Plasmids for prolonged protein expression

Abstract

The present disclosure relates to plasmids that may be used to promote prolonged expression of recombinant proteins in vivo, such as in animal models, and their methods of construction and use, for instance in animals. In some embodiments, the plasmids, following injection into an animal, are converted to minicircles by recombination, such as in the liver of the animal.

Description

PLASMIDS FOR PROLONGED PROTEIN EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 666,866, filed July 2, 2024, the entire contents of which are incorporated by reference herein for all purposes.FIELD

[0002] The present disclosure relates to plasmids that may be used to promote prolonged expression of recombinant proteins in vivo, such as in animal models, and their methods of construction and use, for instance in animals. In some embodiments, the plasmids, following injection into an animal, are converted to mini circles by recombination, such as in the liver of the animal.BACKGROUND

[0003] Recombinant protein production in animals, such as animals used for models of diseases or conditions is often initiated by administering a vector encoding a gene expressing the protein of interest to the animal, for example, by hydrodynamic tail vein injection, or other administration means such as subcutaneous injection or the like. One disadvantage of producing recombinant proteins in animals is the often short duration of protein expression. In many cases, recombinant protein expression is transient and may require multiple administrations to obtain reasonably durable expression of the protein in the animal, for example.

[0004] Thus, there is a need for means to prolong expression of a recombinant gene in an animal, for example through improvements in expression vector systems. There is also a need for vector systems that are simple to prepare and that do not require extensive experimental manipulation prior to administration to an animal.SUMMARY

[0005] Minicircle plasmids have been used for expression of recombinant proteins in animals previously, as a means to prolong expression of the protein, it was first necessary to form such minicircle plasmids in vitro and then to purify the minicircles after formation prior to administration to animals. While this process may allow for longer expression of arecombinant protein in vivo, the process requires a number of time-consuming purification steps. In contrast, the vector systems and methods of the present disclosure require no such purification steps. Instead, the present disclosure provides a vector or set of vectors that comprise the components necessary for formation of minicircle plasmids inside animals after administration, meaning that it is not necessary to obtain and purify minicircles in advance.

[0006] Embodiments included in the disclosure herein include, for example, an expression vector or set of expression vectors comprising an expression cassette located between two recombination sites, and further comprising a recombinase gene encoding a recombinase that recognizes the recombination sites, wherein the expression cassette and the recombinase gene are located on the same vector or on different vectors. Also included is a set of expression vectors comprising a first vector comprising an expression cassette located between two recombination sites and a second vector comprising a recombinase gene encoding a recombinase that recognizes the recombination sites on the first vector. In some embodiments, the recombinase gene is under the control of an inducible promoter, or in other embodiments, the recombinase gene is under the control of a TK promoter, such as a minimal TK promoter. In some cases, the inducible promoter is an arabinose promoter. In some cases, the recombinase gene is Cre and wherein the recombination sites are loxP recombination sites. In some such cases, the loxP recombination sites comprise a mutant loxP LE sequence and a mutant loxP RE sequence that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites. For example, in some cases, the loxP RE and loxP LE comprise the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some cases, the Cre recombinase gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10. In some cases, the coding sequence for the Cre recombinase comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence that is degenerate to SEQ ID NO: 6. In some cases, the expression cassette comprises a gene under the control of a ubiquitin or CMV promoter, and optionally comprises a coding sequence for a poly-A tail. In some cases, the vector or set of vectors are plasmid vectors.

[0007] In some embodiments, an expression vector of any one of claims comprises a plasmid vector comprising an expression cassette located between two loxP recombination sites, which comprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and which plasmid vector further comprises a gene encoding Cre recombinase, optionally wherein thegene encoding Cre recombinase is under the control of an inducible promoter, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter.

[0008] In some cases, a set of expression vectors comprises a first plasmid vector comprising an expression cassette located between two loxP recombination sites, which comprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter; the set further comprising a second plasmid vector comprising a gene encoding Cre recombinase, optionally wherein the gene encoding Cre recombinase is under the control of an inducible promoter.

[0009] In some embodiments herein, recombination at the recombination sites produces a minicircle comprising the expression cassette. In some such cases, recombination produces a minicircle comprising the expression cassette and lacking an origin of replication.

[0010] The disclosure herein also includes a host cell comprising an expression vector or set of expression vectors as described herein. Examples include bacterial cells, such as E. coli cells. The disclosure also includes a composition comprising the expression vector or set of expression vectors described herein and at least one carrier or excipient. The disclosure also includes a liposome or lipid nanoparticle (LNP) comprising the expression vector or set of expression vectors as described herein, or a composition comprising the vector or vectors.

[0011] The disclosure herein also encompasses an animal comprising an expression vector or set of expression vectors described herein, optionally wherein the animal is a laboratory animal, such as a mouse, rat, rabbit, or guinea pig. In some cases, the animal expresses the product of a gene encoded by the expression vector or set of expression vectors for a period of at least 250 days following administration of the vector or set of vectors to the animal. In some cases, the product of the gene is expressed in an animal for a longer period of time than the same gene expressed in an animal from an expression vector or set of expression vectors that encodes the gene but is not capable of recombination. In some cases, the animal is a mouse or rat.

[0012] The disclosure herein further includes a method of expressing a heterologous gene in an animal, comprising administering an expression vector or set of expression vectors as described herein encoding the gene to the animal. In some cases, the animal is a laboratory animal, such as a mouse, rat, rabbit, or guinea pig. In some cases, the animal is a mouse or rat. In some cases, the administration is by hydrodynamic tail vein (HTV) injection, or by other means of administration described elsewhere herein such as subcutaneous orintramuscular injection, or intravenous administration or inhalation or administration to the brain. In some cases, the expression vector or set of expression vectors is administered to the animal by an administration means that allows the vector or set of vectors to transform hepatocyte, lung, or spleen cells. In some cases, the animal expresses the product of a gene encoded by the expression vector or set of expression vectors for a period of at least 250 days following administration of the vector or set of vectors to the animal. In some cases, the product of the gene is expressed in an animal for a longer period of time than the same gene expressed in an animal from an expression vector or set of expression vectors that encodes the gene but is not capable of recombination.

[0013] The disclosure also includes a method of preparing an expression vector or set of expression vectors as described herein, comprising inserting an expression cassette comprising a gene sequence, optional promoter and / or enhancer sequences, and optionally a poly-A tail sequence into a vector such that the expression cassette is located between two recombination sites, and further comprising inserting a polynucleotide comprising a recombinase gene, optional promoter and / or enhancer sequences, and optionally a poly-A tail, into the same or a different vector. In some cases, the method further comprises inserting the recombination sites into the vector, optionally as part of the expression cassette.

[0014] The disclosure further includes, for example, a kit comprising an expression vector or set of expression vectors as described herein, and optionally further instructions for use, for example, for administration to a host cell or to an animal.

[0015] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments and together with the description, serve to further explain certain principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Fig. 1 shows a schematic of the pCMV.lox plasmid and its transformation into a minicircle plasmid and a second plasmid comprising the origin of replication (Ori) segment and ampicillin resistance (Ampr) gene in the presence of Cre recombinase.

[0018] Fig. 2 shows a schematic of SW106 E. coli cells expressing Cre recombinase under an arabinose-inducible promoter, transformed with the pCMV.lox plasmid. The cells are first grown in glucose-containing media to allow for production of the plasmid, the glucose- containing media was washed out, and then the cells were grown in arabinose-containing media, to induce recombination of the plasmid into a minicircle comprising the CMV promoter and its associated expression cassette along with a second plasmid comprising the Ori-Amprcomponents.

[0019] Fig. 3 shows an agarose gel experiment used to detect mini circle formation in the SW106 cells in the presence of arabinose, before and after digestion with EcoRI restriction enzyme. The arrow shows the location of the minicircle plasmid on the gel, which appears 30-120 minutes after the start of arabinose incubation.

[0020] Fig. 4 shows a schematic of hydrodynamic tail vein (HTV) injection of the pCMV.lox plasmid into a mouse strain expressing Cre recombinase under an albumin promoter (Alb-cre mice), resulting in formation of minicircle plasmids in hepatocytes of the mouse.

[0021] Fig. 5 shows prolonged serum expression of IL-22 in Alb-cre mice up to 300 days after HTV injection with the IL-22 / pCMV.lox plasmid, compared to comparatively reduced, and rapidly declining expression of IL-22 after injection of either the IL-22 / pCMV.lox plasmid into mice that do not express Cre (C57BL6 / J mice) or an IL-22 / pCMV plasmid into Alb-cre mice which plasmid does not include loxP recombination sites.

[0022] Fig. 6A-6B is a schematic showing formation of minicircles by recombination, either from a combination of pUb.lox (a plasmid comprising a ubiquitin promoter and loxP sites) and pTK.Cre plasmids (Fig. 6A), or from a pRMNT plasmid, which expresses both a protein of interest behind a ubiquitin promoter in an expression cassette flanked by loxP sites and Cre behind a minimum TK promoter (Fig. 6B).

[0023] Fig. 7A-7B shows expression in vivo in Balb / c mice of either IL-17E (Fig. 7A) or IL- 22 (Fig. 7B) from either an excised expression cassette of pCMV.lox plasmid (i.e., IL- 17E / pCMV.lox or IL-22 / pCMV.lox), in which the gene is expressed from a CMV promoter and which results in rapidly declining expression, or from an excised expression cassette ofpUb.lox plasmid (i.e., IL-17E / pUb.lox or IL-22 / pUb.lox), in which the gene is expressed from a ubiquitin promoter and which results in prolonged expression in mice.

[0024] Fig. 8 compares expression in vivo in Balb / c mice after HTV injection with IL- 22 / pUb.lox and pTK.Cre plasmids, which allow for recombination and minicircle formation and IL-22 expression (top curve), IL-22 / pUb.lox alone, which cannot recombine to form minicircles (third curve from top, showing reducing expression levels over time), IL- 22 / pRMNT, which also allows for recombination and minicircle formation (second from top), or IL-22 / pCMV, which does not contain loxP recombination sites and cannot recombine and which also uses a CMV promoter rather than a ubiquitin promoter (bottom curve, showing very transient expression). Both of the plasmid systems allowing recombination to minicircles in vivo showed sustained, high levels of IL-22 expression up to 250 days post injection.

[0025] Fig. 9A-9C show a schematic (Fig. 9A) of a dextran sulfate sodium (DSS) preclinical mouse model, followed by experimental results in this model. Addition of DSS in the model can provoke damage to colon epithelial cells, which can be rescued in part by IL-22. Fig. 9B shows changes in body weight following addition of either pUb.lox and pTK.Cre plasmids, which do not express IL-22, or IL-22 / pUb.lox and pTK.Cre plasmids, which do express IL- 22, compared to a control group that was not treated with DSS. The changes in body weight indicate that IL-22 was expressed as intended as mice in the IL-22 group had less weight loss than those in the group received the control plasmids. Fig. 9C shows that the colon clinical score of mice in the IL-22 plasmid group was also significantly lower (as indicated by the ***) than mice in the control plasmid group, again indicating that the expression of IL-22 by the plasmid is enough to show the efficacy in this mouse model.

[0026] Fig. 10A-10B shows results of a C. rodentium infection mouse model with obese mice, in which disease is ameliorated by reducing the obesity. Leptin-deficient (ob / ob) mice injected with plasmids allowing for expression of leptin from mini circle plasmids (leptin / pUb.lox and pTK.Cre) survived (Fig. 10A) and lost weight (Fig. 10B), while those injected with plasmids not expressing leptin did not survive (Fig. 10A).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS1. Definitions

[0027] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0028] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. In this application, the article “a” or “the” preceding an item generally means “one or more” of such an item, unless context dictates that only one such item can be present. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0029] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0030] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0031] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0032] The terms “polypeptide” and “protein” are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural and / or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. The terms also include polymers of amino acids that have modifications such as, for example, glycosylation, sialylation, and the like, or that are complexed with other molecules.

[0033] A “polynucleotide” refers to an oligomer of nucleotides, which may be single or double stranded, and which nucleotides may comprise deoxyribonucleotides, or ribonucleotides, for example.

[0034] A “vector” herein refers to a polynucleotide molecule that may be used, for example, to carry a heterologous polynucleotide sequence to a host cell or animal. A vector may be engineered, for example, in order to carry one or more particular polynucleotide sequences to a host cell or animal. An “expression vector” herein refers to a vector that is intended for production or expression of at least one gene product, such as in a host cell in vitro or in an animal in vivo, for example a gene product that is heterologous to the cell or animal. In some embodiments, a “set” of vectors, such as a set of expression vectors, may be employed, i.e., wherein a “set” comprises two or more vectors. Such a set of expression vectors, for example, may be used collectively for expression of a gene product. For example, within a “set of expression vectors,” one vector may comprise the gene to be expressed and a second vector may comprise additional genetic elements, such as a recombinase gene. For instance, the second vector may carry elements that assist in expression of a gene product carried by the first vector. In some cases, a “vector” herein is a “plasmid” or “plasmid vector,” which is a relatively small, extrachromosomal circular DNA molecule. In some cases, a “vector” is a “viral vector,” which is a vector derived from a virus.

[0035] An “expression cassette” refers to the portion of an expression vector that comprises a gene or genes intended for expression. An expression cassette may also include promoter and / or enhancer elements for controlling expression of the gene or genes, as well as a poly-A tail coding sequence.

[0036] A “recombination site” refers to a site in a polynucleotide at which recombination occurs, for example, in the presence of a recombinase protein. For instance, a recombinase protein may recognize a recombination site based on its polynucleotide sequence. In some cases, a vector herein comprises two recombination sites.

[0037] A “recombinase” refers to an enzyme that catalyzes recombination of polynucleotide sequences. For example, in some cases, a recombinase may recognize one or more recombination sites, and thus promote recombination at those sites.

[0038] An “inducible promoter” refers to a promoter whose activity may be induced or controlled based on the presence and concentration of a particular substance, such as a particular co-factor. For example, use of an inducible promoter may allow a researcher to control when and where a particular gene is transcribed and the extent to which it is transcribed.

[0039] A “minicircle” vector refers to a small, circular polynucleotide vector composed of promoter, gene of interest, and poly-A tail sequences, but not including the componentsrequired for replication in bacteria, such as origin of replication or antibiotics resistant genes. A minicircle vector is created by recombination of a larger vector or combination of vectors such as one or more plasmid vectors. In some embodiments, a minicircle is less than 4 kilobases in size. In some embodiments, a minicircle comprises an expression cassette. In some embodiments, a minicircle lacks an origin of replication. In some cases, it comprises an expression cassette and also lacks an origin of replication.

[0040] An “origin of replication,” commonly abbreviated “Ori”, is a polynucleotide sequence that allows for replication of a vector inside a host cell, such as a plasmid vector, or a viral vector or the like.

[0041] A “host cell” as used herein refers to a cell that may be used to carry a vector, such as an expression vector or set of expression vectors, herein. For example, in some cases, a host cell may be used to replicate the vector(s) and / or to express one or more genes comprised in the vector(s).

[0042] A “laboratory animal” herein refers to an animal used for animal models, such as disease models and toxicology models, such as a mouse, rat, rabbit, guinea pig or other small rodent species, or a primate species such as a cynomolgus monkey.

[0043] A “heterologous gene” herein refers to a gene that is different from, such as not native to, a host cell or animal. A heterologous gene, for example, may encode a protein not found in the native host cell or animal, or may alternatively encode a protein that is found in the native host cell or animal, but wherein the gene sequence or structure varies from that found naturally, such as by removal of introns or the presence of a different promoter or the like. For example, a vector or set of vectors may be used to carry a heterologous gene to allow for its expression.

[0044] A “product” or “expression product” of a gene refers to an RNA and / or protein that is produced from a heterologous gene by transcription of the gene and optionally, by translation of the transcription product. For example, an RNA product may also be referred to as a “transcription product” of a gene, while a protein produced from a gene may be referred to as a “protein product.”

[0045] A “means that allows the vector or set of vectors to transform hepatocyte cells” in an animal herein refers to a method of administration of the vector or set of vectors to an animal that allows for the vector or set of vectors to reach the liver of the animal. Examples include hydrodynamic tail vein (HTV) injection of laboratory animals such as mice and rats, or otherforms of systemic administration into the bloodstream such as intravenous infusion, or subcutaneous injection, and their equivalents.2. Exemplary Vectors, Host Cells and Kits

[0046] This disclosure includes, for example, one or more expression vectors that comprise elements sufficient to form minicircle plasmids in an animal following administration to the animal. In some cases, one vector may be sufficient for administration and formation of minicircles in an animal, while in other cases two or more vectors may be administered to the animal for formation of minicircles.

[0047] For example, the disclosure encompasses in some embodiments an expression vector or set of expression vectors, comprising an expression cassette located between two recombination sites, and further comprising a recombinase gene that recognizes the recombination sites, wherein the expression cassette and the recombinase gene are located on the same vector or on different vectors. In some embodiments, the disclosure encompasses a set of expression vectors comprising a first vector comprising an expression cassette located between two recombination sites and a second vector comprising a recombinase gene that recognizes the recombination sites on the first vector. The expression cassette may include a gene that encodes one or more proteins of interest to be expressed in an animal following administration to the animal. The expression vector or the member of the set of expression vectors comprising the recombination sites and expression cassette may form a minicircle in an animal following administration to the animal, allowing for example, for gene expression of the protein or proteins of interest from the expression cassette. In some embodiments, where a set of vectors is used, the other vector or vectors may also comprise gene sequences encoding additional proteins of interest, such as the recombinase gene, and optionally further a selectable marker or the like.

[0048] In some such vector systems, the recombinase gene is under the control of an inducible promoter. In some cases, the inducible promoter is an arabinose promoter. In other cases, the recombinase gene is under the control of a TK promoter, such as a minimal TK promoter. An exemplary TK promoter sequence is that of SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, below:SEQ ID NO: 3 - exemplary TK promoter ATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACTTCGCATA TTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTA A.SEQ ID NO: 4 - exemplary TK promoter CCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCCCCGGAAGAAATATATTTGCA TGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCGAAT TCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGGTCCACTTCGCATATT AAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAA further exemplary TK promoter is a minimal TK promoter sequence such as that of SEQ ID NO: 5:TTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGA CCCGCTTAA, which is the underlined segment in the above SEQ ID NO: 3.

[0049] In some cases, the recombinase gene is Cre and the recombination sites are loxP recombination sites. In some such cases, the loxP recombination sites comprise a mutant loxP LE sequence and a mutant loxP RE sequence that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites. LoxP sites include a central 8 nucleotide segment flanked by inverted repeats of 13 nucleotides in length on each side. For instance, mutant loxP LE and loxP RE sequences have been created that leave the middle eight nucleotides of the sequence (comprising positions 14-21 of SEQ ID NO: 1) intact while mutating one or both of the inverted repeat portions of the sequence in order to cause the recombination reaction to run mainly in the forward direction to prevent reversal of the recombination event. Such mutant loxP LE and loxP RE sequences, therefore, may have improved recombination efficiency compared to wild-type loxP sequences. Exemplary mutant loxP sites are described in Araki et al., BMC Biotechnology 10: 29 (2010), for instance. In some such cases, the loxP RE and loxP LE are mutant loxP sequences and comprise the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively: (ATAACTTCGTATAGCATACATTATACGAACGGTA (SEQ ID NO: 1) and TACCGTTCGTATAGCATACATTATACGAAGTTAT (SEQ ID NO: 2)).

[0050] In some embodiments, the Cre recombinase coding sequence sequence of SEQ ID NO: 6, is shown below.SEQ ID NO: 6 - Cre recombinase nucleic acid sequence ATGCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAAAATTTG CCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGAC ATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCG TTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATACGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAGThe corresponding Cre recombinase amino acid sequence is provided as SEQ ID NO: 9, as follows, which includes a nuclear localization sequence at the N-terminal (residues 2-8; underlined below):MPKKKRKySNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDI RNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDIn other cases, the Cre recombinase has an amino acid sequence according to UniprotP06956, as shown herein in SEQ ID NO: 10, which has no insertion of a nuclear localization sequence at the N-terminal:MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSD SNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGI AYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVR LLEDGDAccordingly, in some embodiments the recombinase comprises a nuclear localization sequence, while in other cases the recombinase does not comprise a nuclear localization sequence. An exemplary nuclear localization sequence comprises residues 2-8 of SEQ ID NO: 9.

[0051] In other cases, a different recombinase and recombination site system may be used, such as the FLP recombinase and FRT recombination sites.

[0052] In some cases, the expression cassette comprises a gene under the control of a ubiquitin promoter, and optionally comprises a coding sequence for a poly-A tail. An exemplary ubiquitin promoter sequence, for example, is that of SEQ ID NO: 7, shown below. SEQ ID NO: 7 - ubiquitin promoter sequenceGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAG CGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGAC GCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCA GCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTT CCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAG GGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTG GCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATC GCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTG GCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGT AGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGC AAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGT GAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTT GAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCAC CATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGT CTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCAGTGCACCCGTACCTTT GGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATG CAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGAC GCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTG GTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTAT CTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGT AATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTT TTTGTTAGAC

[0053] In other cases, a CMV (cytomegalovirus) promoter may be used, such as that of SEQ ID NO: 8, below.SEQ ID NO: 8 - CMV promoter TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGAC TTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCA GTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATG TCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAG

[0054] Genes comprised within the expression cassette herein may include any gene of interest to be expressed from a mini circle plasmid in an animal. For example, expression of a protein, such as a heterologous protein, in an animal may allow for creation of animal models or other animals for experimental use. Example proteins include cytokines, antibodies, hormones, enzymes, and various pharmaceutical proteins such as fusion proteins, antibodies, and the like.

[0055] In some cases, the vector or set of vectors are plasmid vectors. For instance, in some cases, the vector or set of vectors are plasmid(s) that may be grown in bacterial cells. In some cases, the vector or set of vectors are grown in bacterial cells and then purified for administration to an animal.

[0056] Further examples include a plasmid vector comprising an expression cassette located between two loxP recombination sites, which comprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and which plasmid vector further comprises a gene encoding Cre recombinase, optionally wherein the gene encoding Cre recombinase is underthe control of an inducible promoter or a TK promoter or minimal TK promoter, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter. Further examples also comprise a first plasmid vector comprising an expression cassette located between two loxP recombination sites, which comprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter; the set further comprising a second plasmid vector comprising a gene encoding Cre recombinase, optionally wherein the gene encoding Cre recombinase is under the control of an inducible promoter or a TK or minimal TK promoter.

[0057] In any of the vectors or sets of vectors herein, in some embodiments, recombination at the recombination sites produces a minicircle comprising the expression cassette. In some such cases, the recombination produces a minicircle comprising the expression cassette and lacking an origin of replication.

[0058] The disclosure herein also includes a host cell comprising an expression vector or set of expression vectors as described herein. In some cases, the host cell is a bacterial cell, such as E. coli. For example, the vectors may be expanded in a host cell, so that they may be administered in sufficient quantities to an animal.

[0059] The disclosure herein further includes kits comprising, for example, a purified vector or set of vectors as described herein, or a host cell comprising a vector or set of vectors as described herein. In some cases, kits herein may further comprise instructions for use. In some cases, kits herein may further comprise a pharmaceutically acceptable solution for administration to an animal. In some cases, kits herein may further comprise one or more pharmaceutically acceptable excipients or carriers.

[0060] In some cases, vector or set of vectors of a kit herein may be formulated, for example, in a pharmaceutically acceptable carrier such as a liposome or lipid nanoparticle (LNP), such as a cationic lipid complex or a PEGylated lipid complex. In some embodiments, for example, a vector or set of vectors may be administered to an animal, for instance, in a composition comprising at least one carrier or excipient, such as a surfactant, phospholipid, and / or protein such as albumin. In some cases, a polynucleotide vector may be comprised within a liposome or comprised within a lipid nanoparticle (LNP). For example, a number of polynucleotide vectors have been delivered to animals in LNPs, such as a variety of vaccine vectors. See, e.g., Partridge, Trends Mol. Med., 29(5): 343-53 (2023); Zak & Zangi,Pharmaceutics 13: 1675 (2021); Dillard & Sweigart, Nature Rev. Med. 8: 282-300, doi.org / 10.1038 / s41578-022-00529-7 (2023); Kimura et al., J. Controlled Release 330: 753- 64 (2021). In some cases, LNPs allow for tissue or organ-specific delivery of vectors. For example, LNPs tend to be taken up by the liver after systemic administration, such as intravenous or intramuscular administration, while certain LNPs have been administered to the brain, while others may be directed to the spleen. See Id.

[0061] Also included in the disclosure herein is a method of preparing an expression vector or set of expression vectors as described herein, comprising inserting an expression cassette comprising a gene sequence, optional promoter and / or enhancer sequences, and optionally a poly-A tail sequence into a vector such that the expression cassette is located between two recombination sites, and further comprising inserting a polynucleotide comprising a recombinase gene, optional promoter and / or enhancer sequences, and optionally a poly-A tail, into the same or a different vector. In some cases, the method further comprises inserting the recombination sites into the vector, optionally as part of the expression cassette. In some cases, the method further comprises expanding the vector or set of vectors in a host cell, and optionally purifying the vector or set of vectors from the host cell.

[0062] In some methods herein, further steps are included, such as preparing a formulation of the vector or set of vectors in a pharmaceutically acceptable solution optionally including at least one pharmaceutically acceptable excipient or carrier. In some cases, the carrier comprises liposomes or excipients for formation of lipid nanoparticles (LNPs). Thus, in some cases, methods of manufacturing comprise preparation of a liposomal or LNP formulation of a vector or vectors herein, such as for later administration to an animal.3. Exemplary Administration Methods

[0063] The disclosure herein also encompasses methods of administering a vector or set of vectors herein to an animal, for example, for creation of a minicircle plasmid comprising the expression cassette of the vector or set of vectors in the animal. In some embodiments, the vector or set of vectors may be administered in a pharmaceutically acceptable solution, for example in some cases comprising one or more pharmaceutically acceptable excipients or carriers. In some cases, the vector or set of vectors may be formulated in a pharmaceutically acceptable carrier such as a liposome or lipid nanoparticle (LNP).

[0064] Administration methods include, for instance, injection such as hydrodynamic tail vein injection, intramuscular, or subcutaneous injection, intravenous administration, inhalation, or intranasal administration, for example. In some embodiments, administrationresults in uptake of the vector or set of vectors in the lungs, liver, and / or spleen, for example. In some embodiments, it is believed that such uptake by at least one organ of the body of the animal may allow minicircle formation from the vector or set of vectors, and expression of the gene or genes encoded by the expression cassette in the animal.

[0065] Accordingly, methods herein include, for example, a method of expressing a heterologous gene in an animal, comprising administering an expression vector or set of expression vectors described herein, and encoding the gene, to the animal. In some cases, the animal is a laboratory animal. Laboratory animals in some cases include a mouse, rat, rabbit, or guinea pig. In some cases, the animal is an animal such as a pig, goat, sheep, or cow. In some cases, the animal is a mouse. In some cases, the animal is a rat. In some cases, the administration is by hydrodynamic tail vein (HTV) injection, such as in the case of a mouse or rat. In some cases, administration is by subcutaneous injection, intramuscular injection, intravenous administration, intranasal, or inhalation. In some cases, the expression vector or set of expression vectors is administered to the animal by an administration means that allows the vector or set of vectors to transform hepatocyte cells or lung cells or spleen cells. Exemplary means include HTV injection, subcutaneous injection, intramuscular injection, intravenous administration, intranasal, or inhalation, and equivalents thereof. See, e.g., Partridge, Trends Mol. Med., 29(5): 343-53 (2023); Zak & Zangi, Pharmaceutics 13: 1675 (2021); Dillard & Sweigart, Nature Rev. Med. 8: 282-300, doi.org / 10.1038 / s41578-022- 00529-7 (2023); Kimura et al., J. Controlled Release 330: 753-64 (2021); and Cui et al., Pharmaceutics 14: 2428 (2022), for descriptions of various administration methods for genes, vectors, and liposomes or LNP formulations.

[0066] In some cases, the animal expresses the product of a gene encoded by the expression vector or set of expression vectors for a period of at least 150 days following administration, or for a period of at least 200 days following administration, or for a period of at least 250 days following administration, or for a period of at least 300 days following administration. In some cases, the gene is expressed in an animal for a longer period of time than the same gene expressed in an animal from an expression vector or set of expression vectors that encodes the gene but is not capable of recombination, such as where the expression vector or set of expression vectors does not comprise a recombinase gene or does not form a minicircle.

[0067] The disclosure herein also encompasses an animal having been administered with a vector or set of vectors as described herein. In some cases, the animal is a laboratory animal.Laboratory animals in some cases include a mouse, rat, rabbit, or guinea pig. In some cases, the animal is an animal such as a pig, goat, sheep, or cow. In some cases, the animal is a mouse. In some cases, the animal is a rat.

[0068] Further nonlimiting description of embodiments herein is provided in the Examples that follow.EXAMPLESExample 1: Preparation and Use of Plasmids for Recombinant Protein Expression in MiceMethodsPlasmid generation

[0069] A pRK vector was used as a starting point to express a gene of interest under a CMV promoter. This plasmid is named pCMV herein. Next, loxP RE and loxP LE mutant loxP sequences were inserted before and after the expression cassette, respectively, to make pCMV.lox. The mutant loxP sequences were as follows: LoxP RE: ATAACTTCGTATAGCATACATTATACGAACGGTA (SEQ ID NO: 1); LoxP LE: TACCGTTCGTATAGCATACATTATACGAAGTTAT (SEQ ID NO: 2). Next, a ubiquitin promoter sequence was used to replace the CMV promoter to make pUb.lox. A minimum TK promoter with Cre recombinase cDNA was introduced into pUb.lox to make a plasmid called pRMNT. Mouse IL17E, IL22, or leptin cDNA were introduced into each of the plasmids pCMV.lox, pUb.lox and pRMNT, such that the plasmids allowed for expression of murine IL17E, IL22, or leptin.Generating mini circle in bacteria

[0070] A bacterial strain called SW106, that expresses Cre recombinase under arabinose- inducible promoter was transformed with the above plasmids. Specifically, SW106 was transformed with IL22 / pCMV.lox, and cultured overnight at 32°C or 37°C in a regular LB broth containing 0.5% glucose. After the culture, the bacterial pellet was washed with M9 media several times, and then further cultured in M9 media containing 0.5% arabinose. At 0, 30, 60, and 120 min at 32°C, aliquots were taken from the culture and the plasmid was purified by mini-prep or mega-prep commercial kits (Qiagen). Purified plasmids were digested by Eco RI enzyme, and were analyzed on an agarose gel.Hydrodynamic Tail Vein (HTV) injection

[0071] Mice, specifically C57BL / 6, B6.Cg-Tg(Alb-cre)21Mgn / J mice (Alb-Cre mice), Balb / c, or leptin-deficient mice (ob / ob; B6.Cg-Lepob / J), received intravenously in the tailvein approximately 1.6 ml of lactated Ringer’s solution containing 0.4-10 ug of either pCMV, pCMV.lox, pUb.lox, or pRMNT plasmid vectors coding either mouse IL17E, mouse IL22 or mouse Leptin, along with 10 ug pTK.Cre where indicated. In some cases, the expression cassette was digested by appropriate restriction enzymes before the HTV injection. Doses were administered as a bolus intravenous injection (tail vein) over a period of 4-5 seconds (8 seconds maximum) for maximum DNA uptake. Mice were restrained without anesthesia in a conical acrylic restrainer after warming up to increase body temperature and dilate blood vessels. Animals were continuously monitored for any adverse clinical signs for at least 20 minutes post dose. At the indicated time points, the blood samples were obtained via lateral tail vein nick, and the serum samples were analyzed for the expression of the gene of interest by ELISA.DSS colitis model

[0072] C57BL / 6 mice received HTV injection with mouse IL22 / pUb.lox (or control pUb.lox) and pTK.Cre as described above on day -7, and received 3.5% Dextran Sulfate Sodium (DSS, Colitis grade Dextran Sulfate Sodium Salt (36,000-50,000 M.Wt.) MP Grade from MP Biomedicals) solution in a drinking water from day 0 to day 7. From day 7 to day 8, the mice received a regular water. Mice were weighed daily from day 4, and all mice were euthanized by CO2 inhalation using veterinary approved methods on day 8. The colons were assessed by histology for clinical score.C. rodentium infection model

[0073] C57BL / 6 mice and leptin-deficient mice (ob / ob; B6.Cg-Lepob / J) received HTV injection with mouse leptin / pUb.lox (or pUb.lox) and pTK.Cre as described above on day -7. On day 0, the mice were fasted 15 h prior to oral inoculation with 2 x 109colony-forming units of C. rodentuim in 200 ul PBS, and food was replaced back after inoculation. Mice were monitored daily and weighed at least 3 times a week until day 23, and all the remaining mice were euthanized by CO2 inhalation using veterinary approved methods on day 23.Sequences

[0074] Sequences of the promoters, Cre recombinase, and loxP sites are provided in SEQ ID NOs: 1-10 described above. The sequence of the murine IL22 gene is provided in SEQ ID NO: 11 :ATGGCTGTCCTGCAGAAATCTATGAGTTTTTCCCTTATGGGGACTTTGGCCGCCA GCTGCCTGCTTCTCATTGCCCTGTGGGCCCAGGAGGCAAATGCGCTGCCCGTCAA CACCCGGTGCAAGCTTGAGGTGTCCAACTTCCAGCAGCCATACATCGTCAACCGCACCTTTATGCTGGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGATGTCCGG CTCATCGGGGAGAAACTGTTCCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTG ATGAAGCAGGTGCTCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGACA GGTTCCAGCCCTACATGCAGGAGGTGGTGCCTTTCCTGACCAAACTCAGCAATCA GCTCAGCTCCTGTCACATCAGCGGTGACGACCAGAACATCCAGAAGAATGTCAG AAGGCTGAAGGAGACAGTGAAAAAGCTTGGAGAGAGTGGAGAGATCAAGGCGA TTGGGGAACTGGACCTGCTGTTTATGTCTCTGAGAAATGCTTGCGTCTGA Results and Discussion

[0075] Minicircle plasmids were created in E. coli using the Cre / loxP recombination system. A plasmid called pCMV.lox, capable of recombining to form a minicircle in E. coli expressing Cre recombinase was prepared, as shown in Fig. 1. pCMV.lox was designed to generate a minicircle containing an expression cassette including a CMV promoter by recombination induced by Cre recombinase. Cre recombinase recognizes a 34-bp (base pair) element called loxP, comprising two 13-bp inverted repeats that bind to Cre and an 8-bp spacer region that is involved in strand exchange in recombination. The two loxP sites, loxP RE and loxP LE, were placed on either side of the expression cassette in the plasmid, as shown in Fig. 1. In order to skew the equilibrium of the recombination reaction so that the reverse recombination reaction does not occur, each of the two loxP sites contained mutations, according to K. Araki et al., BMC Biotechnology 10(29) (2010). The plasmid both before and after recombination, thus, was intended to be able to express the gene of interest under the CMV promoter.

[0076] However, if the Cre is expressed continuously, the whole pCMV.lox plasmid cannot be replicated, because the gene expression cassette is separated from the components such as Ori and antibiotics resistant gene, which are required for the replication of the plasmid. Thus, it was necessary to grow the E. coli first in the absence of Cre, then express Cre to induce recombination to make a mini circle. For this purpose, an E. coli strain expressing Cre under an arabinose-inducible promoter (called SW106) was utilized. In this E.coli, Cre is expressed only when the E. coli is cultured in the presence of arabinose. Therefore, it was possible to grow the transformed bacteria in the absence of arabinose first, and then incubate the bacteria with arabinose to induce recombination (see Fig. 2).

[0077] The SW106 bacterial line was transformed with either mIL17E / pCMV.lox or mIL22 / pCMV.lox, expressing murine IL17E or murine IL22, respectively. The bacteria was cultured in LB broth containing 0.5% glucose at 32°C overnight. Then the bacteria werewashed with M9 media, and further cultured in M9 media containing 0.5% arabinose for up to 120 min. Culture samples were harvested and plasmids were purified by a mini prep kit (Qiagen). Purified plasmids before and after EcoRI digestion were analyzed by agarose gel, and minicircle generation was confirmed (Fig. 3). However, the yield of minicircle was very low, and changing the culture temperature to 37°C did not improve the yield (data not shown).

[0078] Instead of making minicircle plasmids in vitro, recombination was instead induced in vivo by employing a mouse strain expressing Cre recombinase under an albumin promoter. Since hydrodynamic tail vein (HTV) injection can deliver DNA into hepatocytes, it was hypothesized that delivering plasmid by HTV injection might generate minicircle plasmids in hepatocytes in this mouse strain (Fig. 4). Specifically, B6.Cg-Tg(Alb-cre)21Mgn / J mice (Alb-Cre mice) and regular C57BL / 6J mice were injected with IL22 / pCMV.lox or IL22 / pCMV (which does not have loxP sites), both of which express murine IL22. As shown in Figure 5, only when the Alb-Cre mice received IL22 / pCMV.lox plasmid, was prolonged IL22 expression observed.

[0079] However, this method limits the application to Alb-Cre mice. Thus, two strategies for making the method available to wild-type mice were tested. The first was co-inj ection of the plasmid with a Cre-expressing plasmid (pTK.Cre) (Fig. 6A). The second was adding a Cre expression cassette into the loxP plasmid (pRMNT). For the latter strategy, a minimum TK promoter was selected to minimize the gene induction in the bacteria (Fig. 6B). Additionally, a ubiquitin promoter was selected to induce the gene of the interest, as it was found that CMV promoter was silenced quickly, whereas the ubiquitin promoter expressed for a longer period of time, when tested with a linearized expression cassette (Fig. 7).

[0080] The mice were injected with either mixture of IL22 / pUb.lox and pTK.Cre, or IL22 / pRMNT. As shown in Figure 8, prolonged IL22 expression was confirmed for over 250 days in both groups. The mice receiving only IL22 / pUb.lox, in contrast, showed gradually declining expression. The regular plasmid with CMV promoter, IL22 / pCMV, showed quickly declining protein expression (Fig. 8). This indicated that both options of co-inj ection with a Cre-expressing plasmid and adding a Cre expression cassette to the loxP plasmid allowed for prolonged IL22 expression in the regular wild-type mice.

[0081] Next co-injection with IL22 / pUb.lox and pTK.Cre was tested in a preclinical disease model. Dextran Sulfate Sodium (DSS) damages colon epithelial cells, and is used as a preclinical mouse model for inflammatory bowel disease. IL22 is well-known to amelioratethe disease in this model. Mice were injected with either IL22 / pUb.lox and pTK.Cre, allowing for IL22 expression, or with pUb.lox and pTK.Cre, which does not express IL22. Mice received the plasmids by HTV injection on day -7. On day 0, DSS was added into the drinking water and weight and clinical scores were tracked compared to control mice which were not treated with DSS. (See Fig. 9A-9C.) Mice receiving pUb.lox + pTK.Cre showed body weight loss and high clinical score, whereas mice receiving IL22 / pUb.lox + pTK.Cre showed reduced body weight loss and reduced clinical score. As noted in Fig. 9C, there was a significant difference in the colon clinical score between the two groups.

[0082] The plasmid was also tested in a C. rodentium infection model. Obese mice such as leptin deficient mice (ob / ob mice) are susceptible in this model (J.B. Grace et al., Nature 529: 390-393 (2016); doi: 10.1038 / naturel3564). HTV injection of a plasmid expressing leptin (leptin / pUb.lox) with plasmid pTK.Cre rescued the mice and also reduced obesity to normalize the body weight (Fig. 10A-B), whereas the mice receiving a control plasmid not expressing leptin (pUb.lox) with plasmid pTK.Cre succumbed during the second week of the infection and body weight normalization was not observed.

[0083] These results as a whole show that mini circle plasmids expressing a protein of interest can be generated in vivo in mice from injection of wild-type mice with a parental plasmid including loxP recombination sites in combination with a source of Cre recombinase, either a second plasmid expressing Cre recombinase, or via a further open reading frame in the parental plasmid expressing Cre recombinase, and that the associated minicircle plasmids allow for prolonged gene expression of a desired protein in vivo. Moreover, unlike in other attempts to use minicircle plasmids for protein expression, which required isolation and purification of minicircles produced in vitro, no such isolation or purification is necessary as the minicircles are formed in vivo, possibly in hepatocytes of the liver.

Claims

What is Claimed is:

1. An expression vector or set of expression vectors, comprising an expression cassette located between two recombination sites, and further comprising a recombinase gene encoding a recombinase that recognizes the recombination sites, wherein the expression cassette and the recombinase gene are located on the same vector or on different vectors.

2. A set of expression vectors comprising a first vector comprising an expression cassette located between two recombination sites and a second vector comprising a recombinase gene encoding a recombinase that recognizes the recombination sites on the first vector.

3. The expression vector or set of expression vectors of claim 1 or 2, wherein the recombinase gene is under the control of an inducible promoter, or wherein the recombinase gene is under the control of a TK promoter.

4. The expression vector or set of expression vectors of claim 3, wherein the inducible promoter is an arabinose promoter.

5. The expression vector or set of expression vectors of any one of claims 1-4, wherein the recombinase gene is Cre and wherein the recombination sites are loxP recombination sites.

6. The expression vector or set of expression vectors of claim 5, wherein the loxP recombination sites comprise a mutant loxP LE sequence and a mutant loxP RE sequence that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites.

7. The expression vector or set of expression vectors of claim 6, wherein the loxP RE and loxP LE comprise the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

8. The expression vector or set of expression vectors of any one of claims 1-7 wherein the Cre recombinase gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10, or wherein the Cre recombinase gene comprises the nucleic acid sequence of SEQ ID NO: 6 or a sequence degenerate to SEQ ID NO: 6.

9. The expression vector or set of expression vectors of any one of claims 1-8, wherein the expression cassette comprises a gene under the control of a ubiquitin or CMV promoter, and optionally comprises a coding sequence for a poly-A tail.

10. The expression vector or set of expression vectors of any one of claims 1-9, wherein the vector or set of vectors are plasmid vectors.

11. The expression vector of any one of claims 1 or 3-10, comprising a plasmid vector comprising an expression cassette located between two loxP recombination sites, whichcomprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and which plasmid vector further comprises a gene encoding Cre recombinase, optionally wherein the gene encoding Cre recombinase is under the control of an inducible promoter, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter.

12. The set of expression vectors of any one of claims 2-10, comprising a first plasmid vector comprising an expression cassette located between two loxP recombination sites, which comprise mutant loxP LE and loxP RE sequences that increase yield of recombination products in vitro in a host cell compared to wild-type loxP recombination sites, and optionally wherein the expression cassette comprises a gene under the control of a ubiquitin promoter; the set further comprising a second plasmid vector comprising a gene encoding Cre recombinase, optionally wherein the gene encoding Cre recombinase is under the control of an inducible promoter.

13. The expression vector or set of expression vectors of any one of claims 1-12, wherein recombination at the recombination sites produces a minicircle comprising the expression cassette.

14. The expression vector or set of expression vectors of claim 13, wherein the recombination produces a minicircle comprising the expression cassette and lacking an origin of replication.

15. A composition comprising the expression vector or set of expression vectors of any one of claims 1-14 and at least one carrier or excipient.

16. A liposome or lipid nanoparticle (LNP) comprising the expression vector or set of expression vectors of any one of claims 1-14 or the composition of claim 15.

17. A host cell comprising the expression vector or set of expression vectors of any one of claims 1-14, the composition of claim 15, or the liposome or LNP of claim 16.

18. An animal comprising the expression vector or set of expression vectors of any one of claims 1-14, optionally wherein the animal is a laboratory animal, such as a mouse, rat, rabbit, or guinea pig.

19. The animal of claim 18, wherein the animal expresses the product of a gene encoded by the expression vector or set of expression vectors for a period of at least 250 days following administration of the vector or set of vectors to the animal.

20. The animal of claim 18 or 19, wherein the product of the gene is expressed in an animal for a longer period of time than the same gene expressed in an animal from an expression vector or set of expression vectors that encodes the gene but is not capable of recombination.

21. The animal of claim 19 or 20, which is a mouse or rat.

22. A method of expressing a heterologous gene in an animal, comprising administering the expression vector or set of expression vectors of any one of claims 1-14 encoding the gene, or the composition of claim 15, or the liposome or LNP of claim 16 to the animal.

23. The method of claim 22, wherein the animal is a laboratory animal, such as a mouse, rat, rabbit, or guinea pig.

24. The method of claim 23, wherein the animal is a mouse or rat.

25. The method of any one of claims 22-24, wherein the administration is by hydrodynamic tail vein (HTV) injection, subcutaneous or intramuscular injection, or intravenous administration.

26. The method of any one of claims 22-25, wherein the expression vector or set of expression vectors, or the composition, or the liposome or LNP is administered to the animal by an administration means that allows the vector or set of vectors to transform hepatocyte, lung, or spleen cells.

27. The method of any one of claims 22-26, wherein the animal expresses the product of a gene encoded by the expression vector or set of expression vectors for a period of at least 250 days following administration of the vector or set of vectors to the animal.

28. The method of any one of claims 22-27, wherein the product of the gene is expressed in an animal for a longer period of time than the same gene expressed in an animal from an expression vector or set of expression vectors that encodes the gene but is not capable of recombination.

29. A method of preparing an expression vector or set of expression vectors according to any one of claims 1-14, comprising inserting an expression cassette comprising a gene sequence, optional promoter and / or enhancer sequences, and optionally a poly-A tail sequence into a vector such that the expression cassette is located between two recombination sites, and further comprising inserting a polynucleotide comprising a recombinase gene, optional promoter and / or enhancer sequences, and optionally a poly-A tail, into the same or a different vector.

30. The method of claim 29, wherein the method further comprises inserting the recombination sites into the vector, optionally as part of the expression cassette.

31. A kit comprising an expression vector or set of expression vectors of any one of claims 1- 14, the composition of claim 15, or the liposome or LNP of claim 16, and optionally further comprising instructions for use, for example, for administration to a host cell or to an animal.

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