeutic drug product. The baculovirus expression vector system provided herein comprises a recombinant baculovirus shuttle vector containing two or more sites for insertion of foreign sequences, and one or more sites capable of mediating the insertion of foreign sequences (eg, heterologous genes) Donor vector in the foreign sequence insertion site.

In some embodiments, the therapeutic drug product is a protein. In some embodiments, the therapeutic drug product is a nucleic acid. In some embodiments, therapeutic drug products are recombinant proteins that are useful, for example, in various applications including vaccines, protein replacement therapy (eg, enzyme replacement therapy), and recombinant proteins for basic and applied research. In certain embodiments, the therapeutic drug product is a DNA therapeutic drug substance, which is a plasmid-like, capsid-free nucleic acid molecule encoding a sequence of interest. DNA therapeutic drug substances may be in the form of covalently closed-end DNA (ceDNA). Typically, ceDNA contains a therapeutic protein-coding gene located between the first inverted terminal repeat (5' ITR) and the second ITR (3' ITR).

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 ITR is an ITR obtained from a member of the virus family Parvoviridae. 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 PCT Publication Nos. WO 2019032898A1 , WO 2020033863A1 , and WO 2017152149A1 , 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"). A. Baculovirus Shuttle Vector (Bacmid)

Baculoviruses are the most prominent insect-infecting viruses. More than 500 baculovirus isolates have been identified, most of which are derived from insects of the order Lepidoptera . The two most common isolates are Autographa californica multinucleated polyhedrosis virus (AcMNPV) and silkworm ( Bombyx mori ) nuclear polyhedrosis virus (BmNPV). In the production of viral or nonviral vectors for gene therapy, it is often necessary to infect insect host cells with several baculovirus expression vectors. The production of each baculovirus expression vector is time consuming and drives up production costs, which represents a significant disadvantage of the baculovirus expression vector system prior to the present invention.

In certain embodiments, the baculovirus expression vector systems provided herein comprise a recombinant baculovirus shuttle vector, referred to herein as "BIVVBac." A representative schematic of BIVVBac is shown in Figure 1C . In certain embodiments, BIVVBac is a genetically modified AcMNPV comprising at least two foreign sequence insertion sites. A baculovirus expression vector system comprising a baculovirus shuttle vector containing at least two foreign sequence insertion sites allows reducing the total number of baculovirus expression vectors that need to be produced. In some embodiments, a single baculovirus expression vector of the invention is required to generate a gene therapy vector.

In certain embodiments, BIVVBac comprises a first foreign sequence insertion site and a second foreign sequence insertion site. The first foreign sequence insertion site and the second foreign sequence insertion site can be different, thereby utilizing different mechanisms to drive the insertion of the foreign sequence (eg, heterologous sequence, heterologous gene). Insertion of foreign sequences can be driven by any method known in the art. For example, foreign sequences can be inserted by transposition or site-specific recombination. The foreign sequence insertion site can be designed to be included within the reporter gene such that upon insertion of the foreign sequence, the reporter gene is disrupted. Disruption of the reporter gene can aid in the identification of baculovirus shuttle vector clones into which foreign sequences have been inserted. In such embodiments, the foreign sequence insertion site is fused in frame to the reporter gene, or the reporter gene is fused in frame to the foreign sequence insertion site.

In certain embodiments, the first foreign sequence insertion site and the second foreign sequence insertion site are located in different loci within AcMNPV. In certain embodiments, the first foreign sequence insertion site and the second foreign sequence insertion site are located in different non-essential loci within AcMNPV. Various non-essential loci of AcMNPV are known to those skilled in the art. For example, the polyhedrin gene and the EGT gene are non-essential AcMNPV genes for viral replication in insect cells. Thus, 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.

In certain embodiments, the first foreign sequence insertion site allows insertion of a foreign sequence via translocation. In certain embodiments, the first foreign sequence insertion site comprises a preferential target site for insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a preferential target site for insertion of a transposon. In certain embodiments, the first foreign sequence insertion site is a preferential target site for the attachment site of a bacterial transposon. Suitable bacterial transposons and their corresponding attachment sites are known to those skilled in the art. For example, the transposon Tn7 is known for its ability to translocate at high frequency to a specific site (attTn7) on bacterial chromosomes. Thus, in certain embodiments, the first foreign sequence insertion site is a preferential target site (eg, attTn7) as an attachment site for the Tn7 transposon. In some embodiments, the first foreign sequence insertion site is a preferential target site as an attachment site for a mini-Tn7 transposon (e.g., mini-attTn7, the minimal DNA required for Tn7 transposable element recognition and insertion into a Tn7 transposon sequence).

In certain embodiments, the first foreign sequence insertion site is fused in-frame to the reporter gene. The reporter gene can be any reporter gene known in the art, including, for example, luciferase (luciferase, Luc), green fluorescent protein (GFP), red fluorescent protein (RFP), fluorescent proteins of specific colors, chloride Mycin acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. In certain embodiments, the first foreign sequence insertion site is fused in-frame to a reporter gene encoding an enzyme capable of metabolizing a chromogenic substrate. The enzyme capable of metabolizing the chromogenic substrate may be any enzyme known in the art with the same properties, such as LacZ or a functional part thereof. A reporter gene encoding an enzyme capable of metabolizing a chromogenic substrate can be used for color-based screening of positive insertion events. For example, LacZ can be used in a blue-white screen, where a functional LacZ gene product cleaves the chromogenic substrate X-gal or blue-gal, thereby producing a blue pigment. In a blue-white screen, the presence of functional LacZ as measured by alpha complementation means that the insertion event failed, and blue colonies represent colonies that did not insert the foreign sequence. Alternatively, white colonies represent colonies in which foreign sequences were successfully inserted, disrupting production of a functional LacZ gene product and preventing cleavage of chromogenic substrates such as X-gal.

Thus, in certain embodiments, the first foreign sequence insertion site is a preferential target site for a bacterial transposon fused in-frame to a sequence encoding LacZα or a functional portion thereof. In certain embodiments, the first foreign sequence insertion site is a preferential target site for an attachment site for a Tn7 transposon (eg, attTn7) fused in-frame to a sequence encoding LacZα or a functional portion thereof. After successful transposition, the transposon disrupts the sequence encoding LacZα, thereby disrupting the production of a functional LacZ gene product.

In certain embodiments, the second foreign sequence insertion site allows insertion of foreign sequences via site-specific recombination. In certain embodiments, the second foreign sequence insertion site comprises a preferential target site capable of mediating 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 via Cre recombinase, which recognizes a 34 base pair DNA sequence called a loxP site. Thus, the second foreign sequence insertion site is a preferential target site for Cre-mediated recombination. In certain embodiments, the second foreign sequence insertion site is a preferential target site comprising a loxP site or a variant thereof recognized by Cre recombinase.

The recombinant baculovirus shuttle vectors of the invention contain additional elements required for their ability to propagate in both bacteria (eg, E. coli ) and insect cells. For example, the recombinant baculovirus shuttle vectors of the invention comprise bacterial replicons. In certain embodiments, the baculovirus shuttle vector comprises a bacterial replicon. Various bacterial replicons are known to those skilled in the art and include, for example, F plasmid-derived replicons. In certain embodiments, a suitable bacterial replicon is a miniature F replicon, which is a derivative of an F plasmid composed of the DNA regions oriS and incC required 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.

Other elements of the recombinant baculovirus shuttle vectors of the invention include one or more selectable marker sequences, and other reporter genes. Examples of selectable marker genes known and used in the art include: conferring resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicides, sulfonamides, etc. and genes used as phenotypic markers, i.e. anthocyanin regulatory genes, isopentyltransferase genes, etc. In certain embodiments, the recombinant baculovirus shuttle vector comprises a selectable marker sequence comprising 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 industry include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β - Glucuronidase (Gus), etc. In some cases, selectable markers can also be considered reporters. In certain embodiments, the recombinant baculovirus shuttle vector comprises a reporter gene encoding a fluorescent protein. In certain embodiments, the fluorescent protein is red fluorescent protein.

Those skilled in the art will recognize that the various elements of the recombinant baculovirus shuttle vectors described herein are operably linked to each other. Each of the various coding sequences in the baculovirus shuttle vector may be operably linked to a regulatory region comprising, for example, a promoter sequence. Any promoter sequence known in the art may be suitable.

Thus, the recombinant baculovirus shuttle vector of the present invention comprises a miniature F replicon, an antibiotic resistance gene, LacZα or a functional part thereof comprising an attachment site for a Tn7 transposon, a baculovirus-inducible promoter operably linked to Genes encoding fluorescent proteins and LoxP sites or variants thereof.

In certain exemplary embodiments, the recombinant baculovirus shuttle vector of the present invention is BIVVBac as shown in Figure 1C . In certain embodiments, BIVVBac encodes the AcMNPV C6 genome and has been engineered to encode two foreign sequence insertion sites: 1) mini- att Tn7 in the polyhedrin locus and 2) LoxP in the EGT locus. Combining the miniature att Tn7 insert with the Lac promoter-driven E. coli LacZα fragment for X-gal-mediated blue/white selection of recombinant baculovirus shuttle vectors following Tn7-mediated translocation and for Cre-mediated translocation Guided in-frame fusion of LoxP sites for in vitro or in vivo recombination. BIVVBac also contains the red fluorescent protein gene under the control of the AcMNPV 39K promoter followed by the ets polyadenylation signal.

In certain exemplary embodiments, BIVVBac is introduced into a bacterial strain capable of permissive Tn7-mediated translocation. In certain embodiments, the bacterial strain is an E. coli strain. In certain embodiments, the bacterial strain is E. coli DH10B bacteria. In certain embodiments, helper plasmids encoding Tn7 translocase genes tnsA-E and tetracycline resistance were introduced into DH10B E. coli with BIVVBac. DH10B E. coli with BIVVBac and helper plasmids is referred to herein as BIVVBac ^DH10B .

Any foreign sequence (eg, heterologous sequence) can be introduced into BIVVBac via Tn7-mediated translocation. In certain embodiments, the foreign sequence is introduced into BIVVBac via Tn7-mediated translocation by introducing a Tn7 transfer vector containing the foreign sequence into BIVVBac ^DH10B , thereby generating BIVVBac containing the foreign sequence. In certain embodiments, the foreign sequence-containing BIVVBac comprises a bacterial replicon; a first selectable marker sequence; a foreign sequence (e.g., a heterologous sequence) inserted into a first reporter gene, wherein the inserted foreign sequence disrupts the a reading frame of the first reporter gene; a second reporter gene operably linked to a baculovirus-inducible promoter; and a preferential target site capable of mediating site-specific recombination events.

In certain exemplary embodiments, AAV or non-AAV Rep genes are introduced into BIVVBac via Tn7-mediated translocation for the purpose of generating gene therapy vectors. In certain embodiments, a Tn7 transfer vector comprising an AAV or non-AAV Rep gene is introduced into BIVVBac ^DH10B , thereby generating a BIVVBac comprising a Rep gene. In certain embodiments, the recombinant baculovirus shuttle vector comprises: a miniature F replicon; a first antibiotic resistance gene; a sequence encoding B19 Rep inserted into LacZα or a functional portion thereof, wherein the inserted B19 Rep disrupts A 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 baculovirus shuttle vector comprises: a miniature F replicon; a first antibiotic resistance gene; a sequence encoding a GPV Rep inserted into LacZα or a functional portion thereof, wherein the inserted GPV Rep disrupts A 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 baculovirus shuttle vector comprises: a miniature F replicon; a first antibiotic resistance gene; a sequence encoding AAV2 Rep inserted into LacZα or a functional portion thereof, wherein the inserted AAV2 Rep disrupts A 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.

Any foreign sequence (eg, heterologous sequence) can be introduced into BIVVBac via Cre-mediated recombination. In certain embodiments, foreign sequences are introduced into BIVVBac via CRE-mediated recombination, wherein the foreign sequences are located on the Cre-LoxP transfer vectors described herein. Methods of CRE-mediated recombination are well known to those skilled in the art.

In certain exemplary embodiments, for the purpose of generating gene therapy vectors, a therapeutic protein encoding flanked by symmetric or asymmetric AAV or non-AAV inverted terminal repeats (ITRs) is encoded via CRE-mediated recombination. The gene was introduced into BIVVBac. In certain embodiments, the recombinant baculovirus shuttle vector comprises: a miniature F replicon; an antibiotic resistance gene; LacZα or a functional portion thereof comprising an attachment site for a T7 transposon; A first priority target site (e.g., a first LoxP site); a therapeutic protein-coding gene flanked by symmetric or asymmetric AAV or non-AAV ITRs; and a second priority capable of mediating site-specific recombination events Target site (eg, second LoxP site).

Thus, for the purpose of generating gene therapy vectors, the recombinant baculovirus shuttle vector comprises: a sequence encoding B19 Rep inserted into LacZα or a functional part thereof, wherein the inserted B19 Rep disrupts the reading frame of LacZα or a functional part thereof and a multiplex colony site comprising a heterologous sequence 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 said sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from parvovirus B19.

Thus, for the purpose of generating gene therapy vectors, the recombinant baculovirus shuttle vector comprises: a sequence encoding a GPV Rep inserted into LacZα or a functional part thereof, wherein the inserted GPV Rep disrupts the reading frame of LacZα or a functional part thereof and a multiplex cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises 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 said sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from GPV.

Thus, for the purpose of generating gene therapy vectors, the recombinant baculovirus shuttle vector comprises: a sequence encoding AAV2 Rep inserted into LacZα or a functional part thereof, wherein the inserted AAV2 Rep disrupts the reading frame of LacZα or a functional part thereof and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises 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 said sequence encoding the protein; and a wild-type or truncated 3' inverted terminal repeat sequence derived from AAV2.

Thus, for the purpose of generating gene therapy vectors, the recombinant baculovirus shuttle vector comprises: a sequence encoding human bocavirus (HBoV1) Rep inserted into the miniature attTn7 site, LacZα or a functional part thereof, wherein the inserted HBoV1 Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple colonization site comprising a heterologous sequence comprising from 5' to 3': a wild-type 5' inverted terminal repeat derived from HBoV1 sequence; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type 3' inverted terminal repeat sequence derived from HBoV1.

In certain embodiments, closed-end DNA (ceDNA) is generated using the BIVVBac baculovirus shuttle vector. In some embodiments, a single baculovirus expression vector is used to generate ceDNA. In this "single BAC" approach, a single baculovirus expression vector (e.g., BIVVBac) encodes all the essential elements required to produce ceDNA in a baculovirus system, and potentially in any baculovirus-permissive cell line Used to generate ceDNA. This approach is depicted in Figure 13A .

In certain embodiments, multiple baculovirus expression vectors are used to generate ceDNA. In this "dual BAC" approach, the essential elements required for ceDNA production are inserted into two different baculoviruses (e.g., two BIVVBac baculovirus shuttle vectors) and are potentially used in baculovirus-tolerant Co-infection can be performed in any cell line infected with the virus. This approach is depicted in Figure 13B .

In certain embodiments, ceDNA is produced by stable cell lines. In this approach, the essential elements required for ceDNA production are inserted into two components of the baculovirus system. This approach is depicted in Figure 13C . Stable cell lines can be generated by stably integrating protein coding sequences under the control of a baculovirus gene promoter (eg, a baculovirus constitutive gene promoter). In certain embodiments, the stable cell line is a stable insect cell line. B. Transfer vector

The recombinant baculovirus shuttle vectors described herein comprise at least two foreign sequence insertion sites. In certain embodiments, the at least two foreign sequence insertion sites are 1) miniatt Tn7 for Tn7-mediated translocation, and 2) LoxP for Cre-mediated site-specific recombination. Therefore, the baculovirus expression vector system of the present invention further comprises two or more transfer vectors comprising the foreign sequence insertion site to be inserted into the recombinant baculovirus shuttle vector. foreign sequence. Tn7 transfer vector

In certain embodiments, the transfer vector comprises foreign sequences to be inserted into the miniature attTn7 of the recombinant baculovirus shuttle vector. In certain embodiments, the transfer vector is a Tn7 transfer vector. In certain embodiments, the transfer vector is a miniature Tn7 transfer vector. In certain embodiments, the Tn7 transfer vector comprises the left and right ends of Tn7 (Tn7L and Tn7R) capable of mediating translocation of foreign sequences into miniature attTn7 of the recombinant baculovirus shuttle vector.

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

In certain embodiments, the Rep coding sequence is modified such that the canonical start codon of Rep78 is modified to a non-canonical start codon. In certain embodiments, the other AUG codons preceding the start codon of Rep52 are mutated to carry silent mutations (in case of out-of-frame) or to encode conservative amino acid substitutions (in case of in-frame) to Allows expression of Rep78 and Rep52 polypeptides from a single mRNA transcript by a ribosomal omission scanning mechanism.

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

In certain embodiments, the transfer vector is a Cre-LoxP transfer vector. In certain embodiments, the Cre-LoxP transfer vector is a nucleic acid vector comprising: a first origin of replication for propagating the nucleic acid vector in a first bacterial strain, wherein the first origin of replication is conditional a sexual origin of replication; a second origin of replication for propagating the nucleic acid vector in a second bacterial strain; a multiplex colony site for insertion of foreign sequences (eg, heterologous sequences); a selectable marker sequence; a reporter gene; and/or preferential target sites capable of mediating site-specific recombination events. In certain embodiments, the first origin of replication is conditional on the presence of the pi protein, such as the R6Kγ origin of replication. In certain embodiments, the first origin of replication is the R6Kγ origin of replication. Thus, in certain embodiments, the first bacterial strain comprises a pi protein. In certain embodiments, the second origin of replication is the pUC57 origin of replication. In certain embodiments, the preferential target sites capable of mediating site-specific recombination events comprise LoxP sites or variants thereof, and the site-specific recombination events are mediated by Cre recombinase. The Cre-LoxP transfer vectors described herein may have any other elements suitable for proper identification of the host cell comprising 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 vectors described herein.

In certain embodiments, the Cre-LoxP transfer vector is a pUC57 vector comprising: a LoxP recombination site, the AcMNPV ie1 promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal Enhanced GFP (eGFP) marker gene, ampicillin resistance marker, conditional R6Kγ origin of replication and multiple cloning sites under Features of the Cre-LoxP transfer vector include: 1) multiple selection site (MCS) for insertion of transgenes; 2) LoxP site for Cre-mediated recombination in vitro or in vivo; 3) two origins of replication , the two origins of replication include: i) the ColE1 Ori used to propagate the vector into E. coli strains such as DH5α, NEB Stable, PMC103 and DH10B, and ii) the ColE1 Ori used to propagate the vector to express π ( pi) Conditional R6Kγ Ori in E. coli strains (such as pir+ and pir116 ) of the protein; 4) for use with Kang after insertion of the Cre-LoxP donor vector into the LoxP site in BIVVBac by Cre-mediated recombination ampicillin and ampicillin for selection of the ampicillin resistance gene of recombinant baculovirus shuttle vectors; and 5) for the determination of the stability of the transgene in recombinant BEV while enhancing replication in insect cells through serial passage Type GFP reporter gene. Those skilled in the art will appreciate that whether multiple cloning sites are maintained in a Cre-LoxP transfer vector containing foreign sequences (e.g., transgenes) depends on the manner in which the foreign sequences are inserted (e.g., by using terminal restriction sites point or use of non-terminal restriction sites, or the nature of the restriction enzyme used to colonize the foreign sequence).

In certain exemplary embodiments, the Cre-LoxP transfer vector comprises foreign sequences (eg, heterologous sequences) for the purpose of generating gene therapy vectors. For example, in certain embodiments, the Cre-LoxP transfer vector includes a therapeutic protein-encoding gene flanked by symmetric or asymmetric AAV or non-AAV inverted terminal repeats (ITRs). In certain embodiments, a therapeutic protein-coding gene flanked by symmetric or asymmetric AAV or non-AAV ITRs is located in the position of the multiplex colony site of the Cre-LoxP transfer vector.

Those skilled in the art will recognize that Tn7 transfer vectors and Cre-LoxP transfer vectors are examples of transfer vectors that can be used to insert foreign sequences to generate recombinant baculovirus shuttle vectors of the invention. In certain embodiments, the foreign sequences to be inserted using the transfer vectors described herein are interchangeable between transfer vectors. For example, for the purpose of generating gene therapy vectors, a Rep gene can be introduced into a recombinant baculovirus shuttle vector via a Tn7 transfer vector, and a therapeutic protein-coding gene can be introduced via a Cre-LoxP transfer vector. In another example, for the purpose of generating gene therapy vectors, a Rep gene can be introduced into a recombinant baculovirus shuttle vector via a Cre-LoxP transfer vector, and a therapeutic protein-coding gene can be introduced via a Tn7 transfer vector.

In certain embodiments, the Cre-LoxP transfer vectors of the present invention comprise a LoxP sequence and an enhanced green fluorescent protein (eGFP) encoding gene, and are used for breeding therapeutic protein encoding genes (e.g., with flanking symmetric or hFVIIIco6XTEN with asymmetric ITR; where ITR is non-AAV or AAV).

In certain embodiments, a recombinant baculovirus shuttle vector is provided in which the Rep-encoding gene is inserted via translocation at the miniature att Tn7 site in the polyhedrin locus, and via Cre-mediated recombination at the EGT gene A therapeutic protein-coding gene with symmetric or asymmetric ITR is inserted at the LoxP site in the locus. In certain embodiments, the Rep gene and ITR are non-AAV or AAV.

Accordingly, in certain embodiments, there is provided a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniatt Tn7 site within LacZα ) encoding an AAV rep (e.g., AAV2 rep), wherein the inserted Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiplex cloning site comprising a heterologous sequence, wherein the heterologous sequence ranges from 5 'to 3' comprises: a wild-type or truncated AAV 5' inverted terminal repeat; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated Short form AAV 3' inverted terminal repeat.

In certain embodiments, there is provided a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional part thereof (for example, inserted into the miniature att Tn7 site within LacZα) A sequence encoding a non-AAV Rep, wherein the inserted Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises, from 5' to 3': derived from A wild-type or truncated non-AAV 5' inverted terminal repeat sequence from the first parvoviral non-AAV genome; a protein-encoding sequence; one or more expression control sequences operably linked to said protein-encoding sequence; and Wild-type or truncated non-AAV 3' inverted terminal repeat derived from the second parvovirus non-AAV genome. In certain embodiments, said first parvoviral non-AAV genome and said second parvoviral non-AAV genome are different.

In certain embodiments, provided herein is a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniature att Tn7 site within LacZα) A sequence encoding a B19 Rep , wherein the inserted B19 Rep disrupts the reading frame of LacZα or a functional part thereof; and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat sequence derived from parvovirus B19; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a sequence derived from parvovirus B19 wild-type or truncated 3' inverted terminal repeat.

In certain embodiments, provided herein is a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniattTn7 site within LacZα) A sequence encoding a GPV Rep, wherein the inserted GPV Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': derived A wild-type or truncated 5' inverted terminal repeat sequence from GPV; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated sequence derived from GPV Short 3' inverted terminal repeat.

In certain embodiments, provided herein is a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniattTn7 site within LacZα) A sequence encoding an AAV2 Rep, wherein the inserted AAV2 Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising a heterologous sequence comprising, from 5' to 3': derived from A wild-type or truncated 5' inverted terminal repeat sequence from AAV2; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated sequence derived from AAV2 Short 3' inverted terminal repeat.

In certain embodiments, provided herein is a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniattTn7 site within LacZα) A sequence encoding HBoV1 Rep, wherein the inserted HBoV1 Rep disrupts the reading frame of LacZα or a functional part thereof; and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': derived A wild-type 5' inverted terminal repeat from HBoV1; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type 3' inverted terminal repeat derived from HBoV1 sequence.

In certain embodiments, there is provided a recombinant baculovirus shuttle vector comprising: inserted into LacZα or a functional portion thereof (e.g., inserted into the miniatt Tn7 site within LacZα) A sequence encoding a non-AAV Rep, wherein the inserted Rep disrupts the reading frame of LacZα or a functional portion thereof; and a multiple cloning site comprising a heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': wild type or truncated non-AAV 5' inverted terminal repeat; a sequence encoding a protein; one or more expression control sequences operably linked to said sequence encoding a protein; and a wild-type or truncated non-AAV 3' Inverted terminal repeats. In certain embodiments, the non-AAV Rep and the non-AAV inverted terminal repeat are selected from the ITRs of the non-AAV genome of the family Parvoviridae of the family Parvoviridae. In certain embodiments, the 5' ITR and 3' ITR are of the same parvoviral non-AAV genome. In certain embodiments, the 5' ITR and 3' ITR are of different parvoviral non-AAV genomes. In such embodiments, the non-AAV Rep is a Rep of at least one parvovirus non-AAV genome. In such embodiments, one skilled in the art will be able to determine the most suitable Rep for use in embodiments wherein the 5'ITR and 3'ITR are of different parvovirus non-AAV genomes.

In certain embodiments, the recombinant baculovirus shuttle vector comprises a wild-type or truncated AAV 5' ITR and a wild-type or truncated parvovirus non-AAV 3' ITR. In certain embodiments, the recombinant baculovirus shuttle vector comprises a wild-type or truncated parvovirus 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 for use in embodiments wherein the 5'ITR and 3'ITR are of different parvoviral genomes. III. Foreign sequences

Those skilled in the art will understand 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 agents. Accordingly, the following description of the production of nucleic acid molecules should not be limiting in any way. nucleic acid molecule

In certain embodiments, the baculovirus expression vector systems described herein can be used to produce nucleic acid molecules. For example, generating nucleic acid molecules for use in gene therapy. Accordingly, certain embodiments described herein relate to the generation of plasmid-like, capsid-free nucleic acid molecules encoding sequences of interest. The capsid is the protein coat of the virus that encloses the virus' genetic material. The capsid is known to assist the function of the virion by protecting the viral genome, delivering the genome to the host, and interacting with the host. However, particularly when used in gene therapy, the viral capsid can be a limiting factor in the packaging capacity of the vector and/or in inducing an immune response.

AAV vectors have emerged as a more common type of gene therapy vector. However, the presence of the capsid limits the utility of AAV vectors in gene therapy. Specifically, the capsid itself may limit the size of the transgene included in the vector to as low as less than 4.5 kb. Many therapeutic proteins that can be used in gene therapy can easily exceed this size even before the addition of expression control elements.

In addition, the proteins that make up the capsid may serve as antigens that are targetable by the subject's immune system. AAV is very common in the general population, and most people have been exposed to AAV in their lifetime. Thus, most potential recipients of gene therapy may already have developed an immune response against AAV and are thus more likely to reject the therapy.

In certain embodiments, the invention relates to the production of nucleic acid molecules comprising a first ITR, a second ITR, and a gene cassette (eg, encoding a therapeutic protein and/or miRNA). In some embodiments, the first ITR and the second ITR flank a gene cassette comprising a heterologous polynucleotide sequence. In some embodiments, the nucleic acid molecule does not comprise genes encoding capsid proteins, replication proteins and/or assembly proteins. In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a coagulation 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, nucleic acid molecules also comprise one or more non-coding regions. In certain embodiments, the one or more noncoding regions comprise promoter sequences, introns, post-transcriptional regulatory elements, 3'UTR poly(A) sequences, or any combination thereof.

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

Some aspects of the disclosure relate to a nucleic acid molecule comprising a gene cassette (eg, encoding a therapeutic protein and/or miRNA). In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises a coagulation 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 noncoding region comprises a promoter sequence, an intron, a post-transcriptional regulatory element, a 3'UTR poly(A) sequence, or any combination thereof.

In some embodiments, a nucleic acid molecule disclosed herein comprises 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 the chicken β-actin/rabbit β-globin intron and has been modified to eliminate the five existing ATGs sequence, thereby reducing spurious translation initiation. In certain embodiments, the intron sequence comprises the SV40 small T intron. In some embodiments, an intron sequence is located 5' to a nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, a chimeric intron is positioned 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 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 closed-end double-stranded nucleic acid (ceDNA).

In some embodiments, the gene cassette comprises a nucleotide sequence encoding a FVIII polypeptide, wherein the nucleotide sequence is codon optimized. In some embodiments, the gene cassette comprises a nucleotide sequence encoding codon-optimized FVIII driven by an mTTR promoter and a synthetic intron. In some embodiments, the gene cassette comprises the nucleotide sequence disclosed in International Application No. PCT/US2017/015879, which is incorporated by reference in its entirety. In some embodiments, the gene cassette is the "hFVIIIco6XTEN" gene cassette as described in PCT/US2017/015879. In some embodiments, the gene cassette comprises a codon-optimized cDNA encoding B-domain deleted (BDD) codon-optimized human Factor VIII (BDDcoFVIII) fused to an XTEN 144 peptide.

In some embodiments, the gene cassette comprises a nucleotide sequence encoding 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 chimeric or synthetic introns. In some embodiments, the chimeric intron consists of the chicken β-actin/rabbit β-globin intron and has been modified to eliminate the five existing ATG sequences, thereby reducing spurious translation initiation. In some embodiments, an intron sequence is located 5' to a nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, a chimeric intron is positioned 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 nucleotide sequence of SEQ ID NO: 19.

In some embodiments, the gene cassette comprises a nucleotide sequence encoding codon-optimized FVIII and XTEN peptides driven by a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482), said gene cassette Includes A1MB2 enhancer elements, hybrid synthetic introns (chimeric introns), woodchuck post-transcriptional regulatory elements (WPREs), and bovine growth hormone polyadenylation (bGHpA) signals. In some embodiments, the gene cassette comprises the nucleotide 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. inverted terminal repeat

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 ITR is an ITR obtained from a member of the virus family Parvoviridae. 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 PCT Publication Nos. WO 2019032898A1 , WO 2020033863A1 , and WO 2017152149A1 , 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"). In some embodiments, the ITRs are not derived from the AAV genome. In some embodiments, the ITR is a non-AAV ITR. In some embodiments, the ITR is an ITR from a non-AAV genome from a viral family Parvoviridae selected from, but not limited to: Bocavirus, Dependovirus, Rhodovirus, Aleutian, Parvovirus, Densovirus, Repetorivirus, Contravirus, Avian Parvovirus, Ruminant Parvovirus, Proparvovirus, Parvovirus Type IV, Ambisense Densovirus, Brevedensovirus, Hepatopancreatic densovirus, Shrimp densovirus and any combination thereof. In certain embodiments, the ITR is derived from Rhodovirus Parvovirus B19 (a human virus; also referred to herein as "B19")). In another embodiment, the ITR is derived from a Muscovy Duck Parvovirus (MDPV) strain. In certain embodiments, the MDPV strain is attenuated, such as MDPV strain FZ91-30. In other embodiments, the MDPV strain is pathogenic, such as MDPV strain YY. In some embodiments, the ITRs are derived from porcine parvovirus, such as porcine parvovirus U44978. In some embodiments, the ITR is derived from a mouse parvovirus, eg, mouse parvovirus U34256. In some embodiments, the ITRs are derived from canine parvovirus, eg, canine parvovirus M19296. In some embodiments, the ITRs are derived from mink enteritis virus, such as mink enteritis virus D00765. In some embodiments, the ITR is derived from Dependoparvovirus . In one embodiment, the Parvovirus Reliant is a Goose Parvovirus (GPV) strain of the genus Reliant Virus. In a specific embodiment, the GPV strain is attenuated, such as GPV strain 82-0321V. In another specific embodiment, the GPV strain is pathogenic, such as GPV strain B. Examples of suitable parvovirus ITR sequences are shown in Table 1. Table 1: Parvovirus ITR sequences SEQ ID NO: parvovirus Descriptor sequence 1 B19 B19Δ135 CTCTGGGCCAGCTTGCTTGGGGTTGCCTTGACACTAAGACAAGCGGCGCGCCGCTTGATCTTAGTGGCACGTCAACCCCAAGCGCTGGCCCAGAGCCAACCCTAATTCCGGAAGTCCCGCCCACCGGAAGTGACGTCACAGGAAATGACGTCACAGGAAATGACGTAATTGTCCGCCATCTTGTACCGGAAGTCCCGCCTACCGGCGGCGA CCGGCGGCATCTGATTTGGTGTCTTTCTTTTAAATTTT 2 B19.WT CCAAATCAGATGCCGCCGGTCGCCGCCGGTAGGCGGGACTTCCGGTACAAGATGGCGGACAATTACGTCATTTCCTGTGACGTCATTTCCTGTGACGTCACTTCCGGTGGGCGGGACTTCCGGAATTAGGGTTGGCTCTGGGCCAGCTTGCTTGGGGTTGCCTTGACACTAAGACAAGCGGCGCGCCGCTTGATCTTAGTG GCACGTCAAACCCCAAGCGCTGGCCCAGAGCCAACCCTAATTCCGGAAGTCCCGCCCACCGGAAGTGACGTCACAGGAAATGACGTCACAGGAAATGACGTAATTGTCCGCCATCTTGTACCGGAAGTCCCGCCTACCGGCGGCGACCGGCGGCATCTGATTTGGTGTCTTCTTTTAAATTTT 3 GPV GPVΔ162 CGGTGACGTGTTTCCGGCTGTTAGGTTGACCACGCGCATGCCGCGCGGTCAGCCCAATAGTTAAGCCGGAAACACGTCACCGGAAGTCACATGACCGGAAGTCACGTGACCGGAAACACGTGACAGGAAGCACGTGACCGGAACTACGTCACCGGATGTGCGTCACCGGAAGCATGTGACCGGAACTTGCGTCACTTCCCCCTCCCCTGATTGGCTG GTTCGAACGAACGAACCCTCCAATGAGACTCAAGGACAAGAGGATATTTTGCGCGCCAGGAAGTG 4 GPV.WT CTCATTGGAGGGTTCGTTCGTTCGAACCAGCCAATCAGGGGAGGGGGAAGTGACGCAAGTTCCGGTCACATGCTTCCGGTGACGCACATCCGGTGACGTAGTTCCGGTCACGTGCTTCCTGTCACGTGTTTCCGGTCACGTGACTTCCGGTCATGTGACTTCCGGTGACGTGTTTCCGGCTGTTAGGTTGACCACGCGCATGCCGC GCGGTCAGCCCAATAGTTAAGCCGGAAACACGTCACCGGAAGTCACATGACCGGAAGTCACGTGACCGGAAACACGTGACAGGAAGCACGTGACCGGAACTACGTCACCGGATGTGCGTCACCGGAAGCATGTGACCGGAACTTGCGTCACTTCCCCCTCCCCTGATTGGCTGGTTCGAACGAACGAACCCTCCAATGAGACTCAAGGACAAGAGGATATTTTGCGC GCCAGGAAGTG 5 AAV2 AAV2.WT (5') AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 6 AAV2.WT (3') AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 7 AAV2Δ15 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG 8 AAV2Δ15Δ11 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG 26 HBoV1 HBoV1 5'ITR GTGGTTGTACAGACGCCATCTTGGAATCCAATATGTCTGCCGGCTCAGTCATGCCTGCGCTGCGCGCAGCGCGCTGCGCGCGCATGATCTAATCGCCGGCAGACATATTGGATTCCAAGATGGCGTCTGTACAACCAC 27 HBoV1 3' ITR TTGCTTATGCAATCGCGAAACTCTATATCTTTTAATGTGTTGTTGTTGTACATGCGCCATCTTAGTTTTATATCAGCTGGCGCCTTAGTTATATAACATGCATGTTATATAACTAAGGCGCCAGCTGATATAAAACTAAGATGGCGCATGTACAACAACAACACATTAAAAGATATAGAGTTTCGCGATTGCATAAGCAA therapeutic protein

In some aspects, provided herein is the production of a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a sequence of interest, wherein the sequence of interest encodes a therapeutic protein. In some embodiments, the gene cassette encodes a therapeutic protein. In some embodiments, the gene cassette encodes more than one therapeutic protein. In some embodiments, the gene cassette encodes two or more copies of the same Therapeutic protein. In some embodiments, the gene cassette encodes two or more variants of the same Therapeutic protein. In some embodiments, the gene cassette encodes two or more different therapeutic proteins.

Certain embodiments of the present disclosure relate to a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a therapeutic protein. Any therapeutic protein can be produced by the baculovirus expression vector system of the present disclosure, including but not limited to production of coagulation factors. In some embodiments, the coagulation factor is selected from the group consisting of FI, FII, FIII, FIV, FV, FVI, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, VWF, prekallikrein, high molecular weight kininogen, Fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, α2-antiplasmin, tissue fibrinolysis Zymogen promoter (tPA), urokinase, plasminogen promoter inhibitor-1 (PAI-1), plasminogen promoter inhibitor-2 (PAI2), any zymogen thereof, any active form thereof and any combination thereof. In one embodiment, the coagulation factor comprises FVIII or a variant or fragment thereof. In another embodiment, the coagulation factor comprises FIX or a variant or fragment thereof. In another embodiment, the coagulation factor comprises FVII or a variant or fragment thereof. In another embodiment, the coagulation factor comprises VWF or a variant or fragment thereof. growth factor

In some aspects, provided herein is the production of a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a sequence of interest, wherein the sequence of interest encodes a therapeutic protein, and wherein the therapeutic protein comprises a growth factor. Growth factors can be selected from any growth factors known in the art. In some embodiments, the growth factors are hormones. In other embodiments, the growth factor is a cytokine. In some embodiments, the growth factor is a chemokine.

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 autotaxin. In some embodiments, the growth factor is bone morphogenetic protein (BMP). In some embodiments, the BMP is selected from BMP2, BMP4, BMP5, and BMP7. In some embodiments, the growth factor is a member of the ciliary neurotrophic factor family. In some embodiments, the ciliary neurotrophic factor family member is selected from ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), 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 ephrin. In some embodiments, the ephrinin is selected from the group consisting of 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 stimulating hormone (FBS). In some embodiments, the growth factor is a member of the GDNF family. In some embodiments, the GDNF family member is selected from the group consisting of glial cell line-derived neurotrophic factor (GDNF), neurin, prosefin, and aterin. In some embodiments, the growth factor is growth differentiation factor-9 (GDF9). In some embodiments, the growth factor is hepatocyte growth factor (HGF). In some embodiments, the growth factor is hepatoma-derived growth factor (HDGF). In some embodiments, the growth factor is insulin. In some embodiments, the growth factor is 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 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 wherein 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 neuregulin. In some embodiments 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, 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 nephramine Enzyme (RNLS). In some embodiments, the growth factor is T cell growth factor (TCGF). In some embodiments, the growth factor is thrombopoietin (TPO). In some embodiments, the growth factor is transforming growth factor In some embodiments, the transforming growth factor is transforming growth factor alpha (TGF-alpha) or TGF-beta. In some embodiments, the growth factor is tumor necrosis factor-alpha (TNF-alpha). In some embodiments , the growth factor is vascular endothelial growth factor (VEGF). MicroRNA ( miRNA )

MicroRNAs (miRNAs) are small noncoding RNA molecules (approximately 18-22 nucleotides) that negatively regulate gene expression by inhibiting translation or inducing messenger RNA (mRNA) degradation. Since their discovery, miRNAs have been involved in a variety of cellular processes, including apoptosis, differentiation, and cell proliferation, and they have been shown to have critical roles in carcinogenesis. The ability of miRNAs to regulate gene expression makes miRNA expression in vivo a valuable tool in gene therapy.

In some aspects, provided herein is the production of a nucleic acid molecule comprising a first ITR, a second ITR, and a gene cassette encoding a sequence of interest, wherein the sequence of interest encodes an miRNA, and wherein the first ITR and/or the second ITR is a non- ITRs of adeno-associated virus (eg, first ITR and/or second ITR from non-AAV). The miRNA can be any miRNA known in the art. In some embodiments, the miRNA down-regulates the expression of a target gene. In certain embodiments, the target gene is selected from SOD1, HTT, RHO, or any combination thereof.

In some embodiments, the gene cassette encodes a miRNA. In some embodiments, the gene cassette encodes more than one miRNA. 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.

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 a miHTT-engineered miRNA disclosed in Evers et al., Molecular Therapy 26(9) : 1-15 (electronic version 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 (see Behrman et al., JCB 192(6) :919-27 (2011)).

In some embodiments, the miRNA upregulates the expression of a gene suppressor by downregulating the expression of said gene. In some embodiments, the inhibitor is a natural (eg, wild-type) inhibitor. In some embodiments, the inhibitor is derived from a mutated, heterologous and/or misrepresented gene. performance control element

In some embodiments, nucleic acid molecules or vectors produced by the baculovirus expression vector systems described herein further comprise at least one expression control sequence. An expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, that facilitates the efficient transcription and translation of an encoding nucleic acid to which it is operably linked. For example, an isolated nucleic acid molecule produced by the methods of the present disclosure can be operably linked to at least one transcription control sequence.

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

Other constitutive promoters are known to those of ordinary skill in the art. Promoters useful as gene expression sequences of the present disclosure also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced in the presence of certain metal ions to promote transcription and translation. Other inducible promoters are known to those of ordinary skill in the art.

In one embodiment, the present disclosure includes the expression of a transgene under the control of a tissue-specific promoter and/or enhancer. In another embodiment, a promoter or other expression control sequence selectively enhances expression of the transgene in hepatocytes. Examples of liver-specific promoters include, but are not limited to, mouse transthyretin promoter (mTTR), native human factor VIII promoter, native human factor IX promoter, human alpha-1-antitrypsin promoter (hAAT) , human albumin minimal promoter and mouse albumin promoter. In a specific embodiment, 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 includes any of the mTTR promoters (e.g., mTTR202 promoter, mTTR202opt promoter, mTTR482 promoter), as disclosed in U.S. Patent Publication No. US2019/0048362, which is adopted by References are incorporated herein in their entirety.

In some embodiments, the nucleic acid molecule comprises a tissue-specific promoter. In certain embodiments, a tissue-specific promoter drives expression of a therapeutic protein (eg, a coagulation factor) in the liver (eg, in hepatocytes and/or endothelial cells). In a particular embodiment, 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, triple tetraproline (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, and any combination thereof. In some embodiments, the promoter is selected from liver-specific promoters (e.g., α1-antitrypsin (AAT)), muscle-specific promoters (e.g., muscle creatine kinase (MCK), myosin heavy chain α (αMHC), myoglobin (MB), and desmin (DES)), synthetic promoters (eg, SPc5-12, 2R5Sc5-12, dMCK, and tMCK), and any combination thereof.

Expression levels can be further enhanced using one or more enhancers to achieve therapeutic efficacy. One or more enhancers may be provided alone, or together with one or more promoter elements. Typically, expression control sequences contain multiple enhancer elements and tissue-specific promoters. In one embodiment, the enhancer comprises one or more copies of the alpha-1-microglobulin/bikunin inhibitor (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, enhancers are derived from liver-specific transcription factor binding sites, such as EBP, DBP, HNF1, HNF3, HNF4, HNF6, and Enh1, including HNF1, (sense)-HNF3, (sense)- HNF4, (antisense)-HNF1, (antisense)-HNF6, (sense)-EBP, (antisense)-HNF4(antisense).

In one specific example, a promoter useful in the present disclosure is the ET promoter, also known as GenBank No. AY661265. See also Vigna et al., Molecular Therapy 11(5) :763 (2005). Examples of other suitable vectors and expression control sequences are described in WO 02/092134, EP 1395293 or US Patent Nos. 6,808,905, 7,745,179 or 7,179,903, which are hereby incorporated by reference in their entirety.

In general, expression control sequences should include, as needed, 5' non-transcribed sequences and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed sequences will include a promoter region including a promoter sequence for the transcriptional control of an operably linked encoding nucleic acid. Gene expression sequences optionally include enhancer sequences or upstream promoter sequences as desired.

In certain embodiments, nucleic acid molecules produced by the baculovirus expression vector systems described herein comprise, for example, one or more miRNA target sequences operably linked to a transgene.

In some embodiments, the target sequence is the miR-223 target, which has been reported to block expression most efficiently in myeloid committed progenitors and at least in part in more primitive HSPCs. miR-223 targets can be blocked in differentiated myeloid cells including granulocytes, monocytes, macrophages, and myeloid dendritic cells. miR-223 targets may also be suitable for gene therapy applications that rely on robust transgene expression in lymphoid or erythroid cell lineages. The miR-223 target can also block expression very efficiently in human HSCs.

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 comprising a transgene such that the transcript encoding the transgene is susceptible to miRNA-mediated downregulation . By this approach, transgene expression can be prevented in antigen-presenting cells (APCs) of the hematopoietic lineage while being maintained in non-hematopoietic cells (Brown et al., Nat Med 2006). This strategy can impose tight post-transcriptional control on transgene expression and thus enable 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), and mediates robust immunity to transgene-encoded antigens tolerance.

In some embodiments, the target sequence is a miR181 target. Chen C-Z and Lodish H, Seminars in Immunology (2005) 17(2):155-165 revealed miR-181, a miRNA specifically expressed in B cells in mouse bone marrow (Chen and Lodish, 2005). The literature also reveals that some human miRNAs are associated with leukemia.

The target sequence can be fully or partially complementary to the miRNA. The term "completely complementary" means that the nucleic acid sequence of the target sequence 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 said target sequence, whereby the partially complementary sequence is still recognized by the miRNA. In other words, in the context of the present disclosure, a partially complementary target sequence efficiently recognizes the corresponding miRNA and achieves prevention or reduction of transgene expression in cells expressing said miRNA. Examples of miRNA target sequences are described in WO 2007/000668, WO 2004/094642, WO 2010/055413 or WO 2010/125471, which are hereby incorporated by reference in their entirety. heterologous part

In some embodiments, the transgene encodes a heterologous amino acid sequence. Heterologous amino acid sequences can be linked to the transgene product. In some embodiments, a heterologous amino acid sequence can be linked to the N- or C-terminus of the transgene product. For example, for a transgene encoding FVIII, a heterologous amino acid sequence can be linked to the N- or C-terminus of the FVIII amino acid sequence or 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 of the sites disclosed in International Publication Nos. WO 2013/123457 A1 and WO 2015/106052 A1 or US Publication No. 2015/0158929 A1, said document is hereby incorporated by reference in its entirety.

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

In some embodiments, the heterologous portion comprises one or more XTEN sequences, fragments, variants or derivatives thereof. As used herein, "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring substantially non-repetitive sequence composed primarily of small hydrophilic Little or no secondary or tertiary structure. As a heterologous moiety, XTEN can be used as a half-life extending moiety. Additionally, XTENs may provide desirable properties including, but not limited to, enhanced pharmacokinetic parameters and solubility profiles.

In some embodiments, the XTEN sequences useful in the present disclosure are those having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, A peptide or polypeptide of 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800 or 2000 amino acid residues. In certain embodiments, the XTEN is greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, Greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues residues or peptides or polypeptides of greater than 120 to about 400 residues. In a specific embodiment, the XTEN comprises an amino acid sequence longer than 42 amino acids and shorter than 144 amino acids.

The XTEN sequences of the present disclosure may comprise one or more sequence motifs of 5 to 14 (e.g., 9 to 14) amino acid residues or be at least 80%, 90%, 91%, 92% identical to said sequence motifs. %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, wherein the motif comprises 4 to 6 types of amino acids selected from glycine (G), Amino acids (for example, 5 amino acids) of alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P), consisting essentially of Said amino acid consists of or consists of said amino acid. Examples of XTEN sequences that can be used as heterologous moieties in chimeric proteins of the present disclosure are disclosed, for example, in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al , 2011/0077199 A1 or 2011/0172146 A1, or International Patent Publication No. WO 2010/091122 A1, WO 2010/144502 A2, WO 2010/144508 A1, WO 2011/028228 A1, WO 2011/028229 A1 or WO 201 1/028344 A2, each of said documents is incorporated herein by reference in its entirety. IV. Host cells

Suitable host cells are known to those skilled in the art. "Host cell" refers to any cell that has or is capable of having any substance of interest.

In some embodiments, host cells suitable for use in the invention are of insect origin. In some embodiments, suitable insect host cells include, for example, cell lines isolated from Spodoptera frugiperda (Sf) or cell lines isolated from Trichoplusia ni (Tni). Those skilled in the art will readily be able to determine the suitability of any Sf or Tni cell line. Exemplary insect host cells include, without limitation, Sf9 cells, Sf21 cells, High Five™ cells. Exemplary insect host cells also include, without limitation, any Sf or Tni cell line that is not contaminated with foreign viruses, 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 a specific embodiment, the insect host cell is a Sf9 cell.

In some embodiments, host cells suitable for use in the invention are of bacterial origin. In some embodiments, a suitable bacterial host cell can be any bacterial host cell known to those skilled in the art. In certain embodiments, suitable bacterial host cells are unable to resolve cruciform DNA structures. In certain embodiments, suitable bacterial host strains contain a disruption in the SbcCD complex. In some embodiments, the disruption in the SbcCD complex comprises gene disruption in the SbcC gene and/or the SbcD gene. In certain embodiments, the disruption in the SbcCD complex comprises genetic disruption in the SbcC gene. Various bacterial host strains containing a genetic disruption in the SbcC gene are known in the art. For example and without limitation, bacterial host strain PMC103 comprises genotypes sbcC , recD , mcrA , ΔmcrBCF ; bacterial host strain PMC107 comprises genotypes recBC , recJ , sbcBC , mcrA , ΔmcrBCF ; and bacterial host strain SURE comprises genotypes recB , recJ , sbcC , mcrA , ΔmcrBCF , umuC , uvrC .

In some embodiments, host cells suitable for use in the present invention are of mammalian origin, such as human origin. It is believed that one skilled in the art will be able to preferentially determine the most suitable particular host cell strain for its purpose. Exemplary host cell lines include, but are not limited to, CHO, DG44 and DUXB11 (Chinese hamster ovary line, DHFR negative), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen) , R1610 (Chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), PER.C6®, NS0, CAP, BHK21 and HEK 293 (human kidney).

Introduction of the vectors of the present disclosure into host cells can be accomplished by a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and whole virus infection. See Ridgway, AAG " Mammalian Expression Vectors " Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, eds. (Butterworths, Boston, Mass. 1988).

Host cells comprising a vector of the disclosure are grown in an appropriate growth medium. As used herein, the term "appropriate growth medium" refers to a medium containing the nutrients required for cell growth. Nutrients required for cell growth may include carbon sources, nitrogen sources, 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). Growth media will typically select for cells containing the vector, eg, by drug selection or lack of essential nutrients supplemented by a selectable marker on the vector. V. How to use

The baculovirus expression vector systems provided herein can be used to generate products encoded by foreign sequences inserted into the recombinant baculovirus shuttle vectors described herein. Scalable production of products can be achieved by several methods known in the art.

One method involves infecting a suitable insect host cell that supports the growth of the baculovirus. In certain embodiments, a recombinant baculovirus shuttle vector comprising a foreign sequence as described herein is first propagated in a suitable bacterial host cell (eg, E. coli). Recombinant baculovirus shuttle vectors are then isolated from bacterial host cells and transfected into suitable insect host cells using a suitable transfection reagent (eg, CELLFECTIN). Insect host cells produce recombinant baculovirus particles, which can then be infected into host insect cells for viral amplification of foreign sequences.

In certain embodiments, provided herein is a method of producing a product encoded by a foreign sequence, said method comprising transfecting a recombinant baculovirus shuttle vector described herein into a suitable insect cell under appropriate conditions to produce the recombinant baculovirus; and infecting a second suitable insect cell with the recombinant baculovirus under appropriate conditions to produce a product encoded by the foreign sequence. In certain embodiments, the recombinant baculovirus shuttle vector comprises a Rep coding sequence flanked by a protein-encoding sequence flanked by ITRs for the purpose of generating a gene therapy agent.

In certain embodiments, provided herein is a method of producing a nucleic acid molecule comprising transfecting a recombinant baculovirus shuttle vector described herein into a suitable insect cell under appropriate conditions to produce a recombinant baculovirus and infecting a second suitable insect cell with a recombinant baculovirus under appropriate conditions to produce said nucleic acid molecule. In certain embodiments, provided herein is a method for producing ceDNA, the method comprising transfecting the recombinant baculovirus shuttle vector described herein into suitable insect cells under appropriate conditions to produce recombinant baculovirus; And a second suitable insect cell is infected with the recombinant baculovirus under appropriate conditions to produce the ceDNA.

In another approach, stable cell lines can be generated by stably integrating protein coding sequences under the control of a baculovirus gene promoter (eg, a baculovirus constitutive gene promoter). In certain embodiments, the stable cell line is a stable insect cell line. Stable integration of sequences can be performed by any method known to those skilled in the art. Methods for stably integrating nucleic acids into a variety of host cell lines are known in the art (see Examples below for a more detailed description of exemplary producer cell lines produced by stably integrating nucleic acids). For example, repeated selection (eg, by using a selectable marker) can be used to select cells that have integrated nucleic acid containing the selectable marker (and the AAV cap and rep genes and/or the rAAV genome). In other embodiments, the nucleic acid can be integrated into a 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 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, AC et al. (2000) Proc. Natl. Acad. Sci. 97:5995- 6000).

In the stable cell line approach, in one embodiment, a BEV encoding a complementary protein required for correct expression of the protein coding sequence is introduced into the stable cell line. In certain embodiments, for the purpose of generating gene therapy agents, stable cell lines comprise a therapeutic protein-encoding gene stably integrated therein with flanking symmetric or asymmetric AAV or non-AAV ITRs. BEVs encoding the appropriate Rep are then introduced into stable cell lines under the conditions required for gene therapy production.

Methods to generate specific stable cell lines are described herein (see Example 8). Examples of specific stable cell lines for producing ceDNA include cell lines having the plasmid shown in Figure 8B stably integrated therein. These stable cell lines are listed in Table 5. In some methods, to produce ceDNA, a BEV encoding the appropriate Rep is introduced into a stable cell line under the conditions required for ceDNA production.

In yet another approach, stable cell lines can be used to achieve production of products encoded by foreign sequences in a baculovirus-free manner. In certain embodiments, for the purpose of generating gene therapy agents, stable cell lines comprise a therapeutic protein-encoding gene stably integrated therein with flanking symmetric or asymmetric AAV or non-AAV ITRs. In certain embodiments, baculovirus-free production in a stable cell line comprises transiently expressing a Rep protein in the stable cell line under the control of a baculovirus gene promoter. Suitable baculovirus gene promoters are known to those skilled in the art. In certain embodiments, the baculovirus gene promoter is the immediate early (ie) gene promoter of Orgyia pseudotsugata multinucleated polyhedrosis virus (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 skilled in the art. In certain embodiments, transient gene expression can be achieved by polyethyleneimine (PEI)-mediated transfection.

Downstream purification of the product may involve any method known to those skilled in the art. For example, viral or non-viral vectors for gene therapy purposes can be purified by plasmid DNA separation kits containing silica-based columns that separate low molecular weight DNA from Separation of RNA, high molecular weight DNA, proteins and other impurities.

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

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.

Having generally described this disclosure, a further understanding can be obtained by reference to the examples provided herein. These examples are for illustrative purposes only and are not intended to be limiting. example Example 1: BIVVBac precursor baculovirus shuttle vector

Among expression vectors, baculovirus stands out because of its large genetic cargo capacity (up to tens of kb, 100 kb reported). This transgenic capacity has been exploited for the generation of recombinant AAV vectors (up to a 38 kb expression cassette). It is speculated that the large transgene capacity of baculoviral vectors could also be used for the production of DNA therapeutic drug substances for non-viral gene therapy. Thus, a new multifunctional baculovirus shuttle vector (baculovirus shuttle vector) was generated that was specifically designed to accommodate multiple transgenes, which is not achievable using existing baculovirus shuttle vector tools. This multifunctional baculovirus shuttle vector (termed "BIVVBac") can also be used for the production of rAAV vectors for in vivo gene therapy, as well as the production of any desired proteins, such as recombinant proteins.

The BIVVBac baculovirus shuttle vector was derived from bMON14272 (Invitrogen) encoding the AcMNPV C6 genome ( Fig. 1A ) and was engineered to encode two insertion sites: 1) miniature att Tn7 in the polyhedrin locus, and 2 ) LoxP in the EGT locus. Combining the miniatt Tn7 insert with the Lac promoter-driven E. coli LacZα fragment for X-gal-mediated blue/white selection of recombinant baculovirus shuttle vectors following Tn7-mediated translocation and for Cre-mediated translocation Guided in-frame fusion of LoxP sites for in vitro or in vivo recombination. A LoxP recombination site was inserted in two steps into bMON14272, which contains a miniature att Tn7 insertion in the polyhedrin locus. First, a plasmid was synthesized by GenScript® (Piscataway, NJ) consisting of the Rudolph red fluorescent protein (RFP) gene under the AcMNPV 39K promoter followed by the ets polyadenylation signal , the LoxP recombination site, and the sequence flanking the AcMNPV EGT locus ( FIG. 1B ) (SEQ ID NO: 9). 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 the LoxP sequence were recombined at the EGT locus by homologous recombination in Sf9 cells. Progeny virus produced in cell-free supernatant was plaque purified and RFP+ plaques were expanded in Sf9 cells to generate P1 (passage 1) virus. Baculovirus DNA was then isolated from the P1 virus and used as a template to PCR amplify the region spanning the expected recombination junction using primers inside and outside the transfer plasmid, and the resulting amplicons were sequenced. The recombinant virus was further amplified and used for viral DNA isolation, which was then transformed into E. coli DH10B bacteria (O'Reilly et al. 1992), followed by selection with kamycin, X-Gal and IPTG. Baculovirus shuttle vector DNA isolated from a kamycin-resistant blue E. coli strain was analyzed by restriction enzyme mapping and designated "BIVVBac" ( Figure 1C ). The helper plasmid pMON7124 (Invitrogen) encoding the Tn7 translocase gene tnsA-E and tetracycline resistance was transformed into DH10B E. coli with BIVVBac, followed by selection with kamycin and tetracycline. One of the dual resistant colonies was used to make electrocompetent cells as previously described (Sharma and Schimke, 1996), and the resulting new E. coli strain was designated BIVVBac ^DH10B (Fig. 1D ). This strain was then used to insert the AAV2, B19 or GPV replication (Rep) protein coding gene at the polyhedrin locus by Tn7 translocation as described below. Example 2: Cre-LoxP Donor Vector

A DNA construct consisting of a LoxP recombination site preceded by the transcriptional enhancer hr5 element followed by the AcMNPV p10 polyadenylation signal was synthesized by GenScript® (Piscataway, NJ) Enhanced GFP (eGFP) marker gene, ampicillin resistance marker, conditional R6Kγ origin of replication and multiple colonization site (SEQ ID NO: 10) under the AcMNPV ie1 promoter (Fig. 2A ) . This synthetic DNA was cloned into the pUC57 vector, and the resulting construct was designated "Cre-LoxP Donor Vector" (Figure 2B ) . Key features of the Cre-LoxP donor vector include: (1) multiple colonization site (MCS) for insertion of transgenes; (2) LoxP site for Cre-mediated recombination in vitro or in vivo; (3) ) two origins of replication: (3a) for propagating this vector into the ColE1 Ori in E. coli strains such as DH5α, NEB Stable, PMC103 and DH10B, and (3b) for propagating this vector into expression π (pi ) protein for conditional R6Kγ Ori in E. coli strains (such as pir+ and pir116 ); (4) for use after insertion of the Cre-LoxP donor vector into the LoxP site in "BIVVBac" by Cre-LoxP recombination kamycin and ampicillin for selection of the ampicillin resistance gene of the recombinant baculovirus shuttle vector; and (5) for determining the stability of the transgene in recombinant BEV while replicating in Sf9 cells through serial passages Enhanced GFP reporter gene. Example 3: Replication (Rep) Protein Expression Construct B19.Rep :

The coding sequence of B19 Rep was obtained from B19 strain HV (GenBank Accession No. AF162273), and the wild-type sequence (SEQ ID NO: 11) was synthesized by GenScript® (Piscataway, NJ) ( Fig. 3A ). The synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector under the AcMNPV polyhedrin promoter, and the pFastBac.Polh.B19.Rep transfer vector was generated using a gitamycin-resistant colony. This vector was transformed into BIVVBac ^DH10B E. coli to produce a recombinant BEV, AcBIVVBac.Polh.B19.Rep ^Tn7 , as described below. Titrated BEVs were then used for infection in multiple colonized Sf cell lines encoding hFVIIIco6XTEN with symmetric B19 ITRs, and ceDNA vectors were purified from infected cell pellets as described below. Preliminary results reveal that polyhedrin promoter-driven B19.Rep cannot "rescue" ceDNA. It is hypothesized that high levels of non-splicing B19.Rep expression very late in the baculovirus infection cycle are inefficient and/or cytotoxic. For this hypothesis, the polyhedrin promoter was replaced with the AcMNPV constitutive immediate early ( ie1 ) promoter to generate the pFastBac.IE1.B19.Rep transfer vector (Fig. 3B ) . This vector was used to generate a recombinant BEV, AcBIVVBac.IE1.B19.RepTn7, and then tested in a multiple colonized Sf cell line encoding hFVIIIco6XTEN with ^a symmetric B19 ITR. 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 the stoichiometric representation of the Rep protein is critical for ceDNA production in Sf9 cells, as observed earlier for rAAV production. GPV.Rep :

The coding sequence of GPV Rep was obtained from GPV B strain (GenBank accession number GPU25749). Unlike B19, GPV.Rep generates the spliced proteins (Rep78 and Rep52) from a single mRNA transcript, and to achieve stoichiometric representation of the two proteins, the Rep78 coding sequence was genetically modified to allow for a single mRNA transcript via a ribosomal omission scanning mechanism. The mRNA species expressed Rep78 and Rep52 polypeptides. The AUG start codon of the Rep78 open reading frame (orf), the adjacent proline codon, and twelve downstream AUG triplets that occur before the start codon of the Rep52 orf were altered via gene synthesis. The Rep78 initiation codon and proximal flanking nucleotides were mutated to an inefficient translation initiation signal consisting of CUG triplets presented in the context of the Kozak consensus sequence. Alter the AUG triplet occurring between the start codon of the Rep78 orf and the AUG start codon of the Rep52 orf to carry a silent mutation (in the case of an out-of-frame AUG codon), or to encode a conservative amino acid substitution ( In the case of an in-frame AUG codon). Finally, the modified GPV.Rep coding sequence (SEQ ID NO: 12) was synthesized by GenScript® (Fig. 3C ) , and the synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector under the AcMNPV polyhedrin promoter to generate pFastBac .Polh.GPV.Rep transfer vector (Figure 3D ) . This vector was then transformed into BIVVBac ^DH10B E. coli to produce a recombinant BEV, AcBIVVBac.Polh.GPV.Rep ^Tn7 , as described below. 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 generates splice proteins (Rep78 and Rep52) from a single mRNA transcript, and to achieve stoichiometric representation of the two proteins, the Rep78 coding sequence was genetically modified to allow scanning via ribosomal omissions The mechanism expresses Rep78 and Rep52 polypeptides from a single mRNA transcript, as described earlier (Smith et al., 2009) (Fig. 3E ) . The modified AAV2.Rep coding sequence (SEQ ID NO: 13) (Fig. 3E ) was synthesized by GenScript®, and the synthetic DNA was cloned into the pFastBac1 (Invitrogen) vector under the AcMNPV polyhedrin promoter to generate pFastBac.Polh .AAV2.Rep transfer vector (Figure 3F ) . This vector was then transformed into BIVVBac ^DH10B E. coli to produce a recombinant BEV, AcBIVVBac.Polh.AAV2.Rep ^Tn7 , as described below. Example 4: Human FVIIIco6XTEN Expression Constructs Human FVIIIco6XTEN Expression Constructs with Non- AAV or AAV ITRs :

In PCT Publication No. WO 2019032898A1 a gene construct comprising codon-optimized human FVIII with XTEN 144 peptide (FVIIIco6XTEN), woodchuck post-transcriptional regulatory element (WPRE) under the liver-specific TTPp promoter is described , bovine growth hormone polyadenylation (bGHpA) signaling, and non-AAV or AAV flanking truncated ITRs. Here, these constructs were tested in parallel with new constructs made by replacing the 5' or 3' ITR with non-AAV or AAV wild-type sequences (Table 2). These new constructs were grown and maintained in E. coli strain PMC103 containing a deleted sbcC gene, which encodes an exonuclease that recognizes and eliminates cruciform DNA structures. After optimizing the temperature and growth conditions of the bacterial culture, PMC103 E. coli was able to support the growth of hFVIIIco6XTEN constructs with non-AAV or AAV symmetric or asymmetric ITRs. All new constructs were selected on ampicillin resistant plates and screened by restriction enzyme mapping to confirm the correct genetic structure. The hFVIIIco6XTEN expression construct was then used to make the Cre-LoxP donor vector. Table 2: Human FVIIIco6XTEN Expression Constructs Symmetrical / Asymmetrical 5' ITR 3' ITR construct-1 B19 symmetrical truncated B19Δ135 (SEQ ID NO: 1) B19Δ135 (SEQ ID NO: 1) construct-2 B19 symmetrical wild type B19.WT (SEQ ID NO:2) B19.WT (SEQ ID NO:2) construct-3 GPV symmetrical truncated form GPVΔ162 (SEQ ID NO:3) GPVΔ162 (SEQ ID NO:3) construct-4 GPV symmetric wild type GPV.WT (SEQ ID NO: 4) GPV.WT (SEQ ID NO: 4) construct-5 GPV asymmetric GPVΔ162 (SEQ ID NO:3) GPV.WT (SEQ ID NO: 4) construct-6 AAV2 symmetric wild type AAV2.WT (SEQ ID NO: 5) AAV2.WT (SEQ ID NO: 6) construct-7 AAV2 asymmetric type 1 AAV2Δ15Δ11 (SEQ ID NO:8) AAV2Δ15 (SEQ ID NO: 7) construct-8 AAV2 asymmetric type 2 AAV2.WT (SEQ ID NO: 5) AAV2Δ15 (SEQ ID NO: 7) construct-9 AAV2 asymmetric type 3 AAV2Δ15Δ11 (SEQ ID NO:8) AAV2.WT (SEQ ID NO: 6) Human FVIIIco6XTEN Cre-LoxP Donor Vector with Non- AAV or AAV ITR :

The hFVIIIco6XTEN expression cassettes from constructs -1, -3 and -7 (Table 2) were excised with PstI enzyme and colonized at the same site in the Cre-LoxP donor vector. The resulting construct was transformed into a NEB® stable competent E. coli strain (New England Biolabs), and ampicillin-resistant colonies were selected by restriction enzyme mapping. For the "plasmid Cre-LoxP" in pCLDV-1, -3 and -7, the correct colonies were assigned with the prefix "pCL" (Table 2) (Fig. 4B ) . Similarly, to generate other donor vectors, hFVIIIco6XTEN expression cassettes from constructs -2, -4, -5, -6, -8 and -9 were excised with PstI and/or PvuII enzymes and colonized on Cre- at the same site in the LoxP donor vector. These constructs were transformed into the PMC103 E. coli strain due to the long palindromic repeat of the wild-type ITR sequence, and ampicillin-resistant colonies were selected by restriction enzyme mapping. The resulting Cre-LoxP donor vectors pCLDV-2, -4, -5, -6, -8, and -9 were then inserted at the LoxP recombination site into B19, GPV, or AAV2 Rep-encoding genes at the Tn7 site, respectively. "BIVVBac" in the baculovirus shuttle vector (Table 3). The sequences of the ITRs in Table 3 are shown in Table 1. Table 3: Human FVIIIco6XTEN Cre-LoxP Donor Vectors Cre-LoxP Donor Vector 5' ITR 3' ITR pCLDV-1 pCL.hFVIIIco6XTEN.B19Δ135 B19Δ135 B19Δ135 pCLDV-2 pCL.hFVIIIco6XTEN.B19.WT B19.WT B19.WT pCLDV-3 pCL.hFVIIIco6XTEN.GPVΔ135 GPVΔ162 GPVΔ162 pCLDV-4 pCL.hFVIIIco6XTEN.GPV.WT GPV.WT GPV.WT pCLDV-5 pCL.hFVIIIco6XTEN.GPV.Asy GPVΔ162 GPV.WT pCLDV-6 pCL.hFVIIIco6XTEN.AAV2.WT AAV2.WT AAV2.WT pCLDV-7 pCL.hFVIIIco6XTEN.AAV2.Asy1 AAV2Δ15Δ11 AAV2Δ15 pCLDV-8 pCL.hFVIIIco6XTEN.AAV2.Asy2 AAV2.WT AAV2Δ15 pCLDV-9 pCL.hFVIIIco6XTEN.AAV2.Asy3 AAV2Δ15Δ11 AAV2.WT Example 5: Replication (Rep) Baculovirus Expression Vector (Baculovirus Expression Vector, BEV)

Replication of AAV depends on nonstructural (replication) proteins; Rep78, Rep68, Rep52 and Rep40. Several activities of AAV Rep78 and Rep68 have been characterized that are involved in viral DNA replication, including binding to the AAV ITR, sequence- and strand-specific endonuclease activity, and ATP-dependent DNA helicase activity. Rep68 or Rep78 binds to a specific region within the ITR (the Rep binding site) and nicks one strand of a double-stranded replication intermediate at a unique site called the terminal dissociation site (TRS). The endonuclease reaction results in the covalent attachment of Rep to the newly generated 5' end. The nicked 3' end serves as a primer for ITR extension. The helicase activity of covalently attached Rep molecules can unravel the secondary structure of the ITR. The end dissociation process provides the means for end recovery and generation of progeny viral genomes. Thus, 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, BIVVBac ^DH10B E. coli was first hypertransformed with the transfer vectors pFastBac.IE1.B19.Rep (Figure 3B ), pFastBac.Polh.GPV.Rep (Figure 3D ) and pFastBac.Polh.AAV2.Rep (Figure 3F ) ( FIG. 1D ) , and transformants were then selected on kanamycin, gentamycin, X-Gal and IPTG. Site-specific translocation of the Rep expression cassette and the gintamycin resistance gene at the mini- att Tn7 insertion site in BIVVBac disrupts LacZα (in-frame fusion with mini- att Tn7) and in X-Gal-mediated double Antibiotic selection produces white E. coli colonies. Therefore, recombinant baculovirus shuttle vector DNA was isolated from white E. coli colonies by alkaline lysis miniprep and digested with restriction enzymes to confirm the correct genetic structure. The results of restriction enzyme mapping showed the expected fragments of each recombinant baculovirus shuttle vector, which indicated site-specific translocation of Rep in the polyhedrin locus of BIVVBac ( Fig. 5A ). Further confirmation was obtained by PCR amplifying the region spanning the expected insertion site using primers inside and outside the transfer plasmid and sequencing the resulting amplicon.

Sf9 cells were transfected with the correct recombinant baculovirus shuttle vector encoding B19.Rep, GPV.Rep or AAV2.Rep using Cellfectin® (Invitrogen) transfection reagent according to the manufacturer's instructions. 4–5 days after transfection, progeny baculoviruses were harvested and plaque purified in Sf9 cells as described (Jarvis, 2014). Six plaques of each recombinant BEV, AcBIVVBac.IE1.B19.Rep ^Tn7 (Figure 5B ) , AcBIVVBac.Polh.GPV.Rep ^Tn7 (Figure 5C ) and AcBIVVBac.Polh.AAV2.Rep ^Tn (Figure 5D ) were purified The RFP+ colonies were expanded to P1 (passage 1) in Sf9 cells seeded at 0.5 x 10 ⁶ /mL in T25 flasks with ESF supplemented with 10% heat-inactivated fetal bovine serum (FBS) -921 medium. 4-5 days after infection, P1 virus was harvested by low-speed centrifugation, and anti-B19 NS1 (MyBioSource, Inc.), anti-GPV.REP (GenScript®) and anti-AAV2.REP 303.9 (American Research Products Inc.) monoclonal Strain or polyclonal antibodies were tested by immunoblotting of infected cell pellets to detect B19.REP, GPV.REP, or AAV2.REP. Western blot analysis of AcBIVVBac.IE1.B19.Rep ^Tn7 plaque-purified colonies (Fig. 5E ) revealed a single species of B19.REP with a predicted mass of 74 kDa, suggesting that B19.REP is transcribed from non-spliced mRNA in Sf9 cells performance. Interestingly, equivalent levels of B19.REP expression were observed in all tested virus strains, suggesting a stoichiometric level of B19.REP expression under the AcMNPV IE1 promoter in Sf9 cells. Unlike B19.REP, immunoblot analysis of plaque-purified colonies of AcBIVVBac.Polh.GPV.Rep ^Tn (Fig. 5F ) or AcBIVVBac.Polh.AAV2.Rep ^Tn7 (Fig . 5G ) revealed a predicted mass of 73 kDa for both species. and 49 kDa of REP correspond to Rep78 and Rep52 polypeptides, suggesting that GPV.REP and AAV2.REP are expressed from spliced mRNA transcripts in Sf9 cells. Equivalent levels of Rep78 or Rep52 were observed in all tested virus strains, indicating stable GPV.REP and AAV2.REP expression under the AcMNPV polyhedrin promoter in Sf9 cells. REP78 was expressed at almost half the level of REP52, further suggesting that the correct stoichiometric expression of both peptides is achieved from a single mRNA transcript via a ribosomal omission scanning mechanism. The highest REP expressing colonies of each BEV were selected for further expansion in Sf9 cells and then for infection in stable cell lines encoding hFVIIIco6XTEN flanked by non-AAV and AAV-flanked symmetric or asymmetric ITRs for use with Generated in ceDNA vector. Example 6: Replication (Rep) and Human FVIIIco6XTEN Baculovirus Expression Vector (BEV)

To test whether BIVVBac could be used to accommodate multiple transgenes, a family of derived vectors was generated encoding two transgene expression cassettes: 1) Rep and 2) ITR flanked by non-AAV and AAV symmetric or asymmetric ITRs. hFVIIIco6XTEN. These BEVs are generated in two steps. First, the Rep expression cassette was inserted at the miniatt Tn7 site in the polyhedrin locus via Tn7 translocation as described above. The resulting recombinant baculovirus shuttle vector was then used to insert the hFVIIIco6XTEN expression cassette at the LoxP site in the EGT locus via in vitro Cre-LoxP recombination using Cre recombinase (New England Biolabs). In this process, the Cre-LoxP donor vector (pCLDV-1 to -9) (Table 2) encoding hFVIIIco6XTEN with B19 ITR, GPV ITR and AAV ITR was inserted into AcBIVVBac.IE1.B19.Rep respectively (Figure 5B ) , AcBIVVBac.Polh.GPV.Rep (Figure 5C ) or AcBIVVBac.Polh.AAV2.Rep (Figure 5D ). Recombination reactions were transformed in DH10B E. coli and transformants were selected on kanamycin, gentamycin and ampicillin. Triple antibiotic resistant colonies were screened by PCR amplification of the region spanning the expected insertion site by restriction enzyme mapping and/or using primers inside and outside the transfer plasmid, and the resulting amplicons were sequenced.

The correct recombinant baculovirus shuttle vector encoding the two transgene cassettes was purified by maximal prep and transfected in Sf9 cells using Cellfectin® (Invitrogen) transfection reagent. 4-5 days after transfection, progeny baculoviruses were harvested and plaque purified in Sf9 cells. Six plaque-purified RFP+ and GFP+ colonies of each recombinant BEV (BEV-1 to -9) (Table 4) (Figure 6A , 6B and 6C ) were expanded to P1 (passage 1) in Sf9 cells , the Sf9 cells were inoculated at 0.5 x 10 ⁶ /mL in ESF921 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a T25 flask. At 4-5 days post-infection, all colonies showed infection progression (as determined by the number of GFP+ and RFP+ cells) for each of the recombinant BEVs, indicating that the virus was able to replicate normally and insert multiple transfectants in the same baculovirus genome. The reproductive genes had no adverse effect on progeny virus production. P1 virus was harvested by low-speed centrifugation, and infected cell pellets were processed for REP detection by immunoblotting or for ceDNA isolation using the PureLink Max Prep DNA Isolation Kit (Invitrogen). Immunoblots were performed to detect B19.REP, GPV.REP or AAV2.REP as described above. The results of the immunoblot showed that all six colonies were positive for each REP tested, suggesting that insertion of the hFVIIIco6XTEN cassette at the EGT locus had no adverse effect on immediate early or polyhedrin-driven REP expression. Finally, the top REP expressing colonies of each BEV were further expanded to generate working BEV stocks (P2) before titration in Sf9 cells. Titrated BEVs were used for infection in Sf9 cells to generate the hFVIIIco6XTEN ceDNA vector as described below. Table 4: Rep and human FVIIIco6XTEN BEV BEV ITR BEV-1 AcBIVVBac(B19.Rep)FVIII.B19Δ135.ITR ^LoxP B19 symmetrical truncated Figure 6A BEV-2 AcBIVVBac(B19.Rep)FVIII.B19.WT.ITR ^LoxP B19 symmetrical wild type BEV-3 AcBIVVBac(GPV.Rep)FVIII.GPVΔ162.ITR ^LoxP GPV symmetrical truncated form Figure 6B BEV-4 AcBIVVBac(GPV.Rep)FVIII.GPV.WT.ITR ^LoxP GPV symmetric wild type BEV-5 AcBIVVBac(GPV.Rep)FVIII.GPV.Asy.ITR ^LoxP GPV asymmetric BEV-6 AcBIVVBac(AAV2.Rep)FVIII.AAV2.WT.ITR ^LoxP AAV2 symmetric wild type Figure 6C BEV-7 AcBIVVBac(AAV2.Rep)FVIII.AAV2.Asy1.ITR ^LoxP AAV2 asymmetric type 1 BEV-8 AcBIVVBac(AAV2.Rep)FVIII.AAV2.Asy2.ITR ^LoxP AAV2 asymmetric type 2 BEV-9 AcBIVVBac(AAV2.Rep)FVIII.AAV2.Asy3.ITR ^LoxP AAV2 asymmetric type 3 Example 7: Generation of human FVIIIco6XTEN ceDNA vector from baculovirus

ceDNA production of recombinant BEVs (Table 4) encoding both hFVIIIco6XTEN and Rep genes was tested. Sf9 cells were infected with a titrated working stock (P2) of each BEV at a multiplicity of infection (MOI) of 3 pfu/cell. The cells were tumbled gently at room temperature for 1.5 h, pelleted at 500×g for 5 min, the supernatant was aspirated, and the 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 at 28°C for 72 h in a shaker incubator. At 72 h post-infection, infected cells were harvested, and the pellet was washed once with 1x PBS to remove residual baculovirus particles and/or medium. Then, ceDNA vectors were isolated by PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. The eluted fractions were analyzed by 0.8% to 1.2% agarose gel electrophoresis to determine the yield and purity of each ceDNA vector. As a first round of analysis, ceDNA vectors isolated from Sf9 cells infected with BEV-5 were tested (Table 4, Figure 7A ). All six colonies 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), suggesting that polyhedrin-driven GPV REP was able to rescue the asymmetric ITR-flanked hFVIIIco6XTEN vector DNA (Fig. 7B ) . Samples were re-run at different volumes to visualize the banding pattern and confirm the size of the ceDNA. Multiple high molecular weight bands were observed (Figure 7C ) . This data demonstrates the concept of generating ceDNA from a single recombinant BEV encoding a non-AAV Rep as well as an ITR-flanked hFVIIIco6XTEN transgene. Example 8: Human FVIIIco6XTEN Stable Insect Cell Line

It is speculated that insect cell genomes may also be modified to produce DNA therapeutic drug substances following baculovirus infection. To achieve this, a protein encoding a protein under the AcMNPV immediate early (ie1) promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal was synthesized from GenScript® (Piscataway, NJ ). Neomycin resistance marker plasmid (SEQ ID NO: 14) (Fig. 8A ) . This synthetic DNA was co-transfected in Sf9 cells using Cellfectin® (Invitrogen ^™ ) together with a plasmid encoding the hFVIIIco6XTEN expression cassette flanked by symmetric and asymmetric ITRs for non-AAV or AAV (Table 3). At 24 h post-transfection, cells were visualized under a fluorescent microscope to determine transfection efficiency, and the results showed >80% GFP+ cells, indicating higher transfection efficiency. At 72 h after transfection, cells were selected with G418 antibiotic (Sigma Aldrich) suspended in complete TNMFH medium (Grace 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 the transformed cells were recovered, indicating that the neomycin resistance marker was stably integrated into the cell population. Surviving cells were removed from the selection medium and cultured with fresh complete TNMFH medium until confluent growth. As confluent cells continue to divide, they are gradually expanded as adherent cultures into larger culture vessels. Subsequently, each cell line was adapted to suspension culture by culturing in complete TNMFH for 1 passage and growing in ESF-921 medium supplemented with 10% FBS for 1 passage in shake flasks. Finally, each cell line was adapted as a suspension culture to serum-free ESF-921 in shake flasks. Typically these shake flask cultures were maintained in serum-free ESF-921 medium, passaged every four days, and cell growth was monitored. Each cell line was designated according to the inserted hFVIIIco6XTEN construct and prefixed with " Sf " (Figure 8B ) (Table 5). Table 5: Human FVIIIco6XTEN stable insect cell lines Human FVIIIco6XTEN cell line 5' ITR 3' ITR cell line-1 Sf.hFVIIIco6XTEN.B19Δ135 B19Δ135 B19Δ135 cell line-2 Sf.hFVIIIco6XTEN.B19.WT B19.WT B19.WT cell line-3 Sf.hFVIIIco6XTEN.GPVΔ135 GPVΔ162 GPVΔ162 cell line-4 Sf.hFVIIIco6XTEN.GPV.WT GPV.WT GPV.WT cell line-5 Sf.hFVIIIco6XTEN.GPV.Asy GPVΔ162 GPV.WT cell line-6 Sf.hFVIIIco6XTEN.AAV2.WT AAV2.WT AAV2.WT cell line-7 Sf.hFVIIIco6XTEN.AAV2.Asy1 AAV2Δ15Δ11 AAV2Δ15 cell line-8 Sf.hFVIIIco6XTEN.AAV2.Asy2 AAV2.WT AAV2Δ15 cell line-9 Sf.hFVIIIco6XTEN.AAV2.Asy3 AAV2Δ15Δ11 AAV2.WT Example 9: Generation of human FVIIIco6XTEN ceDNA vector from a stable cell line

To test the stable cell line approach, multiple colonies of each cell line encoding the hFVIIIco6XTEN expression cassette (Table 4) with symmetric or asymmetric ITRs for non-AAV and AAV were used. Cells were infected with a titrated working stock (P2) of each Rep-encoding recombinant BEV (Figure 5B-D ) at a multiplicity of infection (MOI) of 3 pfu/cell. The cells were tumbled gently at room temperature for 1.5 h, pelleted at 500×g for 5 min, the supernatant was aspirated, and the cells were washed once with 10 mL of fresh ESF-921 medium. Finally, the cells were suspended in 50 mL of ESF-921 medium, and then incubated at 28°C for 72 h in a shaker incubator. At 72 h post-infection, infected cells were harvested, and the pellet was washed once with 1x PBS to remove residual baculovirus particles and/or medium. Then, ceDNA vectors were isolated by PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. The eluted fractions were analyzed by 0.8% to 1.2% agarose gel electrophoresis to determine the yield and purity of each ceDNA vector. The results showed DNA bands corresponding to the size (approximately 7.0 kb) of hFVIIIco6XTEN with different intensities as well as high molecular weight bands, as observed previously (Fig. 9A-C ) . Interestingly, all tested cell lines displayed DNA bands corresponding to the size of the hFVIIIco6XTEN expression cassette, independent of flanking symmetric and/or asymmetric ITRs. GPV cell lines -3, -4 and -5 showed multiple bands of different sizes compared to B19 or AAV2 cell lines. Example 10: Human FVIIIco6XTEN expression from ceDNA

To determine whether ceDNA could be used as a non-viral gene therapy vector for the treatment of hemophilia A patients, an in vitro assay was performed to test human FVIII expression from ceDNA in hepatocyte-derived cancerous human Huh7 cells. First, Huh7 cells were seeded at 5 x ¹⁰⁵ cells/mL in 24-well plates in modified IMEM medium (Gibco ^{TM ) supplemented with 10% FBS and 0.1% antibiotic mixture (Invitrogen TM )} , and then incubated with CO ₂ (5%) in a 37ºC incubator for 48 h to achieve monolayer formation due to static cell growth. This growth condition was achieved to mimic hFVIIIco6XTEN expression in the nucleus of non-dividing hepatocytes in vivo. Monolayers were transfected with approximately 1 ug 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 h after transfection, the transfection mixture was aspirated, and the cells were cultured with fresh IMEM medium supplemented with 10% FBS and 0.1% antibiotic mixture. At 48 h and 72 h post-culture, cell-free supernatants were aliquoted from each treatment and secreted hFVIIIco6XTEN was measured by the Chromogenix Coatest® SP Factor VIII Chromogenic Assay according to the manufacturer's instructions. FVIII activity was normalized to the standard and plotted in Figure 10 as U/mL. The assay results showed that except for hFVIII.B19Δ135.ceDNA, the hFVIII expression levels of all tested samples increased with time. This data suggests that FVIII is expressed from a ceDNA vector transfected presumably in the nucleus of a non-dividing monolayer of Huh7 cells. ceDNA obtained from GPV cell lines -3, -4, and -5 showed low levels of FVIII expression compared to AAV lines 6 and 8, suggesting that the GPV ITR was most likely truncated when integrated into the cellular genome, as above. Surprisingly, ceDNA obtained from B19 cell lines-1 and -2 showed no detectable expression of hFVIIIco6XTEN, which was detectable in the plasmid DNA (pCLDV-1, Table 3) used as a control. Overall, GPV and AAV ceDNA vectors showed sustained hFVIII expression at physiological levels in Huh7 cells, suggesting that ceDNA can be used as a non-viral gene therapy vector (Fig. 10 ) . The individual cell lines are described in Table 5. Example 11: Human FVIIIco6XTEN ceDNA vector production by transient transfection

Transient gene expression system is one of the most important technologies for functional analysis in baculovirus-insect cell line system. This system was developed for expressing foreign genes under the control of a baculovirus promoter in a transient expression plasmid. Furthermore, this system offers shorter turnaround times for vector construction and protein synthesis, avoids challenges associated with viral infection and cell lysis, and provides a robust means for observing cellular trafficking of proteins. Baculovirus gene promoters are generally classified as immediate early, early, late, and very late promoters according to their transcription initiation during the infection cycle. Of these, only the immediate early ( ie ) gene promoter is recognized by host RNA polymerase II and is independent of viral transcription factors, making it suitable for baculovirus-free expression of heterologous proteins in insect cells. The most widely and commercially available transient gene expression system was developed based on the immediate early ( ie ) gene promoter of Douglas moth polynuclear polyhedrosis virus (OpMNPV). Compared with other immediate early ( ie ) gene promoters, the OpIE2 promoter of OpMNPV has been shown to confer robust activity for heterologous protein expression in Sf cells (Bleckmann et al. , 2016). Recently, a baculovirus-free system based on polyethyleneimine (PEI)-mediated transient gene expression under the control of the OpIE2 promoter was also described (Puente-Massaguer et al ., 2020).

Therefore, it was hypothesized that transient expression of the Rep protein could "rescue" the human FVIIIco6XTEN ceDNA vector from stable cell lines. To test this hypothesis, PEI-mediated transient Rep protein expression under the OpMNPV immediate early (OpIE2) promoter was utilized for the generation of baculovirus-free ceDNA in stable cell lines. The OpIE2 promoter sequence (SEQ ID NO: 15) from the OpMNPV genome (GenBank accession number NC_001875.2) was synthesized by GenScript® (Piscataway, NJ). Synthetic promoter sequences were cloned to replace the polyhedrin or immediate early promoters in the Rep protein expression constructs (Figures 11A , 11C , 11E ) . The resulting transient expression plasmids pFastBac.OpIE2.B19.Rep (Fig. 11B ) , pFastBac.OpIE2.GPV.Rep (Fig. 11D ) and pFastBac.OpIE2.AAV2.Rep (Fig . 11F ) were used for transfection encoding with non-AAV and Stable cell lines of human FVIIIco6XTEN expressing cassette of symmetric or asymmetric ITR of AAV (Table 5).

Stable cell lines encoding human FVIIIco6XTEN expression cassettes with non-AAV and AAV symmetric or asymmetric ITRs (Table 5) were treated with B19.Rep, GPV.Rep or AAV2.Rep encoded under the OpMNPV immediate ( OpIE2 ) promoter The transient expression plasmid (Figure 11 ) and the plasmid encoding the puromycin resistance marker under the AcMNPV immediate early ( ie1 ) promoter preceded by the transcriptional enhancer hr5 element followed by the AcMNPV p10 polyadenylation signal were superimposed Transformation to generate a new panel of stable cell lines. Transformed cells were selected with puromycin and expanded into shake flask cultures as described in Example 8. These cell lines were used as production cell lines for the production of the baculovirus-free human FVIIIco6XTEN ceDNA vector. Example 12: Modified FVIIIXTEN Expression Cassettes

It is hypothesized that the level of transgene expression can be improved by codon-optimizing the cDNA for the target host. Physiological levels of FVIII expression from the V1.0 FVIIIco6XTEN expression cassette have been demonstrated in previous studies as described in US Pub. No. 20190185543. However, to further improve target specificity and reduce immunogenicity, the FVIIIXTEN cDNA was codon-optimized and CpG repeats were deleted to evade innate immune responses against DNA vectors encoding the FVIIIXTEN expression cassette with parvoviral ITRs. A modified V2.0 FVIIIXTEN expression cassette was generated containing the XTEN 144 peptide (FVIIIXTEN) under the control of the liver-specifically modified mouse transthyretin (mTTR) promoter (mTTR482) and enhancer element (A1MB2) Fused B-domain deleted (BDD) codon-optimized human factor VIII (BDDcoFVIII), hybrid synthetic intron (chimeric intron), woodchuck post-transcriptional regulatory element (WPRE), and bovine growth hormone Polyadenylation (bGHpA) signaling. The V2.0 FVIIIXTEN expression cassette comprises the nucleotide sequence of SEQ ID NO: 19.

Initial in vivo efficacy studies showed a significant 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 the recombinant BIVVBac baculovirus shuttle vector as described above (Example 6). Then, engineered parvovirus ITRs (including AAV2 WT ( FIG. 12A ), B19 WT or minimal (SEQ ID NO. 16) ( FIG. 12B ) and GPVΔ120 (SEQ ID NO. 17) or GPVΔ186 (SEQ ID NO. 18) ( FIG. 12C ) was inserted into the V2.0 FVIIIXTEN expression cassette (SEQ ID NO: 19) and tested for FVIIIXTEN ceDNA vector production in the baculovirus system using three different methods as described below. Example 13: FVIIIXTEN ceDNA Production Methods

In the baculovirus-insect cell lineage, recombinant BEVs deliver the gene of interest under a strong promoter and provide the transcriptional complexes necessary for virus replication in insect cells. This system provides the flexibility to insert the transgene of interest into the baculovirus genome and/or insect cell genome as a stable cell line. These advantages of the baculovirus system were exploited in the design of three different methods for ceDNA generation, thereby providing flexibility in platform selection according to the ease of scalability. Single BAC :

To investigate the use of the single Bac approach for transgene expression, the BIVVBac baculovirus shuttle vector was used. BIVVBac was designed to accept multiple transgene insertions by two different mechanisms at two different sites within the baculovirus genome, as described in Example 1. The optimized FVIIIXTEN expression cassette (SEQ ID NO: 19) was inserted together with the parvoviral ITR at the miniature att Tn7 site in the polyhedrin locus in BIVVBac by Tn7 translocation, and in the same backbone, via Cre-LoxP Recombination inserted the ITR-specific replication (Rep) gene expression cassette at the LoxP site in the EGT locus, as described above (Example 6). Recombinant BEVs were then generated and used for infection in Sf9 cells to produce FVIIIXTEN ceDNA, as depicted in Figure 13A . The concept of the single Bac approach for ceDNA production was demonstrated using different promoters to control the level of Rep expression, as described below. Double BAC :

To investigate the use of the double Bac approach for transgene expression, the optimized FVIIIXTEN expression cassette was inserted by Tn7 transposition together with the parvovirus ITR and/or the ITR-specific replication (Rep) gene expression cassette was inserted in two different At the miniature att Tn7 site in the polyhedrin locus in the BIVVBac baculovirus shuttle vector, as described above in Example 5. Recombinant BEVs were then generated and used for co-infection in Sf9 cells to produce FVIIIXTEN ceDNA, as depicted in Figure 13B . The challenges associated with the dual BAC approach were investigated using different multiplicity of infection (MOI) ratios of the two baculoviruses and fine-tuning of Rep expression levels for reproducible ceDNA productivity, as described in the following experiments. Stable cell line:

To investigate the use of the stable cell line approach for transgene expression, stable cell lines were generated with optimized FVIIIXTEN expression cassettes with parvovirus ITRs, as described in Example 8. Recombinant baculovirus shuttle vectors were also generated by inserting the ITR - specific replication (Rep) gene expression cassette by Tn7 translocation at the miniat tTn7 site in the polyhedrin locus in the BIVVBac baculovirus shuttle vector, As described above in Example 5. Recombinant Rep.BEVs were then generated and used for infection in FVIIIXTEN stable cell lines to produce FVIIIXTEN ceDNA, as depicted in Figure 13C . The challenges associated with the stable cell line approach were investigated by enriching FVIIIXTEN transformants by FACS cell sorting using GFP as a proxy to expedite the process of generating stable cell lines, as described in the experiments below. Example 14: Generation of FVIIIXTEN ceDNA (ceFVIIIXTEN) vectors from single BACs

FVIIIXTEN ceDNA (ceFVIIIXTEN) production of recombinant BEVs encoding V2.0 FVIIIXTEN with AAV2 WT or B19 WT ITRs and its expression under the AcMNPV polyhedrin promoter was tested in Sf9 cells using the single BAC method as described above. The corresponding Rep gene. Approximately 2.0 x 10 ⁶ /mL cells were infected with single BAC BEV at an MOI of 0.1, 0.5, 1.0, 2.0, or 3.0 plaque forming units (pfu)/cell ( FIG. 14A , FIG. 15A ). Cells were suspended in 50 mL of serum-free ESF-921 medium, and then incubated in a 28ºC shaker incubator for 72-96 h or until viability reached 60%-70%. At approximately 96 h post-infection, infected cells were harvested, and pellets were processed for ceFVIIIXTEN isolation using the PureLink Max Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. Final elution fractions were analyzed on 0.8% to 1.2% agarose gel electrophoresis to determine ceFVIIIXTEN productivity. ceFVIIIXTEN AAV2 ITR

Agarose gel analysis of AAV2 single BACs ( Figure 14B ) revealed a DNA band corresponding to the size of the ceFVIIIXTEN AAV2 ITR (approximately 8.5 kb) at all MOIs tested, which was significantly higher at 1.0 pfu/cell compared to other treatments The highest productivity was obtained at an MOI of 0 ( Fig. 14C ). This result contrasts with 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 binding mechanism and endonuclease activity for DNA replication at the terminal dissociation site of the AAV2 WT ITR compared to the HBoV1-NS1 protein, possibly due to the Due to the different hairpin structures (T-shape and U-shape). ceFVIIIXTEN B19 ITR

Agarose gel analysis of B19 single BACs ( Fig. 15B ) showed a DNA band corresponding to the size of the ceFVIIIXTEN B19 ITR (approx. 8.5 kb) at all tested MOIs, but at a lower level of productivity ( Fig. 15C ). Unlike AAV2 or HBoV1, higher viral loads of B19 single BACs did not improve ceFVIIIXTEN productivity, possibly due to higher levels of B19-NS1 expressed under the strong (polyhedrin) baculovirus promoter later in infection. These results suggest that the combination of full-length (WT) parvovirus ITR and stoichiometric REP expression may be crucial for achieving higher ceDNA productivity in the baculovirus system.

Taken together, these experiments show that the single BAC approach indeed proves concept for the generation of ceDNA from a single recombinant BEV encoding the FVIIIXTEN and NS1 transgenes with parvoviral ITRs. It also demonstrates the feasibility and functionality of inserting multiple transgenes at different loci in the baculovirus shuttle vector (BIVVBac), and its potential use for the production of recombinant AAV vectors in baculovirus insect cell lines use. Example 15: Generation of FVIIIXTEN ceDNA (ceFVIIIXTEN) vectors from double BACs

To investigate the double BAC approach for ceDNA transgene expression, several different co-infection conditions were tested by keeping the ratio constant and co-infecting Sf9 cells with double BAC at different MOIs, or by keeping the MOI constant and using Different ratios of double BACs co-infected Sf9 cells. In each case, the viral inoculum was not removed, and the cells were incubated in a 28ºC shaker incubator until viability reached 60%-70%. At approximately 96 h post-infection, infected cells were harvested, and pellets were processed for ceFVIIIXTEN isolation using the PureLink Max Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. Final elution fractions were analyzed on 0.8% to 1.2% agarose gel electrophoresis to determine ceFVIIIXTEN productivity. ceFVIIIXTEN AAV2 ITR

About 2.0 x 10 ⁶ /mL cells inoculated in 50 mL of serum-free ESF-921 medium were titrated with AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITR ^Tn7 BEV working stock solution (P2) (MOI: 0.01, 0.1, 0.3, 1.0, or 3.0 pfu/cell) and a titrated working stock (P2) of AcBIVVBac.Polh.AAV2.RepΔVP80 ^Tn7 BEV (previously described in U.S. Patent Application No. 63/069,115) (MOI of 0.001, 0.01, 0.03, 0.1, or 0.3 pfu/cell to keep the 1:10 ratio constant, respectively) co-transfection ( FIG. 16A to FIG. 16B ). Agarose gel analysis of AAV2 double BACs showed varying degrees of productivity of ceFVIIIXTEN (ceDNA) compared to contaminating baculovirus DNA (vDNA) at different MOIs of co-infection. While MOIs of 0.1-0.01 and 3.0-0.3 pfu/cell showed equivalent levels of ceFVIIIXTEN (ceDNA) productivity, the latter co-infection MOIs showed lower levels of contaminating vDNA ( FIG. 16C ).

These results indicate that while the AAV2 double BAC method showed equivalent levels of ceFVIIIXTEN productivity as the single BAC, there were significant differences in the levels of other impurities co-purified with this purification method. Without being bound by theory, this may further emphasize the importance of stoichiometric AAV2 REP78/REP52 expression for achieving higher ceFVIIIXTEN productivity in Sf9 cells. ceFVIIIXTEN B19 ITR

About 2.0 x 10 ⁶ /mL cells inoculated in 50 mL serum-free ESF-921 medium were titrated with AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITR ^Tn7 BEV working stock solution (P2) (MOI: 0.1, 0.3, 0.5, 1.0, 3.0, or 5.0 pfu/cell) and a titrated working stock (P2) of AcBIVVBac.Polh.B19-NS1 ^Tn7 BEV (MOI of 0.01, 0.03, 0.05, 0.1, 0.3, or 0.5 pfu/cell to maintain 1: 10 ratio or MOI of 0.02, 0.06, 0.1, 0.2 or 0.6 pfu/cell to maintain a 1:5 ratio, respectively) co-transfection ( FIG. 17A to FIG. 17B ). Unlike AAV2 double BACs, agarose gel analysis of B19 double BACs showed equivalent levels of ceFVIIIXTEN (ceDNA) and contaminating baculovirus DNA (vDNA) productivity at different MOIs of co-infection ( Fig. 17C ). Interestingly, none of the conditions tested showed the same improvement in ceFVIIIXTEN B19 ITR productivity as the results obtained with B19 single BAC.

In conclusion, both the B19 single BAC and B19 double BAC methods are proof of concept for the generation of ceDNA from non-AAV parvoviral ITRs in a baculovirus system. ceFVIIIXTEN GPV ITR

About 2.0 x 10 ⁶ /mL cells inoculated in 50 mL of serum-free ESF-921 medium were titrated with AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITR ^Tn7 BEV stock solution (P2) (at MOI of 0.1, 0.3, 0.5 , 1.0, 30 or 5.0 pfu/cell) and a titrated working stock solution (P2) of AcBIVVBac.Polh.GPV-NS1 ^Tn7 BEV (MOI of 0.01, 0.03, 0.05, 0.1, 0.3 or 0.5 pfu/cell to keep 1:10, respectively Ratio or MOI of 0.02, 0.06, 0.1, 0.2 pfu/cell to maintain a 1:5 ratio respectively) co-transfection ( FIG. 18A to FIG. 18B ). Unlike AAV2 or B19 double BACs, agarose gel analysis of GPV double BACs showed no detectable DNA bands corresponding to the size of ceFVIIIXTEN (approximately 8.5 kb) at all MOIs tested, except at 5.0–0.5 pfu Faint bands observed under co-infection at MOI of /cells ( Fig . 18C ). ceFVIIIXTEN productivity obtained with GPV double BAC correlated with GPV single BAC, where V1.0 FVIIIco6XTEN was used with GPV asymmetric ITR, as described above ( Fig. 7B , see Example 7).

In two studies, truncated GPV ITRs were tested due to the reproductive complexity of obtaining full-length WT GPV ITRs at both ends of the FVIIIXTEN transgene cassette. Nonetheless, both the GPV single-BAC and GPV double-BAC approaches proved the concept of ceDNA production and demonstrated the importance of optimal MOI ratio and promoter selection for achieving higher FVIIIXTEN ceDNA productivity in Sf9 cells. Example 16: Generation of FVIIIXTEN ceDNA (ceFVIIIXTEN) Vectors from Stable Cell Lines

To test the stable cell line approach, multiplex colonies of each cell line made with V2.0 FVIIIXTEN flanked by AAV2 WT, B19 minimal, or GPVΔ120 ITR were inoculated at approximately 2.0 x 10 ⁶ /mL in 50 mL Serum-free ESF921 medium and infected with a titrated working stock (P2) of ITR-specific REP.BEV at different MOIs as indicated. In each case, the viral inoculum was not removed, and the cells were incubated in a 28ºC shaker incubator for 72-96 h or until viability reached 60%-70%. At approximately 96 h post-infection, infected cells were harvested, and pellets were processed for ceFVIIIXTEN isolation using the PureLink Max Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. Final elution fractions were analyzed on 0.8% to 1.2% agarose gel electrophoresis to determine ceFVIIIXTEN productivity. ceFVIIIXTEN AAV2 ITR

Cell lines encoding V2.0 FVIIIXTEN with AAV2 WT ITR were developed as previously described (Example 8). The multiplex colonized Sf.FVIIIXTEN.AAV2.WT.ITR cell population was ^then titered with a working stock solution (P2) of 0.001, 0.01 , 0.03, 0.1, 0.3 or 0.5 pfu/cell MOI infection ( Figure 19A , 19B ). Agarose gel analysis of ceFVIIIXTEN isolated from AAV2 stable cell lines showed equivalent levels of productivity at all MOIs tested, except for those that did not show a detectable DNA band corresponding to the size of ceFVIIIXTEN (approximately 8.5 kb) Minimum MOI of 0.001 pfu/cell. Interestingly, cells infected with REP.BEV at MOIs of 0.01 and 0.03 pfu/cell showed higher ceFVIIIXTEN productivity in parallel with contaminating baculovirus DNA (vDNA) compared to higher MOIs ( FIG. 19C ). These results suggest that, unlike the AAV2 single-BAC or AAV2 double-BAC approaches, the stable cell line approach requires a lower viral inoculum to 'rescue' the stably integrated FVIIIXTEN expression cassette flanked by AAV2 WT ITRs in the Sf9 cell genome.

In conclusion, this study demonstrates the concept of producing ceDNA with a cell line stably integrating the FVIIIXTEN expression cassette flanked by parvoviral ITRs. ceFVIIIXTEN B19 ITR

Cell lines encoding V2.0 FVIIIXTEN with the B19 minimal ITR were developed as previously described (Example 8). Then the multi-seed Sf.FVIIIXTEN.B19.minimal.ITR cell population was titrated with AcBIVVBac.Polh.B19-NS1 ^Tn7 BEV working stock solution (P2) at 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0 or an MOI of 5.0 pfu/cell ( Fig. 20A , 20B ). Agarose gel analysis of ceFVIIIXTEN isolated from B19 stable cell line showed that the level of DNA band intensity increased with increasing MOI of REP.BEV. However, the DNA band was slightly below the expected size of ceFVIIIXTEN (approximately 8.5 kb) ( Fig. 20C ). Interestingly, a similar banding pattern was observed for V1.0 ceFVIIIXTEN obtained from a B19 stable cell line (Example 9, Figure 9A ) but with a different ITR. In contrast, ceFVIIIXTEN obtained from Sf9 cells infected with B19 single or double BACs encoding FVIIIXTEN with B19 WT ITRs showed a DNA band of the expected size for V2.0 ceFVIIIXTEN (approximately 8.5 kb) ( Fig. 15C and Fig. 17C ).

These results suggest that while terminal dissociation sites and REP-binding elements are present in truncated or minimal variants of the B19 ITR, the full-length (WT) sequence may be required for B19-NS1 to bind and nick DNA for effectively copied. ceFVIIIXTEN GPV ITR

Cell lines encoding V2.0 FVIIIXTEN with GPVΔ120 ITR were developed as previously described (Example 8). The multiplexed Sf.FVIIIXTEN.GPVΔ120.ITR cell population was then titrated with a working stock solution (P2) of AcBIVVBac.Polh.GPV-NS1 ^Tn7 BEV at 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0 or 5.0 pfu /MOI of cell infection ( Fig. 21A , 21B ). Unlike the B19 cell line, agarose gel analysis of ceFVIIIXTEN isolated from a GPV stable cell line showed a decreasing level of productivity with increasing MOI of infection.

Interestingly, for ceFVIIIXTEN GPV ITRs, an opposite trend was observed in ceFVIIIXTEN productivity compared to other parvovirus ITRs. The lowest MOI (0.01 pfu/cell) for infection in the GPV cell line showed the highest band intensity for ceFVIIIXTEN (ceDNA) compared to other MOIs tested ( FIG. 21C ). These results suggest that very low levels of GPV-NS1 are required for GPV-ITR-mediated ceDNA production, at least in stable cell lines. Furthermore, this data suggests that the lower productivity of ceFVIIIXTEN observed in GPV single or double BACs ( Fig . 7C , Fig. 18C ) may be due to high viral load, which indirectly corresponds to the relative Higher level GPV-NS1 performance than more.

Nonetheless, the stable cell line approach did demonstrate the concept of generating ceDNA from a cell line stably integrated with a FVIIIXTEN expression cassette flanked by non-AAV parvovirus ITRs. Example 17: FVIIIXTEN ceDNA (ceFVIIIXTEN) Vector Purification

In the baculovirus-insect cell lineage, recombinant BEVs deliver the gene of interest under a strong promoter and provide the transcriptional complexes necessary for virus replication in insect cells. Typically, the baculovirus DNA genome replicates in the nucleus and produces tens of millions of progeny virus particles, each containing a full-length DNA genome. Co-purification of baculovirus genomic DNA with ceDNA has been demonstrated while DNA is isolated from insect cells using plasmid DNA-based purification methods such as silica gel columns. Commercial plasmid DNA set columns are generally not designed to separate DNA based on molecular weight, and therefore, generally all forms of DNA present in a sample can bind to these columns. In addition, the binding capacity of large molecular weight DNA may be different from that of low molecular weight DNA, and anion exchange-based column sets are not optimized based on the binding efficiency of DNA of different sizes.

It was hypothesized that the high molecular weight DNA (>20 kb) observed in ceDNA preparations was most likely baculovirus and/or Sf9 cell genomic DNA co-purified with low molecular weight ceFVIIIXTEN (approximately 8.5 kb) (see e.g. Figure 14C to Figure 21C ). Previously, an indirect approach was employed to reduce baculovirus DNA by knocking out baculovirus capsid genes, such as VP80, that are required for viral production of infectious progeny. This approach has shown a dramatic reduction of baculovirus DNA in ceDNA preparations obtained from knockout of BEV (see US Patent Application No. 63/069,115). While this method was effective in reducing baculovirus DNA contamination, it failed to reduce cellular genomic DNA, which is present in significant amounts (approximately 60%) in the total DNA obtained from infected cell pellets.

In this study, a direct method to separate FVIIIXTEN ceDNA from the rest of the unwanted DNA was employed and demonstrated to be highly efficient in obtaining purified FVIIIXTEN (>95% purity) from total DNA preparations from infected cell pellets. This novel method utilizes preparative electrophoresis, which is widely used to separate different protein molecules based on size and charge. See eg Michov, B. (2020) Electrophoresis. Berlin, Boston: De Gruyter, pp. 405-424. For example, a Bio-Rad Model 491 prep cell or other such units can be used to separate complex molecules based on size.

In the current study, preparative electrophoresis was used to separate FVIIIXTEN ceDNA from high-molecular-weight DNA, and purified ceDNA to be used in in vivo studies was successfully obtained, as described below.

The overall workflow for ceDNA purification is shown in Figure 22 , where the process begins with the scale-up of Sf9 cell cultures in serum-free insect cell culture medium from 0.5 L to 1.5 L or higher volumes ( Figure 22A ). After reaching the desired cell density of approximately 2.5 x 10 ⁶ /mL, usually after 2 days of incubation at a seeding density of approximately 1.3 x 10 ⁶ /mL, cells were incubated with single-BAC or double-BAC BEV (depending on the cells used to generate ceDNA method) were infected at an optimized MOI, and the cells were incubated in a shaker incubator at 28ºC until viability reached about 60%-70%, which usually took about 4 days ( Figure 22B ). Once viability reaches approximately 70%, cells are harvested and processed for total DNA purification by anion exchange chromatography kit columns such as PureLink HiPure Expi Plasmid Gigaprep Purification Kit (Invitrogen) according to the manufacturer's instructions. Aliquots of purified DNA material were checked on 0.8% to 1.2% agarose gel electrophoresis to determine DNA productivity and integrity ( Figure 22C ). The purified material was then loaded onto a preparative agarose gel electrophoresis unit containing 0.5% preparative agarose and 0.25% packing agarose, assembled according to the manufacturer's instructions. Run the samples at low voltage (approximately 40 constant volts) at 4ºC for 6–7 days with a buffer recirculation flow rate of approximately 50 mL/min and an elution buffer rate of 50 µL/min to -80 min Collect each fraction in a fraction collection chamber. Following serial resolution electrophoresis, 20 µL of each fraction was checked on a 0.8% to 1.2% agarose gel electrophoresis to determine the purity of the FVIIIXTEN ceDNA ( FIG. 22D ). Desired fractions were combined for precipitation with 3 M NaOAc (pH 5.5) and 100% EtOH at -20C for 1-2 h. Finally, the precipitated FVIIIXTEN ceDNA was precipitated at high speed and washed once with 70% EtOH, then resuspended in TE (pH 8.0) buffer. Purified FVIIIXTEN ceDNA was checked again on 0.8% to 1.2% agarose gel electrophoresis to confirm purity and integrity before being injected into animals for in vivo efficacy studies ( FIG. 22E ). Example 18: FVIIIXTEN ceDNA (ceFVIIIXTEN) In Vivo Efficacy Systemic Administration of ceFVIIIXTEN in HemA Mice

To verify the function of ceDNA in vivo, purified ceFVIIIXTEN with AAV2 or HBoV1 WT ITR was injected at 0.3 µg, 1.0 µg, or 2.0 µg/mouse (equivalent to 12 µg, 40 µg, and 80 µg/kg, respectively) via hydrodynamic Tail vein injection for systemic injection in hFVIIIR593C ^+/+ /HemA mice. Plasma samples from injected mice were collected at 7-day intervals, and FVIII activity was measured by chromogenic assay, as described above.

Plasma FVIII activity normalized to percent of normal for the ceFVIIIXTEN injected cohort is shown in FIG. 23 . Results showed a dose-dependent response in HemA mice, with supraphysiological levels of FVIII expression (>500% of normal) observed at the highest doses of AAV2 or HBoV1 ceDNA tested. However, in the ceFVIIIXTEN AAV2 ITR injected cohort, a gradual decrease in FVIII expression levels was observed until day 140 post-injection, after which levels stabilized. Interestingly, the FVIII expression levels of ceFVIIIXTEN HBoV1 ITRs showed similar expression trends as mice injected with ceFVIIIXTEN AAV2 ITRs (data not shown).

Taken together, these in vivo efficacy studies validate the functionality of ceFVIIIXTEN and demonstrate that the parvoviral ITR can be used to generate functional ceDNA encoding a transgene of interest in a baculovirus insect cell line. Example 19: Generation of FVIIIXTEN HBoV1 ITRs ceDNA vector from single BAC

Single BAC (OneBAC) BEVs encoding the FVIIIXTEN HBoV1 ITR and HBoV1 NS1 genes were tested for FVIIIXTEN ceDNA production in Sf9 cells. Approximately 2.5 x 10 ⁶ /mL of cell lines were infected with titrated working stocks (P2) of each BEV at a multiplicity of infection (MOI) of 0.1, 0.5, 1.0, 2.0, or 3.0 plaque forming units (pfu) /cell ( Figure 24A ). The cells were suspended in 50 mL of serum-free ESF-921 medium, and then cultured in a shaker incubator at 28°C for 72-96 hours or until the cell viability reached 60-70%. At approximately 96 hours post-infection, infected cells were harvested and the pellet was treated with the PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions to isolate the FVIIIXTEN ceDNA vector. The final eluted fractions were analyzed on a 0.8% to 1.2% agarose gel electrophoresis to determine the yield of the FVIIIXTEN ceDNA vector.

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

This result is in contrast to the ceDNA yield obtained with AAV2 ITRs OneBAC, where it was previously observed that the yield decreased with increasing viral load. Without being bound by theory, the HBoV1-NS1 protein may have a unique binding mechanism and endonuclease activity at the terminal resolution site of HBoV1 ITRs for DNA replication, possibly due to REH and LEH Due to the different structures of ITRs.

Taken together, these experiments demonstrate that the single BAC approach does prove proof-of-concept for the production of ceDNA from a single recombinant BEV encoding FVIIIXTEN, ie, harboring the BoV1 ITR and NS1 transgenes. It also shows the feasibility and functionality of inserting multiple transgenes at different sites in a baculovirus shuttle vector (BIVVBac), and its potential for recombinant AAV vector production in a baculovirus insect cell system. Example 20: Generation of FVIIIXTEN HBoV1 ITRs ceDNA Vectors from Double BACs

To study the double BAC approach to express transgenes, clonal recombinant BEVs (clonal recombinant BEVs) encoding FVIIIXTEN HBoV1 ITR with polyhedrin-driven HBoV1-NS1 were injected at different MOIs of 1:10 and 1:5 or FVIIIXTEN ceDNA vector production experiments were performed in Sf9 cells at different ratios of MOI of 0.3, 1.0, 3.0 and 5.0 pfu/cell ( FIG. 25A ). Specifically, about 2.0×10 ⁶ /mL cell line was inoculated in 50 mL serum-free ESF-921 medium, and was titrated with AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRs ^Tn7 BEV working solution (P2) at an MOI of 0.1, 0.3, 0.5, 1.0, 3.0, 5.0 pfu/cell and AcBIVVBac.Polo.HBoV1-NS1 ^Tn7 BEV at a constant 1:10 ratio at MOI of 0.01, 0.03, 0.05, 0.1, 0.3, 0.5 pfu/cell, or at MOI Co-infection was performed separately at a constant 1:5 ratio of 0.02, 0.06, 0.1, 0.2, 0.6, 1.0 pfu/cell. Likewise, cells were also co-infected at a ratio of 1:1, 1:2, 1:5, or 1:10 at a constant MOI of 0.3, 1.0, 3.0, or 5.0 pfu/cell ( Figure 25B ). In each case, the viral inoculum was not removed and the cells were grown in a shaker at 28°C until viability reached 60–70%. Approximately 96 hr after infection, infected cells were harvested, and the cell pellet was treated with the PureLink Maxi Prep DNA Isolation Kit (Invitrogen) according to the manufacturer's instructions to isolate the FVIIIXTEN ceDNA vector. Analyze the final eluted fractions on a 0.8% to 1.2% agarose gel electrophoresis to determine the yield of ceDNA.

As expected, agarose gel analysis revealed that FVIIIXTEN ceDNA was yielded to varying degrees under different conditions. However, at an MOI of 3.0 pfu/cell, double BAC co-infection showed that the yield level of FVIIIXTEN ceDNA increased with increasing virus load, with the highest ratio of 1:10 compared to other tested conditions ( Fig . 25C ). Higher viral load appeared to increase the yield of ceDNA from FVIIIXTEN HBoV1 ITRs, consistent with observations in single BAC BEVs (see Example 6). This further suggests that HBoV1-ITR-dependent FVIIIXTEN ceDNA replication in Sf9 cells requires higher levels of HBoV1-NS1.

The results of single BAC or double BAC indicated that the level of replication of HBoV1-NS1 had a strong influence on the yield of FVIIIXTEN ceDNA in the baculovirus system.

Taken together, these experiments demonstrate that the dual BAC approach does prove proof-of-concept for the generation of ceDNA from two recombinant BEVs encoding FVIIIXTEN and HBoV1 ITR and/or NS1 transgenes. These experiments also demonstrate the importance of optimal MOI ratios and/or promoters for higher FVIIIXTEN ceDNA yields in Sf9 cells. SEQUENCE LISTING 6. Additional nucleotide or amino acid sequences. SEQ ID NO and description Nucleotide or Amino Acid Sequence SEQ ID NO:16 B19 minimal ITR CCAAATCAGATGCCGCCGGTCGCCGCCGGTAGGCGGGACTTCCGGTACAGGCGCGCCGCTGTACCGGAAGTCCCGCCTACCGGCGGCGACCGGCGGCATCTGATTTGGTGTCTTTCTTTTAAATTTT SEQ ID NO. 17 GPVΔ120 ITR: CTAGCGAGCTCATTGGAGGGTTCGTTCGTTCGAACCAGCCAATCAGGGGAGGGGGAAGTGACGCAAGTTCCGGTCACATGCTTCCGGTGACGCACATCCGGTGACGTAGTTCCGGTCACGTGCTTCCTGTCACGTGTTTCCGGTCGCATGCTCACGTGACCGGAAACACGTGACAGGAAGCACGTGACCGGAACTACGTCACCGGATGT GCGTCACCGGAAGCATGTGACCGGAACTTGCGTCACTTCCCCCTCCCCTGATTGGCTGGTTCGAACGAACGAACCCTCCAATGAGACTCAAGGACAAGAGGATATTTTGCGCGCCAGGAAGTGT SEQ ID NO. 18 GPVΔ186 ITR: CTAGCGAGCTCATTGGAGGGTTCGTTCGTTCGAACCAGCCAATCAGGGGAGGGGGAAGTGACGCAAGTTCCGGTCACATGCTTCCGGTGACGCACATCCGGTGACGTAGTTCGCATGCGAACTACGTCACCGGATGTGCGTCACCGGAAGCATGTGACCGGAACTTGCGTCACTTCCCCCTCCCCTGATTGGCTGGTTCGAACGAACGAACCC TCCAATGAGACTCAAGGACAAGAGGATATTTTGCGCGCCAGGAAGTGT SEQ ID NO:19 V2.0 expression cassette mTTR482-intron-coBDDFVIIIXTEN (V2.0)-WPRE-bGHPolyA GGCCCCAGGTTAATTTTTAAAAAGCAGTCAAAGGTCCAAAGTGGCCCTTGGCAGCATTTACTCTCTCTATTGACTTTGGTTAATAATTCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCCTGGACTTATCCTCTGGGCCTCTCCCCACCTTCGATGGCCCCAGGTTAATTTTTAAAAAGCAGTCAAAGGTCAAAGTGGCCCTTGGCAGCATT TACTTCTCTCTATTGACTTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCTGGACTTATCCTCTGGGCCTCTCCCCACCGATATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATAACTCTGTCGGGGCAAAGGTCGGCAGTAGTTTTCATCTTACTCAACATCCTCCCGTGTACGTAGGATCCTGTCT GTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCAAAGCTCCTGCT AGGAATTCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCTGGACTTATCCTCTGGGCCTCTCCCCACCGATATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATACTCTGTCGGGCAAAGGTCGGCAGTAGTTTCCATCTTACTCAACATCCTCCAGTACGTAGGATCCTGTCTGTCTGCACATTTCGTAGAGCGA GTGTTCCGATACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAAGCTCCTGCTAGAGTCGCTGCGCGCTGCCTTC GCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCCAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTATTGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAAGGCCCTTTGTGCGGGGGAGCGGC TCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGT GTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCC GCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAG CGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCTGTTCTTGCCTTC TTCTTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCCATCATTTTGGCAAAGAATTACTCGAGGCCACCATGCAGATTGAACTGTCCACTTGCTTCTTCCTGTGCCTCCTGCGGTTTTGCTTCTCGGCCACCCGCCGGTATTACTTAGGTGCTGTGGAACTGAGCTGGGACTACATGCAGTCCGACCTGGGAGAACTGCCG GTGGACGCGAGATTCCCACCTAGAGTCCCGAAGTCCTTCCCATTCAACACCTCCGTGGTCTACAAAAAGACCCTGTTCGTGGAGTTCACTGACCACCTTTTCAATATTGCCAAGCCGCGCCCCCCCTGGATGGGCCTGCTTGGTCCTACGATCCAAGCAGAGGTCTACGACACCGTGGTCATCACACTGAAGAACATGGCCTCACACCCCGTGTCGCTGCATG CTGTGGGAGTGTCCTACTGGAAGGCCTCAGAGGGTGCCGAATATGATGACCAGACCAGCCAGAGGGAAAAGGAGGATGACAAAGTGTTCCCGGGTGGCAGCCACACTTACGTGTGGCAAGTGCTGAAGGAAAACGGGCCTATGGCGTCGGACCCCCTATGCCTGACCTACTCCTACCTGTCCCATGTGGACCTTGTGAAGGATCTCAACTCGGGACTGA TCGGCGCCCTCTTGGTGTGCAGAGAAGGCAGCCTGGCGAAGGAAAAGACTCAGACCCTGCACAAGTTCATTCTGTTGTTTGCTGTGTTCGATGAAGGAAAGTCCTGGCACTCAGAAACCAAGAACTCGCTGATGCAGGATAGAGATGCGGCCTCGGCCAGAGCCTGGCCTAAAATGCACACCGTCAACGGATATGTGAACAGGTCGCTCCCTGGCCTCA TCGGCTGCCACAGAAAGTCCGTGTATTGGCATGTGATCGGCATGGGTACTACTCCGGAAGTGCATAGTATCTTTCTGGAGGGCCATACCTTCTTGGTGCGCAACCACAGACAGGCCTCGCTGGAAATCTCGCCTATCACTTTCTTGACTGCGCAGACCCTCCTTATGGACCTTGGACAGTTCCTGCTGTTCTGTCACATCAGCTCCCATCAGCATGATG GGATGGAGGCCTATGTCAAAGTGGACTCCTGCCCTGAGGAGCCACAGCTCCGGATGAAGAACAATGAGGAAGCGGAGGATTACGACGACGACCTGACTGACAGCGAAATGGACGTCGTGCGATTCGATGACGACAACAGCCGTCCTTCATCCAAATTAGATCAGTGGCGAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCCGCCGAGGA AGAGGACTGGGACTACGCGCCGCTGGTGCTGGCGCCAGACGACAGGAGCTACAAGTCCCAGTACCTCAACAACGGGCCGCAGCGCATTGGCAGGAAGTACAAGAAAGTCCGCTTCATGGCCTACACTGATGAAACCTTCAAGACGAGGGAAGCCATCCAGCACGAGTCAGGCATCCTGGGACCGCTCCTTTACGGCGAAGTCGGGGATACCCTGC TCATCATTTTTCAAGAACCAGGCATCGCGGCCCTACAACATCTACCCTCACGGGATCACAGACGTGCGCCCGCTCTACTCCCGCCGGCTGCCCAAGGGAGTGAAGCACCTGAAGGATTTTCCCATCCTGCCGGGAGAAATCTTCAAGTACAAGTGGACCGTGACTGTGGAAGATGGCCCTACCAAGTCGGACCTCGCTGTCTGACCCGGTACTATTCCTCGT TTGTGAACATGGAGCGCGACCTGGCCTCGGGGCTGATTGGTCCGCTGCTGATCTGCTACAAGGAGTCCGTGGACCAGCGCGGGAACCAGATCATGTCCGACAAGCGCAACGTGATCCTGTTCTCTGTCTTTGATGAAAACAGATCGTGGTACTTGACTGAGAATATCCAGCGGTTCCTGCCCAACCCAGCGGGAGTGCAACTGGAGGACCCGGAGT TCCAGGCCTCAAACATTATGCACTCTATCAACGGCTATGTGTTCGACTCGCTCCAACTGAGCGTGTGCCTGCATGAAGTGGCATACTGGTACATTCTGTCCATCGGAGCCCAGACCGACTTCCTGTCCGTGTTTCTTCTCCGGATACACCTTCAAGCATAAGATGGTGTACGAGGACACTCTGACCCTCTTCCCATTTTCCGGAGAAACTGTGTTCATGTCCAATGGA AAACCCGGGCTTGTGGATTCTGGGTTGCCATAACTCGGACTTCCGGAATAGAGGGATGACCGCCCTGCTGAAAGTGTCCAGCTGTGACAAGAATACCGGCGATTACTACGAGGACAGCTATGAGGACATCTCCGCTTATCTGCTGTCCAAAGAACAACGCCATTGAACCCAGGTCCTTCTCCAAAACGGTGCACCGACCTCCCGAAAGCGCCACCCCAGAGTC AGGACCTGGCTCGGAACCGGCTACCTCGGGCTCAGAGACACCGGGGACTTCCGAGTCCGCAACCCCCGAGAGTGGACCCGGATCCGAACCAGCAACCTCAGGATCAGAAACCCCGGGAACTTCGGAATCCGCCACTCCCGAGTCGGGACCAGGCACCTCCACTGAGCCTTCCGAGGGAAGCGCCCCCGGATCCCCTGCTGGATCCCCTACC AGCACTGAAGAAGGCACCTCAGAATCCGCGACCCCCTGAGTCCGGCCCTGGAAGCGAACCCGCCACCTCCGGTTCCGAAACCCCTGGGACTAGCGAGAGCGCCACTCCGGAATCGGGCCCAGGAAGCCCTGCCGGATCCCCGACCAGCACCGAGGAGGGAAGCCCCGCCGGGTCACCGACTTCCACTGAGGAGGGAGCCTCATCCCCCCCCGTGCT GAAGCGGCATCAAAGAGAGATCACCAGGACCACTTCTCAGTCCGATCAGGAAGAAATTGACTACGACGATACTATCAGCGTGGAGATGAAGAAGGAGGACTTCGACATCTACGATGAGGATGAGAACCAGTCCCTCGGAGCTTTCAGAAGAAAACCCGCCACTACTTCATCGCTGCCGTGGAGCGCTGTGGGATTACGGGATGTCCAGCTCCACCGCATGT GCTGCGGAATAGAGCGCAGTCAGGATCGGTGCCCCAGTTCAAGAAGGTCGTGTTCCAAGAGTTCACCGACGGGTCCTTCACTCAACCCCTGTACCGGGGCGAACTCAACGAACACCTGGGACTGCTTGGGCCGTATATCAGGGCAGAAGTGGAAGATAACATCATGGTCACCTTCCGCAACCAGGCCTCCCGGCCGTACAGCTTCTACTCTTCACTGATC TCCTACGAGGAAGATCAGCGGCAGGGAGCCGAGCCCCGGAAGAACTTCGTCAAGCCTAACGAAACTAAGACCTACTTTTGGAAGGTCCAGCATCACATGGCCCCGACCAAAGACGAGTTCGACTGTAAAGCCTGGGCCTACTTCTCGATGTGGACCTGGAGAAGGACGTGCACTCGGGACTCATTGGCCCGCTCCTTGTGTCCATACTAATACCCTGAACC CTGCTCACGGTCGCCAAGTCACAGTGCAGGAGTTCGCCCTCTTCTTCACCATCTTCGATGAAACAAAGTCCTGGTACTTTACTGAGAACATGGAACGCAATTGCAGGGCACCCTGCAACATCCAGATGGAAGATCCCACCTTCAAGGAAAACTACCGGTTTCATGCCATTAACGGCTACATAATGGACACGTTGCCAGGACTGGTCATGGCCCAGGACCAGA GAATCCGGTGGTATCTGCTCTCCATGGGCTCCAACGAAAACATTCACAGCATTCATTTTTCCGGCCATGTGTTCACCGTCCGGAAGAAGGAAGAGTACAAGATGGCTCTGTACAACCTTCTACCCTGGAGTGTTCGAGACTGTGGAAATGCTGCCTAGCAAGGCCGGCATTTGGAGAGTGGAATGCCTGATCGGAGAGCATTTGCACGCCGGAATGTCCACC CTGTTTCTTGTGTACTCCAACAAGTGCCAGACCCCGCTGGGAATGGCCTCAGGTCATATTAGGGATTTCCAGATCACTGCTTCGGGGCAGTACGGGCAGTGGGCACCTAAGTTGGCCCGGCTGCACTCTGGCTCCATCAATGCCTGGTCCACAAGGAACCCTTCTCCTGGATTAAGGTGGACCTCCTGGCCCCAATGATTATTCACGGTAT TAAGACCCAGGGTGCCCGACAGAAGTTCTCCTCACTCTACATCTCTCGCAATTCATCATAATGTACAGCCTGGATGGGAAGAAGTGGCAGACCTACCGGGGAAACTCCACTGGAACGCTCATGGTGTTTTTCGGCAACGTGGACTCCTCCGGCATTAAGCACAACATCTTCAACCCTCCGATCATTGCTCGGTACATCCGGCTGCACCCAACTCACTACAGC ATCCGGTCCACCCTGCGGATGGAACTGATGGGTTGTGACCTGAACTCCTGCTCCATGCCCCTTGGGATGGAATCCAAGGCCATTAGCGATGCACAGATCACCGCCTCTTCATACTTCACCAACATGTTCGCGACCTGGTCCCGTCGAAGGCCCGCCTGCACCTCCAAGGTCGCTCCAATGCGTGGCGGCCTCAAGTGAACAACCCAAGGAGTG GCTCCAGGTCGACTTCCAAAAAGACCATGAAGGTCACCGGAGTGACCACCCAGGGCGTGAAGTCCCTGCTGACCTCTATGTACGTTAAGGAGTTCCTCATCCTCCAAGCCAAGACGGACATCAGTGGACCCTGTTCTTCCAAAACGGAAAAGTCAAAGTATTCCAGGGCAACCAGGACTCCTTCACCCCTGTGGTCCAACCATTGCTGACCC GCTACCTCCGCATCCACCCCCAAAGCTGGGTCCACCCAGATCGCACTGCGCATGGAGGTCCTTGGATGCGAAGCCCAAGATCTGTACTAAGCGGCCGCTCATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTAT GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCC GCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGC CTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCTGCCTAGGCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCCACTGTCCTTTCCTAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAAGACCATGGGCGCGCCAGGCCTGTCGACGCCCGGGCGGTACCGCGATCGCTCGCGACGCATAAAG SEQ ID NO:20 Nucleotide sequence encoding coBDDFVIIIXTEN (V2.0) ATGCAGATTGAACTGTCCACTTGCTTCTTCCTGTGCCTCCTGCGGTTTTGCTTCTCGGCCACCCGCCGGTATTACTTAGGTGCTGTGGAACTGAGCTGGGACTACATGCAGTCCGACCTGGGAGAACTGCCGGTGGACGCGAGATTCCCACCTAGAGTCCCGAAGTCCTTCCCATTCAAACACCTCCGTGGTCTACAAAAAGACCCTGTTCGT GGAGTTCACTGACCACCTTTCAATATTGCCAAGCCGCGCCCCCCCTGGATGGGCCTGCTTGGTCCTACGATCCAAGCAGAGGTCTACGACACCGTGGTCATCACACTGAAGAACATGGCCTCACACCCCGTGTCGCTGCATGCTGTGGGAGTGTCCTACTGGAAGGCCTCAGAGGGTGCCGAATATGATGACCAGACCAGCCAGAGGGAAAAGGAGG ATGACAAAGTGTTCCCGGGTGGCAGCCACACTTACGTGTGGCAAGTGCTGAAGGAAAACGGGCCTATGGCGTCGGACCCCCTATGCCTGACCTACTCCTACCTGTCCCATGTGGACCTTGTGAAGGATCTCAACTCGGGACTGATCGGCGCCCTCTTGGTGTGCAGAGAAGGCAGCCTGGCGAAGGAAAAGACTCAGACCCTGCACAAGTTCATTCTGTTGT TTGCTGTGTTCGATGAAGGAAAGTCCTGGCACTCAGAAACCAAGAACTCGCTGATGCAGGATAGAGATGCGGCCTCGGCCAGAGCCTGGCCTAAAATGCACACCGTCAACGGATATGTGAACAGGTCGCTCCCTGGCCTCATCGGCTGCCACAGAAAGTCCGTGTATTGGCATGTGATCGGCATGGGTACTACTCCGGAAGTGCATAGTATCTTTCT GGAGGGCCATACCTTCTTGGTGCGCAACCACAGACAGGCCTCGCTGGAAATCTCGCCTATCACTTTCTTGACTGCGCAGACCCTCCTTATGGACCTTGGACAGTTCCTGCTGTTCTGTCACATCAGCTCCCATCAGCATGATGGGATGGAGGCCTATGTCAAAGTGGACTCCTGCCCTGAGGAGCCACAGCTCCGGATGAAGAACAATGAGGAAGC GGAGGATTACGACGACGACCTGACTGACAGCGAAATGGACGTCGTGCGATTCGATGACGACAACAGCCCGTCCTTCATCCAAATTAGATCAGTGGCGAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCCGCCGAGGAAGAGGACTGGGACTACGCGCCGCTGGTGCTGGCCCAGACGACAGGAGCTACAAGTCCCAGTACCTCAACAACGG GCCGCAGCGCATTGGCAGGAAGTACAAAGAAAGTCCGCTTCATGGCCTACACTGATGAAACCTTCAAGACGAGGGAAGCCATCCAGCACGAGTCAGGCATCCTGGGACCGCTCCTTTACGGCGAAGTCGGGGATACCCTGCTCATCATTTTCAAGAACCAGGCATCGCGGCCCTACAACATCTACCCTCACGGGATCACAGACGTGCGCCCGCTCTACTCCC GCCGGCTGCCCAAGGGAGTGAAGCACCTGAAGGATTTTCCCATCCTGCCGGGAGAAATCTTCAAGTACAAGTGGACCGTGACTGTGGAAGATGGCCCTACCAAGTCGGACCCTCGCTGTCTGACCCGGTACTATTCCTCGTTTGTGAACATGGAGCGCGACCTGGCCTCGGGGCTGATTGGTCCGCTGCTGATCTGCTACAAGGAGTCCGTGG ACCAGCGCGGGAACCAGATCATGTCCGACAAGCGCAACGTGATCCTGTTCTCTGTCTTTGATGAAAACAGATCGTGGTACTTGACTGAGAATATCCAGCGGTTCCTGCCCAACCCAGCGGGAGTGCAACTGGAGGACCCGGAGTTCCAGGCCTCAAACATTATGCACTCTATCAACGGCTATGTGTTCGACTCGCTCCAACTGAGCGTGTGCCTGCATGAAGT GGCATACTGGTACATTCTGTCCATCGGAGCCCAGACCGACTTCCTGTCCGTGTTTCTCTCCGGATACACCTTCAAGCATAAGATGGTGTACGAGGACACTCTGACCCTCTTCCCATTTTCCGGAGAAACTGTGTTCATGTCAATGGAAAACCCGGGCTTGTGGATTCTGGGTTGCCATAACTCGGACTTCCGGAATAGAGGGATGACCGCCCTGCTGAAA GTGTCCAGCTGTGACAAGAATACCGGCGATTACTACGAGGACAGCTATGAGGACATCTCCGCTTATCTGCTGTCCAAGAACAACGCCATTGAACCCAGGTCCTTCTCCAAAACGGTGCACCGACCTCCCGAAAGCGCCACCCCAGAGTCAGGACCTGGCTCGGAACCGGCTACCTCGGGCTCAGAGACACCGGGGACTTCCGAGTCCGCAACCCCCGAGAGT GGACCCGGATCCGAACCAGCAACCTCAGGATCAGAAACCCCGGGAACTTCGGAATCCGCCACTCCCGAGTCGGGACCAGGCACCTCCACTGAGCCTTCCGAGGGAAGCGCCCCCGGATCCCCTGCTGGATCCCCTACCAGCACTGAAGAAGGCACCTCAGAATCCGCGACCCCTGAGTCCGGCCCTGGAAGCGAACCCGCCACCTCCGGTTCCGA AACCCCTGGGACTAGCGAGAGCGCCACTCCGGAATCGGGCCCAGGAAGCCCTGCCGGATCCCCGACCAGCACCGAGGAGGGAAGCCCCGCCGGGTCACCGACTTCCACTGAGGAGGGAGCCTCATCCCCCCCCGTGCTGAAGCGGCATCAAAGAGAGATCACCAGGACCACTTCCAGTCCGATCAGGAAGAAATTGACTACGACGATACTATCAGCGT GGAGATGAAGAAGGAGGACTTCGACATCTACGATGAGGATGAGAACCAGTCCCTCGGAGCTTTCAGAAGAAAACCCGCCACTACTTCATCGCTGCCGTGGAGCGGCTGTGGGATTACGGGATGTCCAGCCACCGCATGTGCTGCGGAATAGAGCGCAGTCAGGATCGGTGCCCCAGTCAAGAAGGTCGTGTTCCAGAGTTCACCGACGGGTCCTT CACTCAACCCCTGTACCGGGGCGAACTCAACGAACACCTGGGACTGCTTGGGCCGTATATCAGGGCAGAAGTGGAAGATAACATCATGGTCACCTTCCGCAACCAGGCCTCCCGGCCGCCGTACAGCTTACTCTTCACTGATCTCCTACGAGGAAGATCAGCGGCAGGGAGCCGAGCCCCGGAAGAACTTCGTCAAGCCTAACGAAACTAAGACCTACTTTTT GGAAGGTCCAGCATCACATGGCCCCGACCAAAGACGAGTTCGACTGTAAAGCCTGGGCCTACTTCTCCGATGTGGACCTGGAGAAGGACGTGCACTCGGGACTCATTGGCCCGCTCCTTGTGTGCCATACTAATACCCTGAACCCTGCTCACGGTCGCCAAGTCACAGTGCAGGAGTTCGCCCTCTTCTTCACCATCTTCGATGAAAACAAAGTCCTGGTACTT TACTGAGAACATGGAACGCAATTGCAGGGCACCCTGCAACATCCAGATGGAAGATCCCACCTTCAAGGAAAACTACCGGTTTCATGCCATTAACGGCTACATAATGGACACGTTGCCAGGACTGGTCATGGCCCAGGACCCAGAGAATCCGGTGGTATCTGCTCTCCATGGGCTCCAACGAAAACATTCACAGCATTCATTTTTCCGGCCATGTGTTCACC GTCCGGAAGAAGGAAGAGTACAAGATGGCTCTGTACAACTCTCTACCCTGGAGTGTTCGAGACTGTGGAAATGCTGCCTAGCAAGGCCGGCATTTGGAGAGTGGAATGCCTGATCGGAGAGCATTTGCACGCCGGAATGTCCACCCTGTTTCTTGTGTACTCCAAACAAGTGCCAGACCCCGCTGGGAATGGCCTCAGGTCATATTAGGGATTTCCAGATCACT GCTTCGGGGCAGTACGGGCAGTGGGCACCTAAGTTGGCCCGGCTGCACTACTCTGGCTCCATCAATGCCTGGTCCACCAAGGAACCCTTCTCCTGGATTAAGGTGGACCTCCTGGCCCCAATGATTATTCACGGTATTAAGACCCAGGGTGCCCGACAAGTTCTCCTCACTCTACATCTCGCAATTCATCATAATGTACAGCCTGGATGGGAAG AAGTGGCAGACCTACCGGGGAAACTCCACTGGAACGCTCATGGTGTTTTTCGGCAACGTGGACTCCTCCGGCATTAAGCACAACATCTTCAACCCTCCGATCATTGCTCGGTACATCCGGCTGCACCCAACTCACTACAGCATCCGGTCCACCCTGCGGATGGAACTGATGGGTTGTGACCTGAACTCCTGCTCCATGCCCCTTGGGATG GAATCCAAGGCCATTAGCGATGCACAGATCACCGCCTCTTCATACTTCACCAACATGTTCGCGACCTGGTCCCGTCGAAGGCCCGCCTGCACCTCCAAGGTCGCTCCAATGCGTGGCGGCCTCAAGTGAACAACCCCAAGGAGTGGCTCCAGGTCGACTTCCAAAAGACCATGAAGGTCACCGGAGTGACCACCCAGGGCGTGAAGTCCCTGCTGACCTCTAT GTACGTTAAGGAGTTCCTCATTCTCCTCAAGCCAAGACGGACATCAGTGGACCCTGTTCTTCCAAAACGGAAAAGTCAAAGTATTCCAGGGCAACCAGGACTCCTTCACCCCTGTGGTCCAACAGCCTGGACCCCCCATTGCTGACCCGCTACCTCCGCATCCACCCCCAAAGCTGGGTCCACCCAGATCGCACTGCGCATGGAGGTCCTTGGATGCGAAGCCCAAG ATCTGTACTAA SEQ ID NO:21 A1MB2 enhancer GGCCCCAGGTTAATTTTTAAAAAGCAGTCAAAGGTCCAAAGTGGCCCTTGGCAGCATTTACTCTCTCTATTGACTTTGGTTAATAATTCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCCTGGACTTATCCTCTGGGCCTCTCCCCACCTTCGATGGCCCCAGGTTAATTTTTAAAAAGCAGTCAAAGGTCAAAGTGGCCCTTGGCAGCATT TACTTCTCTCTATTGACTTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCTGGACTTATCCTCTGGGCCTCTCCCCACC SEQ ID NO:22 mTTR promoter GATATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATACTCTGTCGGGGCAAAGGTCGGCAGTAGTTTCCATCTTACTCAACATCCTCCCAGTGTACGTAGGATCCTGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAA TAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAAGCTCCTGCTAG SEQ ID NO:23 chimeric intron TCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCCTGGACTTATCCTCTGGGCCTCTCCCCACCGATATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATACTCTGTCGGGGCAAAGGTCGGCAGTAGTTTTCATCTTACTCAACATCCTCGTGTAGGATCCTGTCTGTCTGCACATTTCGTAGAGCGAGTGT TCCGATACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGTCCACAAGCTCCTGCTAGAGTCGCTGCGCGCTGCCTTCGCC CCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCCAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTATTGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAAGGCCCTTTGTGCGGGGGGAGCGGCTCG GGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGC GTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCG GTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCTTGTTCTTGCCTTCTTCT TTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTA SEQ ID NO: 24 WPRE TCATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCA CTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTC GCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCTG SEQ ID NO: 25 bGHpA CGACTGTGTGCCTGCTCCCCCCCCCCAGCCCCCCCCCCCCCCCCCCCCCCCCCTGGGGGCCCCCCCCCCCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCTCCTCCCTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC GGTGTCATTCTTCTCTGGGGGGGGGGGGGGGGAggacagggggggggggggggggggggctgggggga SEQ ID NO:28 Nucleotide sequence encoding BDD-co6FVIII (V1.0) (without XTEN) GCCACTCGCCGGTACTACCTTGGAGCCGTGGAGCTTTCATGGGACTACATGCAGAGCGACCTGGGCGAACTCCCCGTGGATGCCAGATTCCCCCCCCGCGTGCCAAAGTCCCTTCCCCTTTAACACCTCCGTGGTGTACAAGAAAACCCTCTTTGTCGAGTTCACTGACCACCTGTTCAACATCGCCAAGCCGCGCCCACCTTGGATGGGCCTCCTGG GACCGACCATTCAAGCTGAAGTGTACGACACCGTGGTGATCACCCTGAAGAACATGGCGTCCCACCCCCGTGTCCCTGCATGCGGTCGGAGTGTCCTACTGGAAGGCCTCCGAAGGAGCTGAGTACGACGACCAGACTAGCAGCGGGAAAAGGAGGACGATAAAGTGTTCCCGGGCGGCTCGCATACTTACGTGTGGCAAGTCCTGAAGGAAAACGGACCTAT GGCATCCGATCCTCTGTGCCTGACTTACTCCTACCTTTCCCATGTGGACCTCGTGAAGGACCTGAACAGCGGGCTGATTGGTGCACTTCTCGTGTGCCGCGAAGGTTCGCTCGCTAAGGAAAAGACCCAGACCCTCATAAGTTCATCCTTTTGTTCGCTGTGTTCGATGAAGGAAAGTCATGGCATTCCGAAACTAAGAACTCGCTGATGCAGGACCGGGATG CCGCCTCAGCCCGCGCCTGGCCTAAAATGCATACAGTCCAACGGATACGTGAATCGGTCACTGCCCGGGCTCATCGGTTGTCACAGAAAGTCCGTGTACTGGCACGTCATCGGCATGGGCACTACGCCTGAAGTGCACTCCATCTTCCTGGAAGGGCACACCTTCTCGTGCGCAACCACCGCCAGGCCTCTCTGGAAATCTCCCCCGATTACCTTTCTGA CCGCCCAGACTCTGCTCATGGACCTGGGGCAGTTCCTTCTCTTCTGCCACATCTCCAGCCATCAGCACGACGGAATGGAGGCCTACGTGAAGGTGGACTCATGCCCGGAAGAACCTCAGTTGCGGATGAAGAACAACGAGGAGGCCGAGGACTATGACGACGATTTGACTCCGAGATGGACGTCGTGCGGTTCGATGACGACAACAGCCCC AGCTTCATCCAGATTCGCAGCGTGGCCAAGAAGCACCCCAAAACCTGGGTGCACTACATCGCGGCCGAGGAAGAAGATTGGGACTACGCCCCGTTGGTGCTGGCACCCGATGACCGGTCGTACAAGTCCCAGTATCTGAACAATGGTCCGCAGCGGATTGGCAGAAAGTACAAAGAAAGTGCGGTTCATGGCGTACACTGACGAAACGTTTAAGACCC GGGAGGCCATTCAACATGAGAGCGGCATTCTGGGACCACTGCTGTACGGAGAGGTCGGCGATACCCTGCTCATCATCTTCAAAAACCAGGCCTCCCGGCCTTACAACATCTACCCTCACGGAATCACCGACGTGCGGCCACTCTACTCGCGGCGCCTGCCGAAGGGCGTCAAGCACCTGAAAGACTTCCCTATCCTGCCGGGCGAAAATCTTCAAGTATAA GTGGACCGTCACCGTGGAGGACGGGCCCACCAAGAGCGATCCTAGGTGTCTGACTCGGTACTACTCCAGCTTCGTGAACATGGAACGGGACCTGGCATCGGGACTCATTGGACCGCTGCTGATCTGCTACAAAGAGTCGGTGGATCAACGCGGCAACCAGATCATGTCCGACAAGCGCAACGTGATCCTGTTCTCCGTGTTTGATGAAAACAGAT CCTGGTACCTCACTGAAAACATCCAGAGGTTCCTCCCAAAACCCCGCAGGAGTGCAACTGGAGGACCCTGAGTTTCAGGCCTCGAATATCATGCACTCGATTAACGGTTACGTGTTCGACTCGCTGCAGCTGAGCGTGTGCCTCCATGAAGTCGCTTACTGGTACATTCTGTCCATCGGCGCCCAGACTGACTTCCTGAGCGTGTTCTTTTCCGGTTACCCTTTAAG CACAAGATGGTGTACGAAGATACCCTGACCCTGTTCCCTTTTCCCGGCGAAACGGTGTTCATGTCGATGGAGAACCCGGGTCTGTGGATTCTGGGATGCCACAACAGCGACTTTCGGAACCGCGGAATGACTGCCCTGCTGAAGGTGTCCTCATGCGAAGGTGTCCTCGGAGACTACTACGAGGACTCCTACGAGGATATCTCAGCCTACCTCCTGTCC AAGAACAACGCGATCGAGCCGCGCAGCTTCAGCCAGAACCCGCCTGTGTGAAGAGGCACCAGCGAGAAATTACCCGGACCACCCTCCAATCGGATCAGGAGGAAATCGACTACGACGACCCATCTCGGTGGAAATGAAGAAGGAAGATTTCGATATCTACGACGAGGACGAAAATCAGTCCCTCGCTCATTCCAAAAAAAACTAGACACTACTTTTATCGCC GCGGTGGAAAGACTGTGGGGACTATGGAATGTCATCCAGCCTCACGTCCTTCGGAACCGGCCCAGAGCGGATCGGTGCCTCAGTTCAAGAAAGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCGCTGTACCGGGAGAACTGAACGAACACCTGGGCCTGCTCGGTCCCTACATCCGCGCGGAAGTGGAGGATAACATCATG GTGACCTTCCGTAACCAAGCATCCAGACCTACTCCTTCTATTCCTCCCTGATCTCATACGAGGAGGACCAGCGCCAAGGCGCCGAGCCCCGCAAGAACTTCGTCAAGCCCAACGAGACTAAGACCTACTTCTGGAAGGTCCAAACACCATATGGCCCCGACCCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCCGACGTGGACCTTGAGAAGGATGTCCAT TCCGGCCTGATCGGGCCGCTGCTCGTGTCACACCAACACCCCTGAACCCAGCGCATGGACGCCAGGTCACCGTCCAGGAGTTTGCTCTGTTTCTTCACCATTTTTGACGAAACTAAGTCCTGGTACTTCACCGAGAATATGGAGCGAAACTGTAGAGCGCCCTGCAATATCCAGATGGAAGATCCGACTTTCAAGGAGAACTATAGATTCCACGCCATCAACGG GTACATCATGGATACTCTGCCGGGGCTGGTCATGGCCCAGGATCAGAGGATTCGGTGGTACTTGCTGTCAATGGGATCGAACGAAAACATTCACTCCATTCACTTCTCCGGTCACGTGTTCACTGTGCGCAAGAAGGAGGAGTACAAGATGGCGCTGTACAATCTGTACCCCGGGGTGTTCGAAACTGTGGAGATGCTGCCGTCCAAGGCC GGCATCTGGAGAGTGGAGTGCCTGATCGGAGAGCACCTCCACGCGGGGATGTCCACCCTCTTCCTGGTGTACTCGAATAAGTGCCAGACCCCGCTGGGCATGGCCTCGGGCCACATCAGAGACTTCCAAGATCACAGCAAGCGGACAATACGGCCAATGGGCGCCGAAGCTGGCCCGCTTGCACTACTCCGGATCGATCAACGCATGGTCCACCAAGGAAC CGTTCTCGTGGATTAAGGTGGACCTCCTGGCCCCTATGATTATCCACGGAATTAAGACCCAGGGCGCCAGGCAGAAGTTTCCTCCCTGTACATCTCGCAATTCATCATCATGTACAGCCTGGACGGGAAGAAGTGGCAGACTTACAGGGGAAACTCCACCGCACCCTGATGGTCTTTTTCGGCAACGTGGATTCCTCCGGCATTAAGCACAACAT CTTCAACCCACCGATCATAGCCAGATATATTAGGCTCCACCCCACTCACTACTCAATCCGCTCAACTCTTCGGATGGAACTCATGGGGTGCGACCTGAACTCCTGCTCCATGCCGTTGGGGATGGAATCAAAGGCTATTAGCGACGCCCAGATCACCGCGAGCCTACTTCACTAACATGTTCGCCACCTGGAGCCCCTCCAAGGCCAGGCTGCACTTGC AGGGACGGTCAAATGCCTGGCGGCCGCAAGTGAACAATCCGAAGGAATGGCTTCAAGTGGATTTCCAAAAAGACCATGAAAGTGACCGGAGTCACCACCCAGGGAGTGAAGTCCCTTCTGACCTCGATGTATGTGAAGGAGTTCCTGATTAGCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAACGGAAAGGTCCAAGGTGTTCCAGGGGAAC CAGGACTCGTTCACACCCGTGGTGAACTCCCTGGACCCCCCACTGCTGACGCGGTACTTGAGGATTCATCCTCAGTCCTGGGTCCATCGATTGCATTGCGAATGGAAGTCCTGGGCTGCGAGGCCCAGGACCTGTACTGA SEQ ID NO:29 Nucleotide sequence encoding coBDDFVIII (V2.0) (without XTEN) GCCACCCGCCGGTATTACTTAGGTGCTGTGGAACTGAGCTGGGACTACATGCAGTCCGACCTGGGAGAACTGCCGGTGGACGCGAGATTCCCACCTAGAGTCCCGAAGTCCTTCCCATTCAACACCTCCGTGGTCTACAAAAAGACCCTGTTCGTGGAGTTCACTGACCACCTTTCAATATTGCCAAGCCGCGCCCCCCCTGGATGGGCCTGC TTGGTCCTACGATCCAAGCAGAGGTCTACGACACCGTGGTCATCACACTGAAGAACATGGCCTCACACCCCGTGTCGCTGCATGCTGTGGGAGTGTCCTACTGGAAGGCCTCAGAGGGTGCCGAATATGATGACCAGACCAGCCAGAGGGAAAAGGAGGATGACAAAGTGTTCCCGGGTGGCAGCCACACTTACGTGTGGCAAGTGCTGAAGGAAAACG GGCCTATGGCGTCGGACCCCCTATGCCTGACCTACTCCTACCCTGTCCCATGTGGACCTTGTGAAGGATCTCAACTCGGGACTGATCGGCGCCCTCTTGGTGTGCAGAGAAGGCAGCCTGGCGAAGGAAAAGACTCAGACCCTGCACAAGTTCATTCTGTTGTTTGCTGTGTTCGATGAAGGAAAGTCCTGGCACTCAGAAACCAAGAACTCGCTGATGCAG GATAGAGATGCGGCCTCGGCCAGAGCCTGGCCTAAAATGCACACCGTCAACGGATATGTGAACAGGTCGCTCCCTGGCCTCATCGGCTGCCACAGAAAGTCCGTGTATTGGCATGTGATCGGCATGGGTACTACTCCGGAAGTGCATAGTATCTTTCTGGAGGGCCATACCTTCTTGGTGCGCAACCACAGACAGGCCTCGCTGGAAATCTCGCC TATCACTTTCTTGACTGCGCAGACCCTCCTTATGGACCTTGGACAGTTCCTGCTGTTCTGTCACATCAGCTCCCATCAGCATGATGGGATGGAGGCCTATGTCAAAGTGGACTCCTGCCCTGAGGAGCCACAGCTCCGGATGAAGAACAATGAGGAAGCGGAGGATTACGACGACGACCTGACTGACAGCGAAATGGACGTCGTGCGATTCGAT GACGACAACAGCCGTCCTTCATCCAAATTAGATCAGTGGCGAAGAAGCACCCCAAGACCTGGGTGCACTACATTGCCGCCGAGGAAGAGGACTGGGACTACGCGCCGCTGGTGCTGGCGCCAGACGACAGGAGCTACAAGTCCCAGTACCTCAACAACGGGCCGCAGCGCATTGGCAGGAAGTACAAAGAAGTCCGCTTCATGGCCTACACTGATGA AACCTTCAAGACGAGGGAAGCCATCCAGCACGAGTCAGGCATCCTGGGACCGCTCCTTTACGGCGAAGTCGGGGATACCCTGCTCATCATTTTCAAAGAACCAGGCATCGCGGCCCTACAACATCTACCCTCACGGGATCACAGACGTGCGCCCGCTCTACTCCCGCCGGCTGCCCAAGGGAGTGAAGCACCTGAAGGATTTTCCCATCCTGCCGGGAGAA ATCTTCAAGTACAAGTGGACCGTGACTGTGGAAGATGGCCCTACCAAGTCGGACCCTCGCTGTCTGACCCGGTACTATTCCTCGTTTGTGAACATGGAGCGCGACCTGGCCTCGGGGCTGATTGGTCCGCTGCTGATCTGCTACAAGGAGTCCGTGGACCAGCGCGGGAACCAGATCATGTCCGACAAGCGCAACGTGATCCTGTTCTCTGTCTTTG ATGAAAACAGATCGTGGTACTTGACTGAGAATATCCAGCGGTTCCTGCCCAACCCAGCGGGAGTGCAACTGGAGGACCCGGAGTTCCAGGCCTCAAACATTATGCACTCTATCAACGGCTATGTGTTCGACTCGCTCCAACTGAGCGTGTGCCTGCATGAAGTGGCATACTGGTACATTCTGTCCATCGGAGCCCAGACCGACTTCCTGTCCGTGTTCTTCT CCGGATACACCTTCAAGCATAAGATGGTGTACGAGGACACTCTGACCCTCTTCCCATTTTCCGGAGAAACTGTGTTCATGTCAATGGAAAACCCGGGCTTGTGGATTCTGGGTTGCCATAACTCGGACTTCCGGAATAGAGGGATGACCGCCCTGCTGAAAGTGTCCAGCTGTGACAAGAATACCGGCGATTACTACGAGGACAGCTATGAGGACATCTCC GCTTATCTGCTGTCCAAGAACAACGCCATTGAACCCAGGTCTTCTCCCAAAACGGTGCACCGGCCTCATCCCCCCCCGTGCTGAAGCGGCATCAAAGAGAGATCACCAGGACCACTTCTCCAGTCCGATCAGGAAGAAATTGACTACGACGATACTATCAGCGTGGAGATGAAGAAGGAGGACTTCGACATCTACGATGAGGATGAGAACCAGTCCCTCGGAGC TTTCAGAAGAAAACCCGCCACTACTTCATCGCTGCCGTGGAGCGGCTGTGGGATTACGGGATGTCCAGCTCACCGCATGTGCTGCGGAATAGAGCGCAGTCAGGATCGGTGTCCCCAGTTCAAGAAGGTCGTGTTCCAAGAGTTCACCGACGGGTCCTTCACTCAACCCCTGTACCGGGGCGAACTCAACGAACACCTGGGACTGCTTGGGCCGTATAT CAGGGCAGAAGTGGAAGATAACATCATGGTCACCTTCCGCAACCAGGCCTCCCGGCCGTACAGCTTCTACTCTTCACTGATCTCCTACGAGGAAGATCAGCGGCAGGGAGCCGAGCCCCGGAAGAACTTCGTCAAGCCTAACGAAACTAAGACCTACTTTTGGAAGGTCCAGCATCACATGGCCCCGACCAAAGACGAGTTCGACTGTAAAGCCTGGGCCTACTT CTCCGATGTGGACCTGGAGAAGGACGTGCACTCGGGACTCATTGGCCCGCTCCTTGTGTGCCATACTAATACCCTGAACCCTGCTCACGGTCGCCAAGTCACAGTGCAGGAGTTCGCCCTCTTCTTCACCATCTTCGATGAAACAAAGTCCTGGTACTTTACTGAGAACATGGAACGCAATTGCAGGGCACCCTGCAACATCCAGATGGAAGATCCCACCTTCA AGGAAAACTACCGGTTTCATGCCATTAACGGCTACATAATGGACACGTTGCCAGGACTGGTCATGGCCCAGGACCAGAGAATCCGGTGGTATCTGCTCTCCATGGGCTCCAACGAAAACATTCACAGCATTCATTTTTCCGGCCATGTGTTCACCGTCCGGAAGAAGGAAGAGTACAAGATGGCTCTGTACAACCTTCTACCCTGGAGTGTTCGAGACTGT GGAAATGCTGCCTAGCAAGGCCGGCATTTGGAGAGTGGAATGCCTGATCGGAGAGCATTTGCACGCCGGAATGTCCACCCTGTTTCTTGTGTACTCCAACAAGTGCCAGACCCCGCTGGGAATGGCCTCAGGTCATATTAGGGATTTCCAGATCACTGCTTCGGGGCAGTACGGGCAGTGGCACCTAAGTTGGCCCGGCTGCACTACTCTG GCTCCATCAATGCCTGGTCCACCAAAGGAACCCTTCTCCTGGATTAAGGTGGACCTCCTGGCCCCAATGATTATTCACGGTATTAAGACCCAGGGTGCCCGACAGAAGTTCTCCTCACTCTACATCTCGCAATTCATCATAATGTACAGCCTGGATGGGAAGAAGTGGCAGACCTACCGGGGAAACTCCACTGGAACGCTCATGGTGTTTTTCGGCAACGT GGACTCCTCCGGCATTAAGCACAACATCTTCAACCCTCCGATCATTGCTCGGTACATCCGGCTGCACCCAACTCACTACAGCATCCGGTCCACCCTGCGGATGGAACTGATGGGTTGTGACCTGAACTCCTGCTCCATGCCCCTTGGGATGGAATCCAAGGCCATTAGCGATGCACAGATCACCGCCTCTTCATACTTCACCAACATGTTCGCGAC CTGGTCCCGTCGAAGGCCCGCCTGCACCTCCAAGGTCGCTCCAATGCGTGGCGGCCTCAAGTGAACAACCCCAAGGAGTGGCTCCAGGTCGACTTCCAAAAAGACCATGAAGGTCCACCGGAGTGACCACCCAGGGCGTGAAGTCCCTGCTGACCCTCTATGTACGTTAAGGAGTTCCTCATCCTCCAAGCCAAGACGGACATCAGTGGACCCTGTTCTTCCAAA ACGGAAAAGTCAAAGTATTCCAGGGCAACCAGGACTCCTTCACCCCTGTGGTCAACAGCCTGGACCCCCCATTGCTGACCCGCTACCTCCGCATCCACCCCCAAAGCTGGGTCCACCAGATCGCACTGCGCATGGAGGTCCTTGGATGCGAAGCCCAAGATCTGTACTAA SEQ ID NO: 30 V1.0 expression cassette TTP-intron-BDDFVIIIco6XTEN (V1.0)-WPRE-bGHPolyA expression cassette ATGCAGATTGAGCTGTCCACTTGTTTCTCCTGTGCCTCCTGCGCTTCTGTTTCTCCGCCACTCGCCGGTACTACCTTGGAGCCGTGGAGCTTTCATGGGACTACATGCAGAGCGACCTGGGCGAACTCCCCGTGGATGCCAGATTCCCCCCCCGCGTGCCAAAGTCCCTCCCTTTAACACCTCCGTGGTGTACAAGAAAACCCTTTGTCGAGTTCAC TGACCACCTGTTCAACATCGCCAAGCCGCGCCCACCTTGGATGGGCCTCCTGGGACCGACCATTCAAGCTGAAGTGTACGACACCGTGGTGATCACCCCTGAAGAACATGGCGTCCCCACCCCGTGTCCCTGCATGCGGTCGGAGTGTCCTACTGGAAGGCCTCCGAAGGAGCTGAGTACGACGACCAGACTAGCAGCGGGAAAAGGAGGACGATAAAGTGT TCCCGGGCGGCTCGCATACTTACGTGTGGCAAGTCCTGAAGGAAAACGGACCTATGGCATCCGATCCTCTGTGCCTGACTTACTCCTACCTTTCCCATGTGGACCTCGTGAAGGACCTGAACAGCGGGCTGATTGGTGCACTTCTCGTGTGCCGCGAAGGTTCGCTCGCTAAGGAAAAGACCCAGACCTCCATAAGTTCCATCCTTTTGTTCGCTGTGTTCGATGA AGGAAAGTCATGGCATTCCGAAACTAAGAACTCGCTGATGCAGGACCGGGATGCCGCCTCAGCCCGCGCCTGGCCTAAAATGCATACAGTCAACGGATACGTGAATCGGTCACTGCCCGGGCTCATCGGTTGTCACAGAAAGTCCGTGTACTGGCACGTCATCGGCATGGGCACTACGCCTGAAGTGCACTCCATCTTCCTGGAAGGGCACACCT TCCTCGTGCGCAACCACCGCCAGGCCTCTCTGGAAATCTCCCCGATTACCTTTCTGACCGCCCAGACTCTGCTCATGGACCTGGGGCAGTTCCTTCTCTTCTGCCACATCTCCAGCCATCAGCACGACGGAATGGAGGCCTACGTGAAGGTGGACTCATGCCCGGAAGAACCTCAGTTGCGGATGAAGAACAACGAGGAGGCCGAGGACTATGACGAC GATTTGACTGACTCCGAGATGGACGTCGTGCGGTTCGATGACGACAACAGCCCCAGCTTCATCCAGATTCGCAGCGTGGCCAAGAAGCACCCCAAAACCTGGGTGCACTACATCGCGGCCGAGGAAGAAGATTGGGACTACGCCCCGTTGGTGCTGGCACCCGATGACCGGTCGTACAAGTCCCAGTATCTGAACAATGGTCCGCAGCGGATTGG CAGAAAGTACAAAGAAAGTGCGGTTCATGGCGTACACTGACGAAACGTTTAAGACCCGGGAGGCCATTCAACATGAGAGCGGCATTCTGGGACCACTGCTGTACGGAGAGGTCGGCGATACCCTGCTCATCATCTTCAAAAACCAGGCCTCCCGGCCTTACAACATCTACCCTCACGGAATCACCGACGTGCGGCCACTCTACTCGCGGCGCCTGCCGAAG GGCGTCAAGCACCTGAAAGACTTCCCTATCCTGCCGGGCGAAATCTTCAAGTATAAGTGGACCGTCACCGTGGAGGACGGGCCCACCAAGAGCGATCCTAGGTGTCTGACTCGGTACTACTCCAGCTTCGTGAACATGGAACGGGACCTGGCATCGGGACTCATTGGACCGCTGCTGATCTGCTACAAAGAGTCGGTGGATCAACGCGGCAAC CAGATCATGTCCGACAAGCGCAACGTGATCCTGTTCTCCGTGTTTGATGAAAACAGATCCCTGGTACCTCACTGAAAACATCCAGAGGTTCCTCCCAAAACCCCGCAGGAGTGCAACTGGAGGACCCTGAGTTTCAGGCCTCGAATATCATGCACTCGATTAACGGTTACGTGTTCGACTCGCTGCAACTGAGCGTGTGCCTCCATGAAGTCGCTTACTGGTACATTCT GTCCATCGGCGCCCAGACTGACTTCCTGAGCGTGTTCTTTTCCGGTTACACCCTTTAAGCACAAGATGGTGTACGAAGATACCCTGACCCTGTTCCCTTTCTCCGGCGAAACGGTGTTCATGTCGATGGAGAACCCGGGTCTGTGGATTCTGGGATGCCACAACAGCGACTTTCGGAACCGCGGAATGACTGCCCTGCTGAAGGTGTCCTCATGCGACAAG AACACCGGAGACTACTACGAGGACTCCTACGAGGATATCTCAGCCTACCTCCTGTCCAAGAACAACGCGATCGAGCCGCGCAGCTTCAGCCAGAACGGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGCACAAGTGAGTCTGCAACTCCCGAGTCCGGACCTGGCTCCGAGC CTGCCACTAGCGGCTCCGAGACTCCGGGAACTTCCGAGAGCGCTACACCAGAAAGCGGACCCGGAACCAGTACCGAACCTAGCGAGGGCTCTGCTCCGGGCAGCCCAGCCGGCTCTCCTACATCCACGGAGGAGGGCACTTCCGAATCCGCCACCCCGGAGTCAGGGCCAGGATCTGAACCCGCTACCTCAGGCAGTGAGACGCCAGGAACGAGC GAGTCCGCTACACCGGAGAGTGGGCCAGGGAGCCCTGCTGGATCTCCTACGTCCACTGAGGAAGGGTCACCAGCGGGCTCGCCCACCAGCACTGAAGAAGGTGCCTCGAGCCCGCCTGTGCTGAAGAGGCACCAGCGAGAAATTACCCGGACCACCCTCCAATCGGATCAGGAGGAAATCGACTACGACGACCATCTCGGTGGAAATGAAGAAGGA AGATTTCGATATCTACGACGAGGACGAAAATCAGTCCCTCGCTCATTCCAAAAGAAAACTAGACACTACTTTTATCGCCGCGGTGGAAAGACTGTGGGACTATGGAATGTCATCCAGCCTCACGTCCTTCGGAACCGGGCCCAGAGCGGATCGGTGCCTCAGTCAAGAAAGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCGCTGTA CCGGGGAGAACTGAACGAACACCTGGGCCTGCTCGGTCCCTAACATCCGCGCGGAAGTGGAGGATAACATCATGGTGACCTTCCGTAACCAAGCATCCAGACCTACTCCTTCTATTCCTCCCTGATCTCATACGAGGAGGACCCAGCGCCAAGGCGCCGAGCCCCGCAAGAACTTCGTCAAGCCCAACGAGACTAAGACCTACTTCTGGAAGGTCCAACACCATAT GGCCCCGACCAAGGATGAGTTTGACTGCAAGGCCTGGGCCTACTTCTCCGACGTGGACCTTGAGAAGGATGTCCATTCCGGCCTGATCGGGCCGCTGCTCGTGTGTCACACCAACACCCCTGAACCCAGCGCATGGACGCCAGGTCACCGTCCAGGAGTTTGCTCTGTTTCTCACCATTTTTGACGAAACTTAAGTCCTGGTACTTCACCGAGAATATGGAGC GAAACTGTAGAGCGCCCTGCAATATCCAGATGGAAGATCCGACTTTCAAGGAGAACTATAGATTCCACGCCATCAACGGGTACATCATGGATACTCTGCCGGGGCTGGTCATGGCCCAGGATCAGAGGATTCGGTGGTACTTGCTGTCAATGGGATCGAACGAAAACATTCACTTCACTTCTCCGGTCACGTGTTCACTGTGCGCAAGAAGGAG GAGTACAAGATGGCGCTGTACAATCTGTACCCCGGGGTGTTCGAAACTGTGGAGATGCTGCCGTCCAAGGCCGGCATCTGGAGAGTGGAGTGCCTGATCGGAGAGCACCTCCACGCGGGGATGTCCACCCTCTTCCTGGTGTACTCGAATAAGTGCCAGACCCCGCTGGGCATGGCCTCGGGCCATCAGAGACTTCCAGATCACAGCAAGCG GACAATACGGCCAATGGGCGCCGAAGCTGGCCCGCTTGCACTACTCCGGATCGATCAACGCATGGTCCACCAAGGAACCGTTCTCGTGGATTAAGGTGGACCTCCTGGCCCCTATGATTATCCACGGAATTAAGACCCAGGGCGCCAGGCAGAAGTTTCCTCCCTGTACATCTCGCAATTCATCATCATGTACAGCCTGGACGGGAAGAAGTGGCAGACTT ACAGGGGAAACTCCACCGGCACCCTGATGGTCTTTTTCGGCAACGTGGATTCCTCCGGCATTAAGCACAACATCTTCAACCCACCGATCATAGCCAGATATATTAGGCTCCACCCCACTCACTACTCAATCCGCTCAACTCTTCGGATGGAACTCATGGGGTGCGACCTGAACTCCTGCTCCATGCCGTTGGGGATGGAATCAAAGGCTATTAGC GACGCCCAGATCACCGCGAGCTCCTACTTCACTAACATGTTCGCCACCTGGAGCCCCTCCAAGGCCAGGCTGCACTTGCAGGGACGGTCAAATGCCTGGCGGCCGCAAGTGAACAATCCGAAGGAATGGCTTCAAGTGGATTTCCAAAAAGACCATGAAAGTGACCGGAGTCACCACCCAGGGAGTGAAGTCCCTTCTGACCTCGATGTATGTGAAGGAGTTCCT GATTAGCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTCCAAAACGGAAAGGTCAAGGTGTTCCAGGGGAACCAGGACTCGTTCACACCCGTGGTGAACTCCCTGGACCCCCCACTGCTGACGCGGTACTTGAGGATTCATCCTCAGTCCTGGGTCCATCAGATTGCATTGCGAATGGAAGTCCTGGGCTGCGAGGCCCAGGACCTGTACT GA

none.

1A to 1D are schematic diagrams of recombinant baculovirus shuttle vectors according to one embodiment of the present invention. Figure 1A shows a schematic diagram of bMON14272, which encodes a kanamycin resistance marker (KanR), a miniattTn7 insertion site fused in-frame with LacZα, and a miniF origin of replication (miniF). Figure 1B shows a schematic linear map of a synthetic transfer vector comprising a gene encoding red fluorescent protein (RFP) under the AcMNPV 39K promoter (p39K), followed by an ets polyadenylation signal ( etsPAS), LoxP recombination sites and EGT flanking gene sequences. Figure 1C shows a schematic diagram of a recombinant baculovirus shuttle vector (BIVVBac) encoding a kanamycin resistance marker (KanR), a mini-attTn7 insertion site fused in-frame with LacZα, a mini-F The origin of replication (miniature F), the gene encoding the red fluorescent protein (RFP) gene under the AcMNPV 39K promoter (p39K), followed by the ets polyadenylation signal (etsPAS), and the LoxP recombination site. Figure 1D shows a schematic diagram of an E. coli strain (BIVVBacDH10B) with a BIVVBac baculovirus shuttle vector and a Tn7 helper plasmid encoding a translocase.

2A to 2B are schematic diagrams of a Cre-LoxP donor vector according to an embodiment of the present invention. Figure 2A shows a schematic linear map of synthetic DNA comprising the AcMNPV immediate early 1 (pIE1) promoter preceded by the AcMNPV transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal (p10 PAS). Genes encoding enhanced GFP, LoxP recombination site (LoxP), ampicillin antibiotic resistance gene, multiple colonization site (MCS), ColE1 and R6Kγ origin of replication (Ori) FIG. 2B shows a schematic map of the Cre-LoxP donor vector prepared by inserting synthetic DNA ( FIG. 2A ) into pUC57 vector according to one embodiment of the present invention.

3A - 3F are schematic diagrams of replicating (Rep) protein expression constructs according to embodiments of the present invention. Figure 3A shows a schematic linear map of synthetic DNA encoding the B19 Rep gene under 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 prepared by inserting B19.Rep synthetic DNA ( FIG. 3A ) into pFastBac1 vector (Invitrogen) according to one embodiment of the present invention. Figure 3C shows a schematic linear map of synthetic DNA encoding the GPV Rep78 and Rep52 splice genes under the AcMNPV polyhedrin promoter, followed by the sv40 polyadenylation signal (SV40 PAS). Modified mRNA transcripts (mRNA) are shown as wavy lines with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) and stop codon (UAA) of Rep52. FIG . 3D shows a schematic map of a Tn7 transfer vector prepared by inserting GPV.Rep synthetic DNA ( FIG. 3C ) into pFastBac1 vector (Invitrogen) according to one embodiment of the present invention. Figure 3E shows a schematic linear map of synthetic DNA encoding the AAV2 Rep78 and Rep52 splice genes under the AcMNPV polyhedrin promoter, followed by the sv40 polyadenylation signal (SV40 PAS). Modified mRNA transcripts (mRNA) are shown as wavy lines with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) and stop codon (UAA) of Rep52. FIG . 3F shows a schematic map of a Tn7 transfer vector prepared by inserting AAV2.Rep synthetic DNA ( FIG. 3E ) into pFastBac1 vector (Invitrogen) according to one embodiment of the present invention.

Figures 4A - 4B are schematic representations of human FVIIIco6XTEN expression constructs. Figure 4A shows a schematic linear map of expression constructs encoding codon-optimized human Factor VIII (FVIIIco6) (FVIIIco6XTEN) comprising the XTEN 144 peptide under the control of the liver-specific TTPp promoter , woodchuck post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation (bGHpA) signaling. The hFVIIIco6XTEN expression cassette is flanked by the following symmetric truncated (Δ), symmetric wild-type (WT) and asymmetric (Asy) ITRs, respectively: B19 (Δ135, WT), GPV (Δ162, WT and Asy) or AAV2 ( WT, Asy1, Asy2, Asy3). The indicated ITR sequences can be found in Table 1. Fig. 4B shows a schematic linear map of the Cre-LoxP donor vector encoding the gene colonized into the Cre-LoxP donor vector as described in Fig. 2B as described in Fig. 4A . hFVIIIco6XTEN expression cassette flanked by the ITR of B19, GPV or AAV2.

Figures 5A to 5G show schematic diagrams of recombinant baculovirus expression vectors (BEVs) comprising sequences encoding Rep, and validation studies thereof. Figure 5A is a recombinant BIVVBac baculovirus shuttle vector encoding B19.Rep, GPV.Rep or AAV2.Rep (respectively BIVVBac.IE1.B19.Rep ^Tn7 , BIVVBac.Polh.GPV.Rep ^Tn7 and BIVVBac.Polh.AAV2. Rep ^Tn7 ) Agarose gel electrophoresis images of restriction enzyme mapping compared to parental BIVVBac and bMON14272 baculovirus shuttle vectors. Figure 5B shows a schematic map of AcBIVVBac.IE1.B19.Rep ^Tn7 . Figure 5C shows a schematic map of AcBIVVBac.Polh.GPV.Rep ^Tn7 . Figure 5D shows a schematic map of AcBIVVBac.Polh.AAV2.Rep ^Tn7 . Figure 5E shows plaque-purified colonies (colonies 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#5, Cl# of recombinant AcBIVVBac.IE1.B19.Rep ^Tn7 BEV 6) B19.REP immunoblot in which multiplexed anti-B19 NS1 (1:2500) was used as primary antibody and IRDye@680RD donkey anti-rabbit LI-COR (1:10,000) was used as secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as standard and is shown on the left. Figure 5F shows plaque-purified colonies of recombinant AcBIVVBac.Polh.GPV.Rep ^Tn7 BEV (colonies 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#5, Cl# 6) Western blot of GPV.REP in which multiplexed anti-GPV REP peptide antibody (1:500) was used as primary antibody and IRDye@680RD donkey anti-rabbit LI-COR (1:10,000) was used as secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as standard and is shown on the left. Figure 5G shows AAV2.REP immunoblotting in a plaque-purified colony of recombinant AcBIVVBac.Polh.AAV2.Rep ^Tn7 BEV with monoclonal anti-AAV2 Rep (1:500) as the primary antibody and IRDye@800CW donkey anti-small Mouse LI-COR (1:10,000) was used as secondary antibody. Precision Plus Protein™ Dual Color (Bio-Rad) was used as standard and is shown on the left.

Figures 6A - 6C are schematic representations of BEVs comprising sequences encoding Rep and hFVIIIco6XTEN. Figure 6A shows a schematic map of recombinant BEVs encoding the B19.Rep expression cassette and the hFVIIIco6XTEN expression cassette flanked by the B19 ITR as indicated (AcBIVVBac(B19.Rep)FVIII.B19.ITR ^LoxP ). Figure 6B shows a schematic map of a recombinant BEV encoding a GPV.Rep expression cassette and a hFVIIIco6XTEN expression cassette flanked by GPV ITRs as indicated (AcBIVVBac(GPV.Rep)FVIII.GPV.ITR ^LoxP ). Figure 6C shows a schematic map of a recombinant BEV encoding an AAV2.Rep expression cassette and an hFVIIIco6XTEN expression cassette flanked by AAV2 ITRs (AcBIVVBac(AAV2.Rep)FVIII.AAV2.ITR ^LoxP ). The ITR sequences indicated in Figures 6A - 6C can be found in Table 1.

7A to 7C illustrate the production 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 a recombinant BEV encoding a GPV.Rep expression cassette and an hFVIIIco6XTEN expression cassette flanked by GPV asymmetric ITRs (AcBIVVBac(GPV.Rep)FVIII.GPV.ITR ^LoxP ) . The indicated ITR sequences can be found in Table 1. Figure 7B is from the plaque purification colonies (colonies 1-6; Cl#1, Cl#2, Cl#3, Cl#4, Cl#4, Cl#4, Cl#4, Cl#4, Cl#1 from AcBIVVBac (GPV.Rep) FVIII.GPV.Asy.ITR ^LoxP BEV #5, Cl#6) Agarose gel electrophoresis images of ceDNA vectors isolated from infected Sf9 cells. DNA bands corresponding to the size of hFVIIIco6XTEN are indicated by arrows. Figure 7C is an agarose gel electrophoresis image of the ceDNA vector obtained from recombinant AcBIVVBac(GPV.Rep) FVIII.GPV.Asy.ITR ^LoxP BEV colony #4 ( as shown in Figure 7B ) loaded at different volumes . DNA bands corresponding to the size of hFVIIIco6XTEN are indicated by arrows.

Figures 8A - 8B show materials used to generate stable human FVIIIco6XTEN-encoding insect cell lines. Figure 8A shows a schematic map of a plasmid encoding a neomycin resistance marker under the AcMNPV immediate early (ie1) promoter preceded by the transcriptional enhancer hr5 element and followed by the AcMNPV p10 polyadenylation signal . Figure 8B shows a schematic map of hFVIIIco6XTEN expression cassettes flanked by B19, GPV or AAV2 ITRs that are stably integrated into the Sf9 cell genome to generate stable cell lines. The list of stable cell lines generated is shown in Table 5, and the sequences of the ITRs are shown in Table 1.

Figures 9A - 9C are images of agarose gel electrophoresis showing the production of the human FVIIIco6XTEN ceDNA vector from a stable cell line. Figure 9A is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf cell lines-1 and -2, the ceDNA vectors encode flanking symmetrical truncated (B19Δ135) or symmetrical wild-type (B19 .WT) hFVIIIco6XTEN of ITR. Figure 9B is an agarose gel electrophoresis image of the ceDNA vectors isolated from Sf cell lines -3, -4 and -5, the ceDNA vectors are flanked by symmetrical truncated (GPVΔ162), symmetrical wild hFVIIIco6XTEN of type (GPV.WT) or asymmetric type (GPV.Asy) ITR. Figure 9C is an agarose gel electrophoresis image of the ceDNA vectors isolated from Sf cell lines -6, -7, -8 and -9, the ceDNA vectors are flanked by symmetrical wild-type (AAV2.WT), hFVIIIco6XTEN of asymmetry 1 (AAV2.Asy1), asymmetry 2 (AAV2.Asy2) or asymmetry 3 (AAV2.Asy3). The stable cell lines used in Figures 9A to 9C are shown in Table 5, and the sequences of the ITRs are shown in Table 1.

Figure 10 is a graph showing hFVIII activity measured by Chromogenix Coatest® SP Factor VIII Chromogenic Assay. ceDNA isolated from a stable cell line was transfected in Huh7 cells, and cell-free supernatants were harvested 48 and 72 h after transfection and tested by chromogenic assay to detect hFVIII. Solid lines indicate ceDNA samples, and dashed lines indicate plasmid DNA samples used as controls.

11A - 11F are schematic diagrams of replicating (Rep) protein expression constructs according to embodiments of the present invention. Figure 11A shows a schematic linear map of synthetic DNA encoding the B19 Rep gene under the OpMNPV immediate early (OpIE2) promoter followed by the sv40 polyadenylation signal (SV40 PAS). Figure 1 IB shows a schematic map of a transient expression plasmid made by replacing the IE1 promoter with OpIE2 in the pFastBac.IE1.B19.Rep construct as shown in Figure 3B . Figure 11C shows a schematic linear map of synthetic DNA encoding the GPV Rep78 and Rep52 splice genes under the OpMNPV immediate early (OpIE2) promoter, followed by the sv40 polyadenylation signal (SV40 PAS). Modified mRNA transcripts (mRNA) are shown as wavy lines with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) and stop codon (UAA) of Rep52. Figure 1 ID shows a schematic map of a transient expression plasmid made by replacing the polyhedrin promoter with OpIE2 in the pFastBac.Polh.GPV.Rep construct as shown in Figure 3D . Figure 11E shows a schematic linear map of synthetic DNA encoding the AAV2 Rep78 and Rep52 splice genes under the OpMNPV immediate early ( OpIE2 ) promoter, followed by the sv40 polyadenylation signal ( SV40 PAS ). Modified mRNA transcripts (mRNA) are shown as wavy lines with the non-canonical start codon (CUG) of Rep78, the canonical start codon (AUG) and stop codon (UAA) of Rep52. Figure 1 IF shows a schematic map of a transient expression plasmid made by replacing the polyhedrin promoter with OpIE2 in the pFastBac.Polh.AAV2.Rep construct as shown in Figure 3F .

12A - 12C are schematic diagrams of a modified FVIIIXTEN expression cassette having a parvovirus ITR, a liver-specific modified mouse transthyretin (mTTR) promoter (mTTR482) and (V2.0) Codon optimized BDDco-FVIIIXTEN transgene (V2.0 FVIIIXTEN) (SEQ ID NO: 19). Figure 12A shows a schematic linear map of the modified FVIIIXTEN expression cassette flanked by AAV2 WT ITRs (SEQ ID NO: 5, SEQ ID NO: 6). Figure 12B shows a schematic linear map of the modified FVIIIXTEN expression cassette flanked by B19 WT (SEQ ID NO: 2) or B19 minimal (SEQ ID NO: 16). Figure 12C shows a schematic linear map of the modified FVIIIXTEN expression cassette flanked by GPVΔ120 (SEQ ID NO: 17) or GPVΔ186 (SEQ ID NO: 18).

13A to 13C are schematic diagrams of a method for producing ceDNA in a baculovirus system according to one embodiment of the present invention. Figure 13A shows a schematic of the single BAC approach, in which a single recombinant BEV encoding the V2.0 FVIIIXTEN and Rep genes at different loci was used for infection in Sf9 cells to produce ceDNA. Figure 13B shows a schematic of the dual BAC approach, in which Sf9 cells were co-infected with recombinant BEVs encoding V2.0 FVIIIXTEN and/or Rep genes to produce ceDNA. Figure 13C shows a schematic of the stable cell line approach, in which the FVIIIXTEN expression cassette was stably integrated into the Sf9 cell genome and rescued by infection of recombinant BEVs encoding the Rep gene to produce ceDNA.

Figures 14A - 14C illustrate the generation of human FVIIIXTEN ceDNA vectors from AAV2 single BACs according to one embodiment of the present invention. Figure 14A is a schematic diagram of a single BAC method for the production of FVIIIXTEN ceDNA vectors in Sf9 cells using recombinant BEVs encoding the AAV2 Rep gene under the AcMNPV polyhedrin promoter and comprising V2.0 FVIIIXTEN flanked by AAV2 WT Human FVIIIXTEN expression cassette for ITR (AcBIVVBac(mTTR.FVIIIXTEN.AAV2.WT.ITR)Polh.AAV2.Rep ^LoxP ). Figure 14B shows a schematic map of AcBIVVBac (mTTR.FVIIIXTEN.AAV2.WT.ITR) Polh.AAV2.Rep ^LoxP BEV. Figure 14C is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with a titrated virus stock (P2) of AcBIVVBac (mTTR.FVIIIXTEN.AAV2.WT.ITR) Polh.AAV2.Rep ^LoxP BEV. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 15A - 15C illustrate the generation of human FVIIIXTEN ceDNA vectors from B19 single BACs according to one embodiment of the present invention. Figure 15A is a schematic diagram of a single BAC method for the production of FVIIIXTEN ceDNA vectors in Sf9 cells using recombinant BEVs encoding the B19 NS1 gene under the AcMNPV polyhedrin promoter and comprising V2.0 FVIIIXTEN flanked by B19 WT Human FVIIIXTEN expression cassette for ITR (AcBIVVBac(mTTR.FVIIIXTEN.B19.WT.ITR)Polh.B19.NS1 ^LoxP ). Figure 15B shows a schematic map of AcBIVVBac (mTTR.FVIIIXTEN.B19.WT.ITR) Polh.B19.NS1 ^LoxP BEV. Figure 15C is an agarose gel electrophoresis image of ceDNA vector isolated from Sf9 cells infected with a titrated virus stock (P2) of AcBIVVBac (mTTR.FVIIIXTEN.B19.WT.ITR) Polh.B19.NS1 ^LoxP BEV. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 16A - 16C illustrate the generation of human FVIIIXTEN ceDNA vectors from AAV2 double BACs according to one embodiment of the present invention. Figure 16A is a schematic representation of the dual BAC approach for FVIIIXTEN ceDNA vector production, wherein Sf9 cells were co-infected with a recombinant BEV encoding a FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by AAV2 WT ITRs (AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITR ^Tn7 ) and/or the AAV2 Rep gene encoded under the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.AAV2.Rep ^Tn7 ). Figure 16B shows schematic maps of AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITR ^Tn7 and AcBIVVBac.Polh.AAV2.Rep ^Tn7 BEVs. Figure 16C is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.AAV2.WT.ITR ^Tn7 and AcBIVVBac.Polh.AAV2.Rep ^Tn7 BEVs as indicated picture. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 17A to 17C illustrate the generation of human FVIIIXTEN ceDNA vectors from B19 double BACs according to one embodiment of the present invention. Figure 17A is a schematic representation of the dual BAC approach for FVIIIXTEN ceDNA vector production, wherein Sf9 cells were co-infected with a recombinant BEV encoding a FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by B19 WT ITRs (AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITR ^Tn7 ) and/or the B19 NS1 gene encoded under the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.B19.NS1 ^Tn7 ). Figure 17B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITR ^Tn7 and AcBIVVBac.Polh.B19.NS1 ^Tn7 BEVs. Figure 17C is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.B19.WT.ITR ^Tn7 and AcBIVVBac.Polh.B19.NS1 ^Tn7 BEVs as indicated picture. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 18A to 18C illustrate the generation of human FVIIIXTEN ceDNA vectors from GPV double BACs according to one embodiment of the present invention. Figure 18A is a schematic representation of the dual BAC approach for FVIIIXTEN ceDNA vector production, wherein Sf9 cells were co-infected with a recombinant BEV encoding a FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by GPVΔ120 ITRs ( AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITR ^Tn7 ) and/or the GPV NS1 gene encoded under the AcMNPV polyhedrin promoter (AcBIVVBac.Polh.GPV.NS1 ^Tn7 ). Figure 18B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITR ^Tn7 and AcBIVVBac.Polh.GPV.NS1 ^Tn7 BEVs. Figure 18C is an agarose gel electrophoresis image of ceDNA vectors isolated from Sf9 cells co-infected with different MOIs of AcBIVVBac.mTTR.FVIIIXTEN.GPVΔ120.ITR ^Tn7 and AcBIVVBac.Polh.GPV.NS1 ^Tn7 BEVs as indicated. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 19A to 19C illustrate the production of human FVIIIXTEN ceDNA vectors from AAV2 stable cell lines according to an embodiment of the present invention. Figure 19A shows a schematic of the stable cell line method for the generation of FVIIIXTEN ceDNA vectors, in which stable cell lines encoding the FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by AAV2 WT ITRs are encoded under the AcMNPV polyhedrin promoter AAV2 Rep gene recombinant BEV (AcBIVVBac.Polh.AAV2.Rep ^Tn7 ) infection. Figure 19B shows a schematic map of AcBIVVBac.Polh.AAV2.Rep ^Tn7 BEV. Figure 19C is an agarose gel electrophoresis image of ceDNA vectors isolated from AAV2 stable cell lines infected with different MOI of AcBIVVBac.Polh.AAV2.Rep ^Tn7 BEV as indicated. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

20A to 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 of the FVIIIXTEN ceDNA vector-generated stable cell line method in which a stable cell line encoding a FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by B19 minimal ITRs is encoded with the AcMNPV polyhedrin promoter Infection with recombinant BEV (AcBIVVBac.Polh.B19.NS1 ^Tn7 ) under the B19 NS1 gene. Figure 20B shows a schematic map of AcBIVVBac.Polh.B19.NS1 ^Tn7 BEV. Figure 20C is an agarose gel electrophoresis image of ceDNA vectors isolated from B19 stable cell lines infected with different MOI of AcBIVVBac.Polh.B19.NS1 ^Tn7 BEV as indicated. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 21A to 21C illustrate the production of human FVIIIXTEN ceDNA vectors from GPV stable cell lines according to one embodiment of the present invention. 21A shows a schematic diagram of the stable cell line method for FVIIIXTEN ceDNA vector generation, wherein the stable cell line encoding the FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN and flanked by GPVΔ120 ITRs is encoded with the AcMNPV polyhedrin promoter. Recombinant BEV ( ^{AcBIVVBac.Polh.GPV.NS1Tn7} ) infection of GPV NS1 gene. Figure 21B shows a schematic map of AcBIVVBac.Polh.GPV.NS1 ^Tn7 BEV. Figure 21C is an agarose gel electrophoresis image of ceDNA vectors isolated from GPV stable cell lines infected with different MOIs of AcBIVVBac.Polh.GPV.NS1 ^Tn7 BEV as indicated. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

Figures 22A to 22E show a workflow for generating and purifying FVIIIXTEN ceDNA vectors using a dual BAC method according to one embodiment of the present invention. Figure 22A shows a schematic diagram of Sf9 cell expansion and duration (days 0-2), in which cells were sequentially scaled up from small scale (0.5 L) to large scale culture (1.5 L) flasks to achieve in serum-free ESF921 medium Cell density of 2.5 to 3.0 x 10 ⁶ /mL. Figure 22B shows a schematic diagram of the infection and duration of incubation (days 2-6) of Sf9 large culture (1.5 L) flasks in which cells were treated with recombinant BEV at MOIs of 0.1 and 0.01 plaque forming units (pfu)/cell, respectively. Co-infection with recombinant BEVs encoding a FVIIIXTEN expression cassette comprising V2.0 FVIIIXTEN flanked by HBoV1 ITRs (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITR ^Tn7 ) and/or encoding HBoV1 NS1 under the AcMNPV polyhedrin promoter Gene (AcBIVVBac.Polh.HBoV1.NS1 ^Tn7 ). Figure 22C shows images of the plasmid Giga Prep purification kit and agarose gel electrophoresis and duration of treatment (days 6-7), where cell density and viability of infected cells were measured daily and once cell viability reached 70 %-80%, the cells were pelleted by low-speed centrifugation. FVIIIXTEN ceDNA vectors were purified from infected cell pellets by PureLink ^™ HiPure Expi Plasmid Gigaprep Kit (Invitrogen), and aliquots were run on agarose gel electrophoresis to determine FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA ) and/or the productivity of genomic DNA (gDNA) in Sf9 cells. Figure 22D shows images of Bio-Rad Model 491 Prep Cell and agarose gel electrophoresis and duration of treatment (days 7-12) where Giga-prep purified DNA was loaded into the prep cell FVIIIXTEN ceDNA (approximately 8.5kb fragment) was separated from high molecular weight DNA on a preparative agarose gel. The purity of the FVIIIXTEN ceDNA was determined by analyzing the eluted fractions collected from the preparative agarose gel electrophoresis at 70-80 min intervals on a 0.8% to 1.2% agarose gel. Figure 22E shows an image of agarose gel electrophoresis in which fractions collected from the preparation tank were pooled and precipitated with 1/10 volume of 3M NaOAc (pH 5.5) and 3 volumes of 100% ethanol to obtain purified FVIIIXTEN ceDNA. Gel images showing the purity of FVIIIXTEN ceDNA compared to the starting material, where arrows indicate DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) bring.

Figure 23 shows a graphical representation of plasma FVIII activity levels measured by the Chromogenix Coatest® SP Factor VIII Chromogenic Assay. Figure 23 shows a graphical plot of plasma FVIII activity levels measured at different intervals in blood samples collected from hFVIIIR593C ^+/+ /HemA mice injected systemically via hydrodynamic tail vein injection for 80 , 40 or 12 µg/kg of FVIIIXTEN HBoV1 (wtHBoV1) or AAV2 (wtAAV2) ITR ceDNA.

24A to 24C illustrate the generation of human FVIIIXTEN ceDNA vectors using the One BAC (One BAC ) method according to one embodiment of the present invention. 24A shows a schematic diagram of the One BAC method for producing FVIIIXTEN ceDNA vectors in Sf9 cells using recombinant BEVs encoding the HBoV1 NS1 gene under the AcMNPV polyhedrin promoter and the human FVIIIXTEN expression cassette flanked by HBoV1 ITRs ( AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS1 ^LoxP ). Figure 24B shows a schematic map of AcBIVVBac (mTTR.FVIIIXTEN.HBoV1.ITRs) Polh.HBoV1.NS1 ^LoxP BEV. Figure 24C is agarose of ceDNA vectors isolated from Sf9 cells infected with titrated virus stock (P2) of (AcBIVVBac(mTTR.FVIIIXTEN.HBoV1.ITRs)Polh.HBoV1.NS1 ^LoxP ) BEV Gel electrophoresis image. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

25A to 25C illustrate the generation of human FVIIIXTEN ceDNA vectors using the Two BAC (Two BAC) method according to one embodiment of the present invention. 25A is a schematic diagram of the One BAC method for producing FVIIIXTEN ceDNA vectors, wherein the Sf9 cell line is flanked by human FVIIIXTEN expression cassettes of HBoV1 ITRs (AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRs ^Tn7 ) and/or recombinant BEV encoded in AcMNPV Co-infection with recombinant BEV of the HBoV1 NS1 gene under the polyhedrin promoter. Figure 25B shows a schematic map of AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRs ^Tn7 and AcBIVVBac.Polh.HBoV1.NS1 ^Tn7 BEVs. Figure 25C is an image of agarose gel electrophoresis of ceDNA vectors, the ceDNA vectors are obtained from AcBIVVBac.mTTR.FVIIIXTEN.HBoV1.ITRs ^Tn7 and AcBIVVBac.Polh.HBoV1.NS1 ^Tn7 BEV with constant ratios of different MOIs or different ratios of constant MOIs isolated from co-infected Sf9 cell lines. DNA bands corresponding to the sizes of FVIIIXTEN ceDNA vector (ceDNA), baculovirus DNA (vDNA) and Sf9 cell genomic DNA (gDNA) are indicated by arrows.

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Claims (195)

A baculovirus shuttle vector, the baculovirus shuttle vector comprising: Bacterial replicons; selectable marker sequence; a first reporter gene comprising a first priority target site for insertion of the transposon; a second reporter gene operably linked to a baculovirus-inducible promoter; and A second preferential target site capable of mediating site-specific recombination events. The baculovirus shuttle vector according to claim 1, wherein the bacterial replicon is a low copy number replicon. The baculovirus shuttle vector according to claim 2, wherein the low copy number replicon is a miniature F replicon. The baculovirus shuttle vector of any preceding claim, wherein the selectable marker sequence comprises an antibiotic resistance gene. The baculovirus shuttle vector according to claim 4, wherein the antibiotic resistance gene is a kanamycin resistance gene. The baculovirus shuttle vector according to any preceding claim, wherein the first reporter gene encodes an enzyme capable of metabolizing a chromogenic substrate. The baculovirus shuttle vector according to claim 6, wherein the enzyme is LacZα or a functional part thereof. The baculovirus shuttle vector according to claim 6 or 7, wherein the chromogenic substrate is blue-gal or X-gal. The baculovirus shuttle vector of any preceding claim, wherein said first preferential target site for insertion of a transposon does not disrupt the reading frame of said first reporter gene. The baculovirus shuttle vector of any preceding claim, wherein the first preferential target site is an attachment site for a bacterial transposon. The baculovirus shuttle vector of any preceding claim, wherein the first preferential target site is the attachment site of the T7 transposon. The baculovirus shuttle vector according to claim 11, wherein the transposon is a T7 transposon. The baculovirus shuttle vector of any preceding claim, wherein the second reporter gene encodes a fluorescent protein. The baculovirus shuttle vector according to claim 13, wherein the fluorescent protein is red fluorescent protein. The baculovirus shuttle vector of any preceding claim, wherein the baculovirus-inducible promoter is a 39K promoter. The baculovirus shuttle vector of any preceding claim, wherein the second preferential target site comprises a LoxP site or a variant thereof. The baculovirus shuttle vector of any preceding claim, wherein the site-specific recombination event is mediated by Cre recombinase. A baculovirus shuttle vector, the baculovirus shuttle vector comprising: mini F replicon; Antibiotic resistance genes; the LacZα gene or a functional part thereof comprising an attachment site for the T7 transposon; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: 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 Preferential target sites capable of mediating site-specific recombination events. The recombinant baculovirus shuttle vector according to claim 19, wherein the heterologous sequence comprises a heterologous gene. The recombinant baculovirus shuttle vector according to claim 19 or 20, wherein the heterologous gene comprises a protein-coding sequence. The recombinant baculovirus shuttle vector according to claim 20 or 21, wherein the heterologous sequence comprises an expression control sequence. The recombinant baculovirus shuttle vector according to claim 22, wherein the expression control sequence is operably linked to the sequence encoding the protein. The recombinant baculovirus shuttle vector according to claim 22 or 23, wherein the expression control sequence comprises a baculovirus promoter. The recombinant baculovirus shuttle vector according to claim 24, wherein the baculovirus promoter is an immediate early, early, late or very late gene promoter. The recombinant baculovirus shuttle vector as described in claim item 24 or 25, wherein the baculovirus promoter is selected from the polyhedrin promoter, immediate early 1 promoter (immediate-early1 promoter) and immediate early 2 promoter ( immediate-early2 promoter). The recombinant baculovirus shuttle vector according to any one of claims 22-26, wherein the expression control sequence comprises a polyadenylation signal. The recombinant baculovirus shuttle vector according to any one of claims 21-27, wherein the protein is a Rep protein isolated from the genome of members of the family Parvoviridae . The recombinant baculovirus shuttle vector according to any one of claims 21-28, wherein the protein is parvovirus Rep protein. The recombinant baculovirus shuttle vector according to claim 29, wherein the parvovirus Rep protein is selected from B19 Rep, AAV2 Rep and GPV Rep. The recombinant baculovirus shuttle vector according to any one of claims 19-30, wherein said heterologous sequence comprises a second selectable marker sequence. The recombinant baculovirus shuttle vector according to claim 31, wherein the second selectable marker sequence comprises a gentamycin resistance gene. The recombinant baculovirus shuttle vector according to any one of claims 19-32, wherein the bacterial replicon is a miniature F replicon. The recombinant baculovirus shuttle vector according to any one of claims 19-33, wherein the first selectable marker sequence comprises a kamycin resistance gene. The recombinant baculovirus shuttle vector according to any one of claims 19-34, wherein the first reporter gene encodes LacZα or a functional part thereof. The recombinant baculovirus shuttle vector according to any one of claims 19-35, wherein the second reporter gene encodes red fluorescent protein. The recombinant baculovirus shuttle vector according to any one of claims 19-36, wherein the baculovirus-inducible promoter is a 39K promoter. The recombinant baculovirus shuttle vector according to any one of claims 19-37, wherein said second preferential target site comprises a LoxP site or a variant thereof. The recombinant baculovirus shuttle vector according to any one of claims 19-38, wherein the site-specific recombination event is mediated by Cre recombinase. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: a heterologous sequence inserted into the mini-attTn7 (mini-attTn7) site, wherein the heterologous sequence encodes a Rep, and wherein the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; and LoxP sites or variants thereof. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: Bacterial replicons; the first antibiotic resistance gene; a heterologous sequence inserted into the miniature attTn7 site, wherein the heterologous sequence encodes a Rep, and wherein the inserted Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: Bacterial replicons; the first antibiotic resistance gene; a heterologous sequence inserted into the miniature attTn7 site, wherein the heterologous sequence encodes a B19 Rep, and wherein the inserted B19 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: Bacterial replicons; the first antibiotic resistance gene; a heterologous sequence inserted into the miniature attTn7 site, wherein the heterologous sequence encodes a GPV Rep, and wherein the inserted GPV Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: Bacterial replicons; the first antibiotic resistance gene; a heterologous sequence inserted into the miniature attTn7 site, wherein the heterologous sequence encodes an AAV2 Rep, and wherein the inserted AAV2 Rep disrupts the reading frame of the LacZα gene or a functional portion thereof; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP sites or variants thereof. A nucleic acid carrier, said nucleic acid carrier comprising: A first origin of replication for propagating the nucleic acid vector in a first bacterial strain, wherein the first origin of replication is a conditional origin of replication; a second origin of replication for propagating said nucleic acid vector in a second bacterial strain; Multiple cloning sites for insertion of heterologous sequences; selectable marker sequence; reporter gene; and Preferential target sites capable of mediating site-specific recombination events. The vector according to claim 45, wherein the first origin of replication is conditional on the presence of pi protein. The vector according to claim 45 or 46, wherein the first origin of replication is R6Kγ. The vector according to any one of claims 45-47, wherein said first bacterial strain comprises pi protein. The vector of any one of claims 45-48, wherein the second origin of replication is pUC57. The vector of any one of claims 45-49, wherein the selectable marker sequence comprises an antibiotic resistance gene. The vector according to claim 50, wherein the antibiotic resistance gene is an ampicillin resistance gene. The vector according to any one of claims 45-51, wherein the reporter gene encodes a fluorescent protein. The vector according to claim 52, wherein the fluorescent protein is green fluorescent protein. The vector according to any one of claims 45-53, wherein said preferential target site comprises a LoxP site or a variant thereof. The vector according to any one of claims 45-54, wherein the site-specific recombination event is mediated by Cre recombinase. A nucleic acid carrier, said nucleic acid carrier comprising: A first origin of replication for propagating the nucleic acid vector in a first bacterial strain, wherein the first origin of replication is a conditional origin of replication; a second origin of replication for propagating said nucleic acid vector in a second bacterial strain; Multiple cloning sites containing heterologous sequences; selectable marker sequence; reporter gene; and Preferential target sites capable of mediating site-specific recombination events. The vector according to claim 56, wherein said heterologous sequence comprises a heterologous gene. The vector according to claim 57, wherein the heterologous gene comprises a protein-coding sequence. The vector according to claim 57 or 58, wherein the heterologous sequence comprises an expression control sequence. The vector according to claim 59, wherein said expression control sequence is operably linked to said sequence encoding a protein. The vector according to claim 59 or 60, wherein said expression control sequence comprises a tissue-specific promoter. The vector according to claim 61, wherein the tissue-specific promoter is a triple tetraproline (tristetraprolin, TTP) or murine transthyretin (mTTR) promoter. The vector according to any one of claims 59-62, wherein said expression control sequence comprises a polyadenylation signal. The vector of claim 63, wherein the polyadenylation signal is bovine growth hormone polyadenylation signal. The vector of any one of claims 59-64, wherein the expression control sequence comprises a post-transcriptional regulatory element. The vector according to claim 65, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The carrier of any one of claims 58-66, wherein the protein is a therapeutic protein. The carrier of claim 67, wherein the therapeutic protein is a coagulation factor. The carrier of claim 66, wherein the coagulation factor is Factor VIII (FVIII). The carrier according to claim 68 or 69, wherein the blood coagulation factor is FVIII-XTEN. The vector of any one of claims 56-70, wherein the heterologous sequence comprises a 5' inverted terminal repeat (ITR). The vector of any one of claims 56-71, wherein the heterologous sequence comprises a 3' inverted terminal repeat (ITR). The vector according to claim 71 or 72, wherein said 5' ITR and said 3' ITR are derived from parvoviruses. The vector according to claim 73, wherein the parvovirus is selected from B19, GPV, HBoV1 and AAV2. The vector of any one of claims 71-74, wherein the 5'ITR is a wild-type or variant 5'ITR derived from B19. The vector of any one of claims 71-74, wherein the 5'ITR is a wild-type or variant 5'ITR derived from GPV. The vector of any one of claims 71-74, wherein the 5'ITR is a wild-type or variant 5'ITR derived from AAV2. The vector of any one of claims 71-74, wherein the 5'ITR is a wild-type 5'ITR derived from HBoV1. The vector according to any one of claims 75-77, wherein the variant 5'ITR is a truncated 5'ITR. The vector of any one of claims 71-79, wherein the 3'ITR is a wild-type or variant 3'ITR derived from B19. The vector of any one of claims 71-78, wherein the 3'ITR is a wild-type or variant 3'ITR derived from GPV. The vector of any one of claims 71-78, wherein the 3'ITR is a wild-type or variant 3'ITR derived from AAV2. The vector of any one of claims 71-78, wherein the 3'ITR is a wild-type 3'ITR derived from HBoV1. The vector according to any one of claims 80-82, wherein the variant 3'ITR is a truncated 3'ITR. The vector according to any one of claims 56-76, wherein said first origin of replication is conditional on the presence of pi protein. The vector of claim 85, wherein the first origin of replication is R6Kγ. The vector according to claim 85 or 86, wherein said first bacterial strain comprises pi protein. The vector of any one of claims 56-87, wherein the second origin of replication is pUC57. The vector of any one of claims 56-88, wherein the selectable marker sequence comprises an antibiotic resistance gene. The vector of claim 89, wherein the antibiotic resistance gene is an ampicillin resistance gene. The vector according to any one of claims 56-90, wherein the reporter gene encodes a fluorescent protein. The vector according to claim 91, wherein the fluorescent protein is green fluorescent protein. The vector of any one of claims 56-92, wherein said preferential target site comprises a LoxP site or a variant thereof. The vector according to any one of claims 56-93, wherein the site-specific recombination event is mediated by Cre recombinase. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: 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 priority target site capable of mediating site-specific recombination events; a multiple cloning site comprising a second heterologous sequence; and A second preferential target site capable of mediating site-specific recombination events. The recombinant baculovirus shuttle vector according to claim 95, wherein the first heterologous sequence comprises a first heterologous gene. The recombinant baculovirus shuttle vector according to claim 96, wherein the first heterologous sequence comprises an expression control sequence operably linked to a sequence encoding a protein. The recombinant baculovirus shuttle vector according to claim 97, wherein the expression control sequence comprises a baculovirus promoter. The recombinant baculovirus shuttle vector according to claim 98, wherein the baculovirus promoter is an immediate early, early, late or very late promoter. The recombinant baculovirus shuttle vector according to claim 99, wherein the baculovirus promoter is selected from the group consisting of polyhedrin promoter, immediate early 1 promoter and immediate early 2 promoter. The recombinant baculovirus shuttle vector according to any one of claims 97-100, wherein the protein is a Rep protein isolated from the genome of a member of the family Parvoviridae. The recombinant baculovirus shuttle vector according to any one of claims 97-101, wherein the protein is a parvovirus Rep protein. The recombinant baculovirus shuttle vector according to claim 102, wherein the parvovirus Rep protein is selected from B19 Rep, AAV2 Rep and GPV Rep. The recombinant baculovirus shuttle vector according to any one of claims 95-103, wherein the second heterologous sequence comprises a second heterologous gene. The recombinant baculovirus shuttle vector according to claim 104, wherein the second heterologous sequence comprises an expression control sequence operably linked to a sequence encoding a protein. The recombinant baculovirus shuttle vector according to claim 105, wherein the expression control sequence comprises a tissue-specific promoter, a polyadenylation signal and/or a post-transcriptional regulatory element. The recombinant baculovirus shuttle vector according to claim 106, wherein the tissue-specific promoter is triple tetraproline (TTP) or mouse thyroxine transporter (mTTR) promoter. The recombinant baculovirus shuttle vector according to claim 107, wherein the polyadenylation signal is bovine growth hormone polyadenylation signal. The recombinant baculovirus shuttle vector according to claim 107, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The recombinant baculovirus shuttle vector according to any one of claims 105-109, wherein the protein is a therapeutic protein. The recombinant baculovirus shuttle vector according to claim 110, wherein the therapeutic protein is a coagulation factor. The recombinant baculovirus shuttle vector according to claim 111, wherein the coagulation factor is factor VIII (FVIII). The recombinant baculovirus shuttle vector according to claim 111 or 112, wherein the blood coagulation factor is FVIII-XTEN. The recombinant baculovirus shuttle vector according to any one of claims 95-113, wherein the second heterologous sequence comprises a 5' inverted terminal repeat (ITR). The recombinant baculovirus shuttle vector according to any one of claims 95-114, wherein the second heterologous sequence comprises a 3' inverted terminal repeat (ITR). The recombinant baculovirus shuttle vector of claim 115, wherein the 5' ITR is derived from the genome of a first member of the family Parvoviridae, and the 3' ITR is derived from the second member of the family Parvoviridae. member's genome. Claim 116. The recombinant baculovirus shuttle vector, wherein said first member and said second member are identical. The recombinant baculovirus shuttle vector of claim 116, wherein said first member and said second member are different. The recombinant baculovirus shuttle vector according to any one of claims 114-118, wherein said 5' ITR and said 3' ITR are derived from a parvovirus selected from B19, GPV, HBoV1 and AAV2 . The recombinant baculovirus shuttle vector according to claim 119, wherein the 5' ITR is a wild-type or truncated 5' ITR derived from B19. The recombinant baculovirus shuttle vector according to claim 119, wherein the 5' ITR is a wild-type or truncated 5' ITR derived from GPV. The recombinant baculovirus shuttle vector according to claim 119, wherein the 5' ITR is a wild-type or truncated 5' ITR derived from AAV2. The recombinant baculovirus shuttle vector as claimed in claim 119, wherein the 5' ITR is a wild-type 5' ITR derived from HBoV1. The recombinant baculovirus shuttle vector according to claim 119, wherein the 3' ITR is a wild-type or truncated 3' ITR derived from B19. The recombinant baculovirus shuttle vector as claimed in claim 119, wherein the 3' ITR is a wild-type or truncated 3' ITR derived from GPV. The recombinant baculovirus shuttle vector as claimed in claim 119, wherein the 3' ITR is a wild-type or truncated 3' ITR derived from AAV2. The recombinant baculovirus shuttle vector as claimed in claim 119, wherein the 3' ITR is a wild-type 3' ITR derived from HBoV1. The recombinant baculovirus shuttle vector according to any one of claims 95-126, wherein the recombinant baculovirus shuttle vector further comprises a bacterial replicon. The recombinant baculovirus shuttle vector of claim 128, wherein the bacterial replicon is a miniature F replicon. The recombinant baculovirus shuttle vector according to any one of claims 95-129, wherein the recombinant baculovirus shuttle vector further comprises one or more selectable marker sequences. The recombinant baculovirus shuttle vector of claim 130, wherein the one or more selectable marker sequences comprise one or more antibiotic resistance genes. The recombinant baculovirus shuttle vector according to claim 131, wherein the one or more antibiotic resistance genes are selected from ampicillin resistance gene, kanamycin resistance gene and gitamycin resistance gene. The recombinant baculovirus shuttle vector according to any one of claims 95-132, wherein the first reporter gene encodes LacZα or a functional part thereof. The recombinant baculovirus shuttle vector according to any one of claims 95-133, wherein the recombinant baculovirus shuttle vector further comprises at least a second reporter gene and a third reporter gene. The recombinant baculovirus shuttle vector according to claim 134, wherein each of the second reporter gene and the third reporter gene encodes a fluorescent protein. The recombinant baculovirus shuttle vector according to claim 135, wherein the fluorescent protein is green fluorescent protein or red fluorescent protein. The recombinant baculovirus shuttle vector of any one of claims 95-136, wherein said first preferential target site and said second preferential target site each comprise a LoxP site or a variant thereof. The recombinant baculovirus shuttle vector according to any one of claims 95-137, wherein the site-specific recombination event is mediated by Cre recombinase. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding Rep, wherein the inserted Rep sequence disrupts the reading frame of the LacZα gene or a functional part thereof; and A heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat derived from the first genome of a member of the family Parvoviridae; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats derived from the second genome of members of the family Parvoviridae. The recombinant baculovirus shuttle vector according to claim 139, wherein the first genome and the second genome of the family Parvoviridae are the same. The recombinant baculovirus shuttle vector of claim 140, wherein the first genome and the second genome of the family Parvoviridae are different. The recombinant baculovirus shuttle vector of any one of claims 139-141, wherein the Rep is derived from the first genome or the second genome of a member of the family Parvoviridae. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding B19 Rep, wherein the inserted B19 Rep sequence disrupts the reading frame of the LacZα gene or a functional part thereof; and A heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': Wild-type or truncated 5' inverted terminal repeats derived from B19; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats derived from B19. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding a GPV Rep, wherein the inserted GPV Rep sequence disrupts the reading frame of the LacZα gene or a functional part thereof; and A heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat sequence derived from GPV; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats derived from GPV. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding an AAV2 Rep, wherein the inserted AAV2 Rep sequence disrupts the reading frame of the LacZα gene or a functional portion thereof; and A heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat derived from AAV2; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats derived from AAV2. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding HBoV1 Rep, wherein the inserted HBoV1 Rep sequence disrupts the reading frame of the LacZα gene or a functional part thereof; and A heterologous sequence, wherein the heterologous sequence comprises from 5' to 3': Wild-type or truncated 5' inverted terminal repeats derived from HBoV1; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats derived from HBoV1. The recombinant baculovirus shuttle vector according to any one of claims 139-146, wherein the protein is a therapeutic protein. The recombinant baculovirus shuttle vector according to claim 147, wherein the therapeutic protein is a coagulation factor. The recombinant baculovirus shuttle vector according to claim 148, wherein the coagulation factor is factor VIII (FVIII). The recombinant baculovirus shuttle vector according to claim 148 or 149, wherein the blood coagulation factor is FVIII-XTEN. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding a Rep protein, wherein the Rep disrupts the reading frame of a reporter gene or a functional portion thereof; and A heterologous sequence, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 20. The recombinant baculovirus shuttle vector according to claim 147, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 20. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: A sequence encoding a Rep protein, wherein the Rep disrupts the reading frame of a reporter gene or a functional portion thereof; and A heterologous sequence, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 29. A host cell comprising the baculovirus shuttle vector according to any one of claims 1-18. A host cell comprising the recombinant baculovirus shuttle vector according to any one of claims 19-44. A host cell comprising the vector according to any one of claims 45-55. A host cell comprising the vector according to any one of claims 56-94. A host cell comprising the recombinant baculovirus shuttle vector according to any one of claims 95-153. A method of producing the recombinant baculovirus shuttle vector according to any one of claims 19-44, said method comprising: Introduce the following into bacterial cells: The baculovirus shuttle vector as described in any one of claims 1-18; and A donor nucleic acid molecule comprising a bacterial replicon operably linked to a transposon comprising a heterologous sequence as described in any one of claims 19-44; growing the bacterial cell under conditions in which translocation occurs; and The recombinant baculovirus shuttle vector is isolated from the bacterial cells. A method of producing a recombinant baculovirus shuttle vector as described in any one of claims 95-153, said method comprising: Introduce the following into bacterial cells: The recombinant baculovirus shuttle vector as described in any one of claims 19-44; the carrier of any one of claims 56-94; and Cre recombinase; growing the bacterial cell under conditions such that site-specific recombination occurs; and The recombinant baculovirus shuttle vector is isolated from the bacterial cells. A method for producing recombinant baculovirus, said method comprising transfecting the recombinant baculovirus shuttle vector according to any one of claims 95-153 into insect cells under appropriate conditions. A method of producing a closed-end DNA (ceDNA) molecule, the method comprising: Infect the insect cells with the recombinant baculovirus comprising the baculovirus shuttle vector according to any one of claims 1-44 and 95-153. A method of producing a closed-end DNA (ceDNA) molecule, the method comprising: Under appropriate conditions, the recombinant baculovirus shuttle vector as described in any one of claims 139-153 is transfected into insect cells to produce recombinant baculovirus; and The recombinant baculovirus is used to infect a second insect cell under appropriate conditions to produce ceDNA molecules. The method of any one of claims 161-163, wherein the recombinant baculovirus comprises a single baculovirus shuttle vector. A method of producing a closed-ended DNA (ceDNA) molecule comprising a wild-type or truncated B19 inverted terminal repeat, the method comprising: Transfecting the recombinant baculovirus shuttle vector as described in claim 139 into insect cells under appropriate conditions to produce recombinant baculovirus; and The recombinant baculovirus is used to infect a second insect cell under appropriate conditions to produce ceDNA molecules. A method of producing a closed-ended DNA (ceDNA) molecule comprising a wild-type or truncated GPV inverted terminal repeat, the method comprising: Transfecting the recombinant baculovirus shuttle vector as described in claim 139 into insect cells under appropriate conditions to produce recombinant baculovirus; and The recombinant baculovirus is used to infect a second insect cell under appropriate conditions to produce ceDNA molecules. A method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated AAV2 inverted terminal repeat, the method comprising: Transfecting the recombinant baculovirus shuttle vector as described in claim 139 into insect cells under appropriate conditions to produce recombinant baculovirus; and The recombinant baculovirus is used to infect a second insect cell under appropriate conditions to produce ceDNA molecules. A method of producing a closed-ended DNA (ceDNA) molecule comprising a wild-type HBoV1 inverted terminal repeat, the method comprising: Transfecting the recombinant baculovirus shuttle vector as described in claim 139 into insect cells under appropriate conditions to produce recombinant baculovirus; and The recombinant baculovirus is used to infect a second insect cell under appropriate conditions to produce ceDNA molecules. A kind of plasmid, described plasmid comprises nucleic acid sequence, and described nucleic acid sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat derived from the genome of a first member of the family Parvoviridae; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeat derived from the genome of the second member of the family Parvoviridae. The plasmid of claim 169, wherein the one or more expression control sequences comprise tissue-specific promoters, polyadenylation signals and/or post-transcriptional regulatory elements. The plasmid according to claim 170, wherein the tissue-specific promoter is a triple tetraproline (TTP) or murine transthyretin (mTTR) promoter. The plasmid of claim 169 or 170, wherein the polyadenylation signal is bovine growth hormone polyadenylation signal. The plasmid according to any one of claims 169-172, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The plasmid of any one of claims 169-173, wherein the protein is a therapeutic protein. The plasmid of claim 174, wherein the therapeutic protein is a coagulation factor. The plasmid of claim 175, wherein the coagulation factor is Factor VIII (FVIII). The plasmid according to claim 175 or 176, wherein the coagulation factor is FVIII-XTEN. The plasmid according to any one of claims 175-177, wherein the coagulation factor comprises the nucleotide sequence of SEQ ID NO: 20. The plasmid according to any one of claims 169-178, wherein said first member and/or said second member are selected from B19, GPV, HBoV1 and AAV2. The plasmid of claim 179, wherein said first member and/or said second member are identical. The plasmid of claim 179, wherein said first member and/or said second member are different. A stable cell strain comprising the nucleic acid sequence according to any one of claims 170-181, wherein the nucleic acid sequence is stably integrated into the genome of the stable cell strain. The stable cell line of claim 182, wherein said stable cell line is a stable insect cell line. The stable cell line of claim 183, wherein the stable insect cell line is Sf9. A method of producing a closed-end DNA (ceDNA) molecule comprising a wild-type or truncated inverted terminal repeat, said method comprising introducing a baculovirus encoding a Rep protein as described in any one of claims 182-184 in a stable cell line. The method of claim 185, wherein the stable cell line is a stable insect cell line. The method of claim 186, wherein the insect cell line is Sf9. A recombinant baculovirus shuttle vector set, the recombinant baculovirus shuttle vector set comprising a first baculovirus shuttle vector and a second baculovirus shuttle vector, wherein said first baculovirus shuttle vector comprises a Rep-encoding sequence inserted into the miniature attTn7 site, wherein the inserted Rep disrupts the reading frame of the reporter gene or a functional portion thereof; and Wherein said second baculovirus shuttle vector comprises a heterologous sequence, wherein said heterologous sequence comprises from 5' to 3': A wild-type or truncated 5' inverted terminal repeat (ITR) derived from the first genome of a member of the family Parvoviridae; protein-coding sequences; one or more expression control sequences operably linked to said sequence encoding the protein; and Wild-type or truncated 3' inverted terminal repeats (ITRs) derived from the second genome of members of the virus family Parvoviridae. The set of recombinant baculovirus shuttle vectors as claimed in claim 182, wherein said 5' ITR and said 3' ITR are derived from a parvovirus selected from B19, GPV, HBoV1 and AAV2. The recombinant baculovirus shuttle vector set as described in claim 188 or 189, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 20. The recombinant baculovirus shuttle vector set as described in claim 188 or 189, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 19. The recombinant baculovirus shuttle vector set as described in claim 188 or 189, wherein the heterologous sequence comprises the nucleotide sequence of SEQ ID NO: 29. A method of producing a closed-end DNA (ceDNA) molecule, the method comprising: Insect cells are infected with a recombinant baculovirus comprising a recombinant baculovirus shuttle vector set according to any one of claims 188-192. A method of producing a closed-end DNA (ceDNA) molecule, the method comprising: Transfecting the recombinant baculovirus shuttle vector set as described in any one of claims 188-192 into insect cells under appropriate conditions to produce recombinant baculoviruses; and The recombinant baculovirus is infected under appropriate conditions to a second insect cell to produce ceDNA molecules. A recombinant baculovirus shuttle vector, the recombinant baculovirus shuttle vector comprises: Bacterial replicons; the first antibiotic resistance gene; Sequence encoding HBoV1 Rep, wherein the inserted HBoV1 Rep disrupts the reading frame of the LacZα gene or its functional part; a gene encoding a fluorescent protein operably linked to a baculovirus-inducible promoter; and LoxP site or its variants