WO2006076288A2 - Dna constructs for long-term expression of intravascularly injected naked dna - Google Patents

Abstract

Hydrodynamic intravascular injection of recombinant DNA molecules that can be expressed in the liver provides sustained and/or high-level expression of genes in a simple, safe, inexpensive, and reproducible fashion. These DNA molecules include a first sequence with the promoter of a liver-expressed gene operably linked to a second sequence encoding a protein of interest. The DNA molecules can be expressed in vivo to produce functionally active proteins. Methods of the invention provide tools to study these proteins in vivo and therapeutic applications for the expressed DNA molecules.

Description

DNA CONSTRUCTS FOR LONG-TERM EXPRESSION OF INTRAVASCULARLY INJECTED NAKED DNA

SEQUENCE LISTING

[001] The instant application contains a "lengthy" Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. The CD-Rs, recorded on January 6, 2006, are labeled CRF, "Copy 1," "Copy 2" and "Copy 3", respectively, and each contains only one identical 506 KB file (89452304. APP).

PRIORITY CLAIM

[002] This application claims the benefit of priority to U.S. Application No.

60/642,604, "DNA constructs for long-term expression of intravenously injected naked DNA," filed January 11, 2005, which is incorporated by reference in its entirety.

TECHNICAL FIELD

[003] The present invention relates to DNA molecules that contain the promoter of a liver-expressed gene operably linked to a gene sequence encoding a protein of interest other than a reporter gene, and that can be introduced into an animal to express a functionally active protein of interest in vivo. The DNA molecules can be used for a variety of purposes, including studying the in vivo dynamics, functions, and interactions of one, or more than one, expressed protein; identifying in vivo targets of an expressed protein; and providing therapeutic treatments. The invention also relates to methods of transforming liver cells in vivo with these DNA molecules.

BACKGROUND ART

[004] Introduction of Proteins into Animals

[005] Introduction of proteins into animals is one of the most important methodologies in biomedical research and drug development. This methodology is at the core of an increasingly important branch of the biotechnology and pharmaceutical industries which focuses on the development of protein-based therapeutics. [006] There are different ways a protein can be introduced into an animal. In rodents, a gene sequence encoding the protein can be integrated into the genome of embryonic cells, giving rise to transgenic animals which stably express the protein. Alternatively, the protein can be expressed in vitro, for example in cell culture, then purified by biochemical means, and introduced into the animal by injecting a composition containing the purified protein. A third approach is to introduce a DNA molecule encoding the protein into juvenile or adult animals, leading to transient expression of the protein. The latter approach has important advantages compared to the two former. Unlike transgenic technology, it can potentially be applied to humans, and unlike the in vitro expression approach, it circumvents the often difficult, time-consuming, and expensive production of a purified protein preparation. [007] Gene Transfection

[008] Introduction of a protein-encoding DNA molecule into an animal can result in the uptake of the DNA molecule by target cells in a process called gene transfection. Such gene transfection provides for the delivery of genetic information to a cell, which can result in expression of a protein that can inhibit, eliminate, augment, or alter the expression of an endogenous nucleotide sequence or the function of an endogenous protein, or can result in a biological characteristic not naturally associated with the cell. Delivery of genes to cells in vitro has been widely used and has generated useful information about the function of proteins within a cell, or within a simplified system of cells (Luo and Saltzman, Nat. Biotechnol. 18:33-37 (2000)). The techniques used for in vitro transfection are well developed. However, most of the techniques used for in vitro transfection, such as calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA, cannot be used in vivo (Rodύiϊz, Swiss Med. WkIy. 131:4-9 (2001)).

[009] Efforts have been made to develop new techniques or variations of in vitro techniques for in vivo transfection. DNA transfer in vivo can be accomplished by viral and non- viral delivery. As set forth below, viral methods include adenovirus, adeno-associated virus, retrovirus, and lentivirus vectors; and non-viral methods include polylysine conjugates, various polymers, liposomes, and naked DNA. However, technical barriers still limit the use of in vivo transfection because the various known techniques have either low transfer efficiency, do not lead to sustained and/or high-level gene expression, are dangerous to the host, or have a combination of these drawbacks (Nyuyen and Ferry, Gene Ther. Suppl 1:576-84 (2004)). Solutions to such technical barriers would be valuable because in vivo transfection is a potentially powerful technique. It can be used to conduct basic research to understand the function of a protein within an intact organism and to deliver therapeutic proteins to patients.

[010] Viral Gene Transfection Methods

[011] Viral delivery techniques make use of the machinery viruses have evolved to transfer foreign DNA into cells (Luo and Saltzman, Nat. Biotech. 18:33-7 (2000)). Viral vectors can typically transfer genes into cells in an efficient manner (Luo and Saltzman, Nat. Biotech. 18:33-7 (2000)). In addition, retroviral and adeno- associated virus vectors can integrate into mammalian genomes, thereby leading to prolonged expression (Relph et al., Brit. Med. J. 329:839-42 (2004)). However, these vectors can only carry a relatively small amount of foreign DNA (Liu et al., MoI. Interv. 1:168-72 (2001)). hi addition, integration may result in inactivation of genes required for cell viability or in activation of proto-oncogenes (Liu et al., MoI. Interv. 1:168-72 (2001)). Lastly, retroviral vectors can only transfect proliferating cells (Rochlitz, Swiss Med. WkIy. 131:4-9 (2001)). Even the viral vectors that do not integrate into the host genome have potentially dangerous side effects. For example, they may mutate to reacquire infectious ability. Herpes and adenoviral vectors may retain viral promoters and genes that could be expressed in human cells under certain conditions, causing immune or other harmful effects (Nguyen and Ferry, Gene Titer. Suppl. 1 :S76-84 (2004)). In practical terms, viral vectors are difficult and costly to make in large quantities, further limiting their potential use in both large-scale basic research projects and in gene therapy (Nguyen and Ferry, Gene Ther. Suppl. 1 :S76-84 (2004)).

[012] Non-Viral Gene Transfection Methods

[013] Non-viral methods transfer purified plasmid or linear DNA, either with or without the help of transfection reagents such as liposomes, lipids, and polyamines (U.S. Patent Application No. 10/206,747). When the DNA is transferred without the help of transfection reagents, the DNA is referenced to as "naked." hi contrast to viral vectors, linear and plasmid DNA is easy and inexpensive to make and purify (Young and Dean, Microcirculation 9:35-49 (2002)). In addition, naked DNA is one of the safest in vivo polynucleotide delivery systems (Young and Dean, Microcirculation 9:35-49 (2002)).

[014] Injection of naked DNA into tissue has long been known to result in the expression of foreign genes. For example, injection of plasmid DNA into muscle has been reported to result in gene expression by muscle cells (Wolff et al, Science 247:1465-8 (1990)). Naked DNA was also reportedly expressed following its injection into cardiac muscle (Acsadi et al., New Biol. 3:71-81 (1991)). However, the efficiency of gene transfer of non- viral vectors is thought to be generally lower than that of viral vectors. This relatively low in vivo transformation efficiency is a limiting factor in performing methods of non-viral transformation (Kobayashi et al., J. Pharmacol. Exp. Ther. 297:853-60 (2001)). It would be desirable to improve the efficiency of non- viral in vivo gene transfection methods, for example, by improving the transformation efficiency or the duration of gene expression. [015] Recent advances have started to bridge the gap between the transformation efficiency of viral vectors and naked DNA. For example, the intravascular delivery of naked plasmid DNA into muscle and liver tissues has reportedly lead to expression in up to 20% of the cells, approaching the transfection levels observed with viral delivery systems (Zhang et al., Hum. Gene Ther. 12:427-38 (2001); Budker et al., Gene Ther. 5:272-6 (1998); Zhang et al., Hum. Gene Ther. 8:1763-72 (1997); Budker et al., Gene Ther. 3:593-8 (1996)). [016] One particularly powerful technique is the hydrodynamic tail vein method. This technique entails injecting naked DNA into a blood vessel, for example, the tail vein. The injections are typically performed under high pressure, with a high volume, and in a short time frame, usually several seconds (Liu et al., Gene Ther. 6:1258-66 (1999); Zhang et al., Hum. Gene Ther. 10:1735-7 (1999)). Using this method, the delivery efficiency to liver cells is reportedly approximately 40%. Although this method has been used in many laboratories and has been reported to allow efficient transduction of liver cells, in most instances expression is limited to a short period of time (Nguyen and Ferry, Gene TJier. Suppl. l:S76-84 (2004)). For example, the cytomegalovirus (CMV) promoter has been used in DNA injection studies, but its expression was reported to be limited to a duration of 1-2 days, an insufficient time to observe the biological effects of many gene products. Contributing to the short duration is the degradation of circulating DNA by blood nucleases and by macrophages (Kobayashi et al., J. Pharmacol. Exp. Ther. 297:853- 60 (2001)).

[017] While efficient transfer of genes into liver cells using non- viral vectors has become less problematic with the advent of the hydrodynamic tail vein method, the challenge of achieving sustained expression of genes transfected with non- viral techniques remains.

[018] Use of Viral Promoters to Express Genes of Interest

[019] Viral regulatory elements typically used to express or overexpress proteins include those derived from CMV and Rous sarcoma virus (RSV). Results using these viral promoters/enhancers for in vivo gene expression have been variable and inconsistent.

[020] Several studies have reported long-term expression following intramuscular injection of naked DNA containing a variety of genes driven by these viral promoters/enhancers. For example, Feiger et al., U.S. Patent No. 6,413,942; Rizzuto et al., Proc. Natl. Acad. ScL USA 96:6417-22 (1999); Lefesvre et al., BMC MoI. Biol. 3:12 (2002); and Tripathy et al., Proc. Natl. Acad. Sd. USA 93:10876-80 (1996), reported that intramuscular injection of naked plasmids containing genes under the control of the CMV or RSV promoter can lead to sustained gene expression in mice. Furthermore, some studies have reported that viral enhancers support expression of proteins of interest when the constructs containing them are injected intravenously. Kameda et al., Biochem. Biophys. Res. Commun. 309:929-36 (2003), and Jiang et al., Biochem. Biophys. Res. Commun. 289:1088-92 (2001), reported that an expression plasmid encoding the CMV enhancer and the chicken β-actin promoter can sustain long-term expression of genes of interest following tail vein injection by a hydrodynamics-based procedure.

[021 ] In contrast, numerous other studies using viral-derived regulatory elements have been unable to show sustained expression of the gene of interest. He et al., World J. Gastroenterol. 10:567-72 (2004), injected naked plasmid DNA encoding an engineered human proinsulin driven by the CMV promoter into the tail vein of diabetic mice using a hydrodynamics-based procedure. Blood glucose levels reportedly decreased to a level close to that of normal mice on the first day after treatment, but returned to elevated levels within three days of treatment. Serum insulin levels in the diabetic mice were reported to be correspondingly higher than those of normal mice at day one following treatment with the plasmid, but to have returned to pretreatment levels within seven days of treatment. Injecting human growth hormone, under the control of the CMV promoter, into the portal vein of mice reportedly failed to result in detectable human growth hormone serum levels unless the mice were also treated with dexamethasone and cyclosporine (U.S. Patent Application No. 2001/0009904). Human factor IX driven by the CMV promoter was undetectable after day 7 following injection of a supercoiled plasmid encoding the gene into the tail vein of mice (U.S. Patent Application No. 2002/0061861). Herwijer et al., J. Gene Med. 3:280-91 (2001), have suggested that the CMV promoter cannot sustain long-term expression of transferred DNA after intravascular delivery because it is silenced only a few days after delivery. However, Zhang et al., Gene Ther. 7:1344-9 (2000), reported biphasic expression of human αl -antitrypsin administered by tail vein injection of a DNA plasmid with a viral CMV promoter with a 100-fold decline in expression from day 1 to day 30, then a persistent low level of expression lasting six months following injection.

[022] Use of Eukaryotic Promoters to Express Genes of Interest

[023] Researchers have also used eukaryotic-derived promoters, such as the ubiquitous β-actin promoter and promoters that are only active in the targeted cell type or tissue, to express proteins in vivo, with differing results. Miao et al., MoI. Ther. 3:947-57 (2001), and Miao et ύ., Hum. Gene Ther. 14:1297-1305 (2003), reported that injecting human factor IX driven by the alpha 1 -antitrypsin promoter into mice leads to gene expression for at least six months. Zhang et al., Hum. Gene Ther. 15:770-82 (2004), reported stable expression of dystrophin in mouse muscle following intraarterial delivery of plasmid DNA expressing the mouse dystrophin gene under the control of the muscle-specific human desmin gene control region. Injecting a plasmid containing the human growth hormone gene under the control of the metallothionein promoter reportedly led to a low level of expression of human growth hormone inducible by zinc sulfate in the drinking water (U.S. Patent No. 6,413,942). [024] In summary, various different systems for in vivo gene delivery and expression have been developed and tested. However, none of the systems has been proven to be simple, safe, inexpensive, and reproducible in facilitating sustained and/or high-level gene expression, and easy-to-use so as to allow rapid, routine application. Hence, there is a need for a system that combines these properties. Such a system would make it possible to test and study the function of many different genes in a more time- and cost-efficient manner in animal models, including disease models, and to harness the therapeutic activities of genes or combinations of genes for human treatment.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES [025] Brief Description of the Drawings

[026] Figure 1 shows a schematic representation of the DNA constructs used herein, including the Examples. "3A4 promoter" denotes the cytochrome P450 3A4 promoter. "EPO" is the gene encoding erythropoietin and "lacZ" is the gene encoding β-galactosidase. "bPolyA" denotes a bovine polyadenylation sequence. [027] Figure 2 shows a graph showing serum EPO levels, as determined by

ELISA, in three mice injected with the lacZ construct (bgal#l - #3) and three mice injected with the EPO construct (EPO#1 - #3). The EPO levels were determined for each mouse at each of three time points: 1 day after injection (day 1), 3 days after injection (day 3), and 7 days after injection (day 7) and expressed as International Units per liter (IU/1).

[028] Figure 3 shows the livers of two mice injected with the lacZ construct shown in Fig. 1, two mice injected with the EPO construct shown in Fig. 1, and two uninjected wild-type (WT) mice. All six livers were fixed in paraformaldehyde and stained with X-gal. The punctate staining in the livers of mice injected with the lacZ construct demonstrates the expression of lacZ protein in these mice. There was no staining observed in either the control livers of mice injected with the EPO construct or the control livers of uninjected wild-type mice.

[029] Figure 4a shows a graph showing increased hematocrits (HCT) in the blood of three mice injected with the EPO construct 13 days prior to measurement. [030] Figure 4b shows splenomegaly resulting from EPO over-expression in mice receiving the EPO construct by tail vein injection, as compared to mice that received the lacZ construct.

[031] Figure 5 shows a diagram of a DNA molecule for expressing genes of interest in vivo. It includes a promoter upstream of an intron and a bovine poly A (bpA) sequence, all of which are flanked by an attB and an attP sequence. The restriction sites used to construct the DNA molecule are also shown. [032] Figure 6 shows the results of an ELISA assay of the serum protein level of a gene of interest (FGFRl -IHc-Fc (described in an U.S. Provisional Patent Application filed on January 10, 2006)) following the injection of a minicircle construct into the tail vein of a mouse, as described in greater detail in Example 6. The serum protein level rose to a peak approximately 9 days post-injection and maintained a sustained level for at least 44 days post-injection. [033] Brief Description of the Tables

[034] Table 1 provides an annotated list of liver-expressed genes with promoters useful for practicing the present invention. Each is identified by an internal reference number (FP ID); a Reference ID that can be used to access information on the gene in the National Center for Biotechnology Information (NCBI) database; and annotation from the NCBI database (Genes Containing Useful Promoters). [035] Table 2 provides the internal identification numbers (FP ID) and the sequence identification numbers (SEQ. ID. NOS.) for the promoter and intron sequences of the invention. It includes SEQ. ID. NOS.: 1-122, each of which sequence provides the 5' untranslated region (utr), the transcription start site (TSS) and the genomic sequence of about 1000 bp upstream of the TSS of the identified gene; SEQ. ID. NOS.: 123-244, each of which sequence provides the 5' utr of the identified gene; and SEQ. ID. NOS.:245-392, each of which sequence provides 1500 bp genomic sequence upstream of the translation start site, including intron sequence and at least part of the 5' utr of the identified gene. Each sequence is also associated with a Reference ID of Table 1. Table 2 further includes SEQ. ID. NOS.:393 and 394, which are introns useful in the present invention.

[036] Table 3 provides the coordinates of selected intronic sequences of the invention. It includes the NCBI Reference ID, the number of untranslated introns (No. UTR Introns), the human chromosome on which the gene is located, a designation as to whether the intron belongs to the plus or the minus strand, and a designation of the nitron's genomic coordinates within the untranslated region (UTR Intron Coordinates).

DETAILED DESCRIPTION OF THE INVENTION

[037] The invention provides DNA molecules with a promoter of a liver- expressed gene operably linked to a gene encoding a protein, which can be expressed in vivo to produce a functionally active protein. An intron sequence may be included as an additional functional component. The invention also provides methods for the sustained and/or high-level expression of these DNA molecules in the livers of animals. The compositions and methods of the invention provide simple, safe, and reproducible gene delivery systems to introduce and study the function of many different genes in animal models as well as for therapeutic intervention. [038] Definitions

[039] The terms used herein have their ordinary meanings, as set forth below, and can be further understood in the context of the specification. [040] The term "gene" refers to a nucleic acid sequence that comprises coding sequences necessary for the production of a polypeptide or polypeptide precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties of the full-length polypeptide or a fragment thereof are retained. The term also encompasses the coding region of a gene and the sequences located adjacent to the coding region on both the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. The term "gene" encompasses both cDNA and genomic forms.

[041] The terms "liver-expressed gene" and "gene expressed in the liver," as used interchangeably herein, refer to a gene that is transcribed and/or translated in one or more cells of the liver, for example, hepatocytes, blood vessels, lymph vessels, Kupffer cells, and cells of the bile canaliculi and bile ducts. A "liver-expressed gene" may also be expressed in tissues other than liver.

[042] A "reporter gene" encodes a gene product commonly used in the art to detect a gene of interest, and which can be easily assayed for expression. Reporter gene products are typically enzymes or other proteins with activity that can be easily visualized or measured. Examples of reporter genes include β-galactosidase, chloramphenicol acetyltransferase (CAT), luciferase, and green fluorescent protein. [043] The terms "gene expression," "expression of a DNA molecule," and to

"express" a gene or nucleic acid, used interchangeably herein, refer to the conversion of the information contained in a gene into a gene product, for example, RNA or protein, by transcription and/or translation. The conversion of gene information into a protein is also referred to as "expression of a protein."

[044] The term "sustained expression" refers to the expression of a gene product for more than 2 to 4 days. For example, the expression of a protein for 5 to 15 days, 16 to 25 days, 26 to 35 days, or 36 to 45 days constitutes sustained expression. Expression for more than 45 days also constitutes sustained expression. [045] The term "overexpression" or "high-level expression" refers to the expression of a gene product at a level that exceeds the normal or baseline expression levels of the gene product. For example, expression of a secreted gene product at 1 to 2 μg/ml serum, 3 to 5 μg/ml serum, 6 to 10 μg/ml serum, or 11 to 20 μg/ml serum can constitute high-level expression. Expression at more than 20 μg/ml serum can also constitute high-level expression.

[046] A "vector" is a nucleic acid molecule originating from a virus, a plasmid, a synthetic source, or a cell, into which another nucleic acid fragment of appropriate size can be integrated without losing the capacity for self-replication. Vectors can introduce nucleic acids into host cells, where they may be reproduced. [047] "Recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

[048] A "host cell" is an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention may be called a "recombinant host cell." [049] The term "operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors, etc., are present. Such a promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and a coding sequence, as can translated introns, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[050] A "promoter," as used herein, is a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of an operably linked downstream (3' direction) coding sequence. For purposes of the present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Promoters of the invention include those that are naturally contiguous to a nucleic acid molecule and those that are not naturally contiguous to a nucleic acid molecule. Additionally, promoters of the invention include inducible promoters, conditionally active promoters, such as a cre- lox promoter, constitutive promoters, and tissue specific promoters. [051 ] An "intron" is a non-coding region of a gene which is transcribed into nuclear RNA. They are removed, or spliced, from the nuclear RNA transcript, and are therefore absent in the mRNA transcript. Introns may contain regulatory elements such as enhancers. "Heterologous introns" are those derived from any source that is not naturally occurring. Heterologous introns may be derived, for example, from a tissue other than the source of the intron, or from an animal of a different species. [052] A "transcription start site" is the location of the first DNA nucleotide transcribed to RNA. The nucleotide at which transcription begins can be designated +1, and nucleotides numbered from this reference point. Negative numbers can indicate upstream nucleotides and positive numbers downstream nucleotides. [053] An "origin of replication" is the sequence at which synthesis begins in the DNA replication process. An origin of replication may be sufficient for replication of a circular DNA molecule in a host cell from a prokaryotic source. [054] A "DNA complexing agent" is an agent that specifically binds to DNA.

It may, for example, perturb the secondary structure of the DNA. A DNA complexing agent may, for example, label DNA, improve the ability of DNA to cross a cell membrane, intercalate into DNA, cross-link DNA, inhibit DNA synthesis, and/or inhibit DNA regulation.

[055] A "liposome" is an artificial phospholipid bilayer vesicle formed from an aqueous suspension of phospholipid molecules. It may comprise one or more concentric phospholipid bilayers. Liposomes may be used medically, especially to convey therapeutics, for example, vaccines, drugs, enzymes, or other substances to targeted cells or organs.

[056] The terms "protein," "peptide," and "polypeptide," used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones. The term includes single chain proteins as well as multimers. The term also includes aptamers. It further includes conjugated proteins, fusion proteins, including, but not limited to, fetuin A, fetuin B, a leucine zipper domain, a tetranectin trimerization domain, a mannose binding protein, or an Fc region. Also included in this term are variations of naturally-occurring proteins, where such variations are homologous or substantially similar to the naturally-occurring protein, as well as corresponding homologs from different species. Variants of polypeptide sequences include insertions, additions, deletions, or substitutions as compared with the subject polypeptides.

[057] "Transmembrane proteins" extend into or through the cell membrane's lipid bilayer; they can span the membrane once, or more than once. Transmembrane proteins, having part of their molecules on either side of the bilayer, have many and widely variant biological functions. Transmembrane proteins are often involved in cell signaling events; they can comprise signaling molecules, or can interact with signaling molecules. Extracellular domains of transmembrane proteins may be cleaved to produce soluble receptors.

[058] "Secreted proteins" are generally capable of being directed to the endoplasmic reticulum, secretory vesicles, or the extracellular space as a result of a secretory leader, signal peptide, or leader sequence. They may be released into the extracellular space, for example, by exocytosis or proteolytic cleavage, regardless of whether they comprise a signal sequence. A secreted protein may in some circumstances undergo processing to a mature polypeptide. Secreted proteins may comprise leader sequences of amino acid residues, located at the amino terminus of the polypeptide and extending to a cleavage site, which, upon proteolytic cleavage, result in the formation of a mature protein.

[059] As used herein, "extracellular" refers to the region outside a cell. The extracellular fragment of a transmembrane protein extends to the cell exterior. [060] As used herein, "intracellular" refers to the region of the cell contained within its plasma membrane. The intracellular fragment of a transmembrane protein extends into the cell interior.

[061] A "functionally active" entity, "biologically active" entity, or an entity having "biological activity," is an entity having structural, regulatory, or biochemical functions of a naturally occurring molecule or any function related to or associated with a metabolic or physiological process. Functionally active polynucleotide fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a polynucleotide of the present invention. The functional activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates functional activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that can, for example, be detected as unique for the polynucleotide molecule, or that can be used as a primer in a polymerase chain reaction. A functionally active polypeptide or fragment thereof includes one that can participate in a biological reaction, for example, one that can serve as an epitope or immunogen to stimulate an immune response, such as production of antibodies, or that can participate in stimulating or inhibiting signal transduction by binding to ligands receptors or other proteins, or nucleic acids; or activating enzymes or substrates. Also by way of example, functionally active erythropoietin stimulates the production of red blood cells. [062] A "naturally-occurring" molecule is one that exists in nature and without artificial aid. It can exist in any species, and includes all allelic and splice variants.

[063] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type. A pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

[064] A "composition" herein refers to a mixture that usually contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a cell culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium. For example, compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders. [065] A "buffer" is a system that tends to resist change in pH when a given increment of hydrogen ion or hydroxide ion is added. A buffered solution will demonstrate a lesser change in pH than an unbuffered solution in response to addition of an acid or base. Any conventional buffer can be used with the compositions herein including but not limited to, for example, Tris, phosphate, imidazole, and bicarbonate. [066] 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 invention, as claimed. Moreover, it must be understood that the invention is not limited to the particular embodiments described; such embodiments may, of course, vary. Further, the terminology used to describe particular embodiments is not intended to be limiting, since the scope of the present invention will be limited only by its claim. [067] Nucleic Acids

[068] The present invention provides recombinant DNA molecules that do not contain sequences derived from viruses that infect eukaryotic cells. These DNA molecules comprise a first sequence operably linked to a second sequence, in which the first sequence comprises a promoter of a gene expressed in the liver and the second sequence encodes a protein that is not a reporter, such that the DNA molecule can be expressed in an animal to produce a protein that is functionally active in vivo. The promoter of the first sequence can comprise a transcription start site, such as those included in the listing of 5' untranslated regions in the tables, and may provide specificity to the expression of the functionally active protein. For example, the promoter may confer a particular spatial or temporal expression pattern to the animal protein.

[069] The DNA molecule can also comprise a third sequence element that is operably linked to the first and second sequence elements, and that comprises an intron sequence. This third sequence can be a heterologous intron or an intron comprising a nucleotide sequence of the invention, including those listed in the tables. The DNA molecule can comprise an attB sequence, a promoter, an intron, a bpA sequence, and an attP sequence arranged as shown in Figure 5 and constructed using the restriction sites shown in Figure 5.

[070] The present invention also provides nucleic acids that are related to the above DNA molecules and derived by processes such as transcription, splicing, processing, mutation, synthesis, chemical modification, or recombinant modification. Non-limiting embodiments or fragments of such nucleic acid molecules include genes or gene fragments, exons, introns, rnRNA, tRNA, rRNA, siRJNA, ribozymes, antisense nucleotide sequences, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe sequences, and primer sequences. Such nucleic acid molecules or fragments thereof include splice variants of an mRNA; naturally occurring nucleotide sequences, for example DNA or RNA; or synthetic analogs of purines and pyrimidines, as known in the art. Synthetic analogs may demonstrate increased stability under assay conditions. A nucleic acid molecule can also comprise modified nucleotides, such as methylated nucleotides or nucleotide analogs. [071 ] The present invention further relates to variants of the herein described nucleic acid molecules, which may occur naturally, such as a natural allelic variant, such as one of several alternate forms of a gene occupying a given locus on a chromosome of an organism, as described in, for example, Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using mutagenesis techniques known in the art. [072] Such variants include those produced by nucleotide substitutions, deletions, or additions. The substitutions, deletions, or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the non-coding regions may be such that the properties or activities of the gene regulatory elements, or portions thereof, are substantially the same. Alterations in the coding regions may produce conservative or non- conservative amino acid substitutions, deletions or additions. These may take the form of silent substitutions, additions, or deletions which do not alter the properties or activities of the encoded proteins, or portions thereof.

[073] The present invention also relates to such polynucleotides which hybridize to the herein described sequences if there is at least 91%, at least 92%, or at least 95% identity between the sequences. The present invention relates to polynucleotides which hybridize under stringent conditions to the herein described polynucleotides. Stringent conditions generally include conditions under which hybridization will occur only if there is at least 95%, or at least 97% identity between the sequences. For example, overnight incubation at 42⁰C in a solution containing 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O. Ix SSC at about 65°C, constitute stringent conditions.

[074] The polynucleotides which hybridize to the polynucleotides shown in the Tables and Sequence Listing can retain substantially similar biological function or activity as the shown polynucleotide. Using the information provided herein, such as the nucleotide sequences set forth in the Tables and Sequence Listing, nucleic acid molecules of the invention may be obtained using standard cloning procedures. [075] Promoters of Liver-Expressed Genes

[076] The liver plays a central role in metabolism and the production of serum proteins. It has two circulatory systems, a systemic circulation that brings oxygenated blood directly from the heart and a portal circulation that brings nutrients from the intestines. In addition, it has a system of ducts that transports metabolites, drugs toxins and other materials out of the liver via bile into the small intestine. Thus, liver cells are readily accessible via the bloodstream, and particles injected into the blood circulation can quickly reach the liver. In addition, the liver plays a role in many inborn errors of metabolism and acquired disorders such as hemophilia, hypercholesterolemia, and hepatitis.

[077] Because of these features, those in the art recognize that inducing liver cells to express a heterologous gene of interest provides a useful system for studying the function of the protein encoded by that gene of interest. This system allows for studying the function of proteins that remain within the liver cells, and of proteins that are secreted into the circulation. Moreover, expression of therapeutic genes in the liver permits treatment strategies for diseases requiring delivery and functional expression of missing or defective genes in the liver, or requiring delivery and functional expression of missing or defective genes encoding secreted proteins. For example, supplying the gene encoding the LDL surface receptor to hepatocytes may lower blood LDL and cholesterol levels (Hussain et al., Annu. Rev. Nutr. 19:141-72 (1999)). Examples of therapeutic secreted genes include, but are not limited to, hormones, insulin, interleukins, interferons, growth factors, and erythropoietin (Bonin-Debs et al., Expert Opin. Biol. Ther. 4:551-8 (2004)). Liver-expressed genes also provide a model system for studying protein function throughout an organism, as such genes and their gene products can also be expressed and functional in other parts of the body. Similarly, the use of gene expression in the liver to supply therapeutic gene products of interest provides wide-ranging therapeutic benefits, as the gene products can be expressed or transported throughout the organism, thereby allowing them to exert their therapeutic effects throughout the organism. [078] The invention provides promoters of liver-expressed genes that are utilized in the disclosed nucleic acids to express genes of interest in the liver of injected animals, including humans. Many of these promoters are derived from a large family of liver-expressed genes, the cytochrome P450 gene family. Cytochrome P450 proteins are a group of heme-thiolate monooxygenases that perform a variety of oxidation reactions, often as part of the body's mechanism to dispose of harmful substances by making them more water-soluble. Much of the body's total mass of cytochrome P450 proteins is found in the liver, specifically, in the microsomes of hepatocytes. There are over a thousand different cytochrome P450 proteins. However, only 49 genes and 15 pseudogenes have been sequenced in humans, hi humans, cytochrome P450 3A4 is the most prevalent cytochrome P450 protein in the body, and it is expressed in an inducible fashion.

[079] Stable expression of proteins from recombinant naked DNA under the control of cytochrome P450 promoters has not yet been achieved. The instant invention provides recombinant nucleic acids that operably link the promoter sequences of cytochrome P450 genes (such as cytochrome P450 3A4) in such a fashion to a gene of interest that allows for the expression of the gene of interest in the liver and any other site where the promoter is active, following injection of the naked nucleic acids into an animal. Exemplary promoters of cytochrome P450 genes used in the nucleic acids of the invention are listed in the tables.

[080] In addition, promoters of other liver-expressed genes are provided, including, but not limited to, genes encoding c-jun; jun-b; c-fos; c-myc; serum amyloid A; apolipoprotein B editing catalytic subunit; liver regeneration factors, such as LRF-I; signal transducers; activators of transcription, such as STAT-3; serum alkaline phosphates (SAP); insulin-like growth factor-binding proteins, such as IGFBP-I; cyclin Dl; active protein- 1; CCAAT enhancer core binding protein; ornithine decarboxylase; phosphatase of regenerating liver- 1; early growth response gene-1; hepatocyte growth factors; hemopexin; insulin-like growth factors (IGF), such as IGF-I and IGF-2; hepatocyte nuclear family 1; hepatocyte nuclear family 4; hepatocyte Arg-Ser-rich domain-containing proteins; glucose 6-phosphatase; acute phase proteins, such as serum amyloid A and serum amyloid P (SAA/SAP); steroid hydroxylases; leukotriene hydroxylases; fatty acid hydroxylases; desmolase; peptidyl isomerases; and sterol demethylases. Such exemplary promoter sequences of the invention are listed in the tables.

[081] To enhance the levels and duration of expression from the above described promoters, a compatible enhancer sequence and/or locus control region can be coupled with the promoter. The invention provides an enhancer and/or locus control region operably linked with a promoter and gene sequence of interest, hi an embodiment, a liver-specific locus control region from the apolipoprotein E (ApoE)- encoding gene locus is employed upstream of the promoter. Associating a promoter with a locus control region can prevent transcriptional silencing by chromosomal proteins, thereby maintaining a transcriptionally active chromatin structure. Also, inherent enhancer elements, which contain binding sites for various transcription factors, can help drive high-level expression from the promoter. In addition, the locus control region may contain matrix attachment regions that increase the nuclear retention of a DNA molecule of interest. Various locus control regions, including those from the CD2, beta-globin, and ApoE loci, have been tested in gene therapy vectors (Ellis and Pannell, Clin. Genet. 59:17-24 (2001); Kowolik et al., J. Virol. 75:4641-4648 (2001); Miao et al., Hum. Gene Ther. 14:1297-1305 (2003)). Alternatively, instead of a locus control region, the above described promoters can be coupled with a suitable enhancer sequence. Enhancers provide a common mechanism of transcriptional activation in eukaryotic cells, as described, for example, by Guarente, Cell 52:303-5 (1988). [082] Introns

[083] hi an embodiment, the invention provides a third operably linked sequence element. Introns may enhance the in vivo expression of the polypeptide encoded by the second sequence element. The ability of an intron to enhance the expression of a transcriptional unit relates to several factors, including the intron' s positive effects on the stability of the transcript, more efficient assembly into spliceosome complexes, and increased synthesis of a mature transcript. Moreover, some introns also comprise enhancer sequences, which, in addition, increase the rate of transcription. However, not all introns seem to share these positive effects on gene expression. Several reports, for example, by Garcia de Veas Lovillo et al., Eur. J. Biochem. 270:206-12 (2003), have observed inhibitory effects of introns on gene expression from artificially assembled expression cassettes. Intron sequence, size, location within the expression cassette, and the presence of enhancer elements can determine the effects of an intron on gene expression, and are properly considered when incorporating an intron into a gene therapy vector. [084] hi an embodiment, nucleic acids of the invention include the first intron, or fragments thereof, of the human Factor IX gene. This intron has previously been shown to increase the in vivo expression of a linked sequence, for example a sequence encoding Factor IX (Wang et al., Hum. Gene Ther. 7:1743-56 (1996)). Inclusion of the intron in a transgenic mouse also strongly increased transgene expression (Jallat et al., EMBO J. 9:3295-301 (1990)). Furthermore, this intron has been reported to mediate high level expression from a minicircle expression cassette, as shown by Chen et al., MoI. Ther. 8:495-500 (2003). [085] The invention also provides intron sequences that differ from previously tested intron sequences and that are compatible with the promoters used herein. Hence, in an embodiment, nucleic acids of the invention include introns derived from cytochrome P450 genes, including those listed in the Tables and the Sequence Listing. These introns are compatible with the liver-expressed promoters described above and, together with such promoters, they facilitate sustained and/or high-level expression of a gene of interest in the liver. [086] Minicircle Producing PIasmids and Minicircle Vectors

[087] A limiting factor for the use of non- viral gene therapy vectors is the often relatively short duration of transgene expression in vivo. After an initially high level of transgene expression following the delivery of the vector to the target cells, transgene expression is often reduced to non-therapeutic levels within a relatively short time, such as one or two weeks, even though the vector DNA persists much longer in the target cells. This phenomenon has been explained by transcriptional silencing of vector DNA, mediated by the bacterial DNA sequences that are generally contained in the vector DNA for purposes of convenient selection and amplification in bacteria (Chen et al., Gene Ther. 11:856-864 (2004)).

[088] More recently, the development of a vector system was reported that circumvents the problem of transcriptional silencing by removing the bacterial sequences from the vector prior to its use in vivo. This vector system is described in Chen et al., MoI. Ther. 8:495-500 (2003), and U.S. Pat. App. No. 2004/0214329 Al. In brief, an expression cassette for a gene of interest is flanked by attachment sites for a recombinase, which is expressed in an inducible fashion in a portion of the vector sequence outside of the expression cassette. Upon induction of recombinase expression, the vector DNA is recombined, resulting in two distinct circular DNA molecules. One of the circular DNA molecules is relatively short, forming a minicircle that comprises the expression cassette for the gene of interest. This minicircle DNA is devoid of any bacterial DNA sequences. The second circular DNA sequence contains the remaining vector sequence, including the bacterial sequences and the sequence encoding the recombinase. The minicircle DNA can be isolated and purified, and then administered as a gene therapy vector in vivo. Sustained and high- level expression of a gene of interest was reported in mice injected with such purified minicircle DNA (Chen et al., MoI. Ther. 8:495-500 (2003)). Expression levels from the minicircle DNA were significantly higher than from the unrecombined vector DNA, consistent with the loss of the bacterial silencing effect. [089] In an embodiment, the nucleic acids of the invention comprise the herein disclosed novel expression cassettes in minicircle-producing DNA vectors. These nucleic acids feature unique combinations of the provided promoters, genes of interest, and introns. Embodiments of the expression cassettes include combinations of promoters and introns derived from the human cytochrome P450 genes, such as those listed in the Tables and Sequence Listing. The use of these sequence elements from the strongly liver-expressed cytochrome P450 genes in minicircle vectors has not been previously described.

[090] In an embodiment of the invention, minicircle DNA vectors are prepared as described by Chen et al., MoI. Ther. 8:495-500 (2003). Briefly, plasmids similar to pBAD.φC31.hFTX and pBAD.φC31.RHB, with the same or different genes of interest, are used to transform E. coli. Following recombination, the E. coli produce a minicircle comprising an expression cassette with the gene of interest, as described in more detail herein. Minicircle DNA vectors of the invention can also be produced from other recombinases, for example, lambda and ere. [091] Expression cassettes of the invention may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs can include a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. These cassettes can be used for gene therapy. Purified expression cassettes of the invention express the transgene persistently more sustained than plasmids comprising expression cassettes and bacterial DNA (Chen et al., Gene Ther. 11:856-864 (2004)).

[092] The minicircle-producing plasmids may include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culturing in E. coli and other bacteria.

[093] The minicircle producing plasmids may include at least one origin of replication. Such origins of replication allow for the multiplication of the vector in a suitable host cell which can be either a eukaryotic or a prokaryotic cell. Origins of replication are known in the art, as described, for example, in Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).

[094] Minicircle DNA vectors of the invention are less labor-intensive to produce than purified linear vectors. They are also safer than linear vectors, which have detectable and higher levels of integration into liver host chromosomal DNA compared to circular plasmids (Chen et al., MoI. Ther. 8:495-500 (2003)). [095] Specific embodiments of the provided nucleic acids are described in the Examples and Figures. The provided nucleic acids have improved properties for reproducible, sustained, high-level expression of genes of interest and for cost- effective, easy production of vector DNA suitable for in vivo administration. Thereby, the invention provides a vector system that addresses important unmet needs for in vivo gene expression systems, as, for example, applied to gene therapy or drug development. In particular, the invention provides an improved in vivo gene expression system. In an embodiment, the system expresses proteins in vivo in a high-throughput manner, thereby facilitating the fast evaluation of secreted protein function. Furthermore, the provided in vivo expression system is ideally suited to study in vivo dynamics of fusion proteins, to test the function of single-chain antibodies, to co-express two or more molecules, and to express tagged proteins to identify their target cells in vivo.

[096] In an embodiment, the invention provides a DNA molecule comprising a promoter and an intron in a configuration in which the promoter sequence is entirely upstream of the intron sequence and the sequence encoding the gene of interest can be easily inserted on either side of the intron without splitting it (Figure 5). This configuration allows genes of interest to be more easily and routinely inserted into the DNA molecule to obtain expression-competent vectors and provides that the genes of interest can be expressed in vivo in a high throughput manner. [097] Host Cells

[098] The invention also provides recombinant host cells comprising a nucleic acid molecule described herein. The host cell can be a prokaryotic or eukaryotic cell. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above- described host cells are known in the art.

[099] To identify the host cells of the invention, the invention provides as selectable markers genes that confer a phenotype on a cell expressing the marker. Generally, a selectable marker allows the selection of transformed cells based on their ability to thrive in the presence or absence of a chemical or other agent that inhibits an essential cell function. Suitable markers, therefore, include genes coding for proteins which confer drug resistance or sensitivity thereto, impart color to, or change the antigenic characteristics of those cells transfected with a molecule encoding the selectable marker, when the cells are grown in an appropriate selective medium. For example, selectable markers include cytotoxic markers and drug resistance markers, whereby cells are selected by their ability to grow on media containing one or more of the cytotoxins or drugs; auxotrophic markers by which cells are selected for their ability to grow on defined media with or without particular nutrients or supplements, such as thymidine and hypoxanthine; metabolic markers for which cells are selected, for example, their ability to grow on defined media containing the appropriate sugar as the sole carbon source, and markers which confer the ability of cells to form colored colonies on chromogenic substrates or cause cells to fluoresce. [0100] Introduction of a nucleic acid of the invention containing a selectable marker into a host cell can be effected by calcium phosphate transfection, DEAE- dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, J., et al. (2001) Molecular Cloning, A Laboratory Manual. 3^rd ed. Cold Spring Harbor Laboratory Press. [0101] The host cells of the invention include prokaryotic hosts cells for use, for example, for the amplification of the herein described plasmid constructs. Plasmids can be amplified in E. coli (DH5α) grown in LB medium and purified using MAXI prep columns (Qiagen, Mississauga, Ontario, Canada). To quantify, plasmids can be subsequently diluted in, for example, 50 mM Tris-HCl pH 7.4, and absorbencies can be measured at 260 nm and 280 nm. Plasmid preparations with A_26o/A₂₈o ratios between about 1.75 and about 2.00 are suitable for further use. [0102] The invention also includes eukaryotic host cells for use, for example, to express a protein of interest for examination of its biological activity in vitro, preliminary to the injection of the DNA construct in vivo. The polypeptides may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. [0103] Expression of Polypeptides

[0104] The invention provides nucleic acids comprising therapeutic genes that may have therapeutic effects upon entry into a cell. These effects may be mediated following transcription (for example, by an anti-sense nucleic acid) or following expression of the nucleic acid as a protein. A therapeutic effect of a protein can be accomplished by the protein remaining within the cell, remaining attached to the cell membrane, or by being secreted and dissociated from the cell where it can enter the interstitial space, extracellular matrix, and/or vascular system. Hence, the protein encoded by the second sequence element of the nucleic acids of the invention can be a secreted protein, a transmembrane protein, an extracellular or intracellular fragment of a transmembrane protein, or an intracellular protein. In addition, the second sequence element can encode a protein from various types of animals, including humans and mice, as well as from plants, fungi, or bacteria.

[0105] Secreted proteins that can be therapeutic include, but are not limited to, hormones, cytokines, growth factors, clotting factors, anti-proteases, angiogenic proteins (for example, vascular endothelial growth factor and fibroblast growth factors), antiangiogenic proteins (for example, endostatin and angiostatin), and other proteins present in the blood. Transmembrane proteins that can be therapeutic include, but are not limited to, receptors, transporters, channels, and signal transduction proteins. Intracellular proteins that can be therapeutic proteins include, but are not limited to, signal transduction proteins, transcription factors, translation factors, kinases and other enzymes.

[0106] Examples of polypeptides that can be expressed in the practice of the invention include, but are not limited to, cytokines and growth factors, such as interleukins 1-18, interferons, lymphokines, hormones, Regulated on Activation, Normal T Expressed and Secreted (RANTES), lymphotoxin-β, Fas ligand, flt-3 ligand, ligand for receptor activator of NF-kappa B (RANKX), soluble receptors, TNF-related apoptosis-inducing ligand (TRAIL), CD40 ligand, Ox40 ligand, 4-1BB ligand (and other members of the TNF family), thymic stroma-derived lymphopoietin, stimulatory factors, for example, granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), inhibitory factors, mast cell growth factor, stem cell growth factor, epidermal growth factor, growth hormone, tumor necrosis factor (TNF), leukemia inhibitory factor (LIF), oncostatin- M, hematopoietic factors such as erythropoietin and thrombopoietin, and splice variants of any of these. [0107] Descriptions of some proteins that can be expressed according to the invention may be found in, for example, Human Cytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993) and The Cytokine Handbook (A. W. Thompson, ed.; Academic Press, San Diego Calif.; 1991). [0108] Receptors for any of the aforementioned proteins may also be expressed according to the invention, including, for example, both forms of tumor necrosis factor receptor (referred to as p55 and p75), interleukin-1 receptors (type 1 and 2), interleukin-4 receptor, interleukin-15 receptor, interleukin-17 receptor, interleukin-18 receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK), receptors for TRAIL, and receptors that comprise death domains, such as Fas or apoptosis-inducing receptor (AIR).

[0109] Other proteins that can be expressed according to the invention include, but are not limited to, cluster of differentiation antigens (referred to as CD proteins), for example, those disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference; Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD molecules disclosed in subsequent workshops. Examples of such molecules include CD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligand and CD40 ligand). Several of these are members of the TNF receptor family, which also includes 41BB and OX40; the ligands are often members of the TNF family (as are 4- IBB ligand and OX40 ligand); accordingly, members of the TNF and TNFR families can also be expressed using the present invention. [0110] Enzymatically active proteins that can be expressed according to the invention include, but are not limited to, metalloproteinase-disintegrin family members, various kinases (including streptokinase and tissue plasminogen activator as well as death associated kinase containing ankyrin repeats, and IKR 1 and 2), TNF- alpha converting enzyme, and numerous other enzymes. Ligands for enzymatically active proteins can also be expressed by applying the instant invention. [0111] The invention can also be applied to the expression of various types of recombinant proteins, including, but not limited to, immunoglobulin molecules or portions thereof, and chimeric antibodies (antibodies having a human constant region coupled to a murine antigen binding region) or fragments thereof. Numerous techniques are known by which DNA encoding immunoglobulin molecules can be manipulated to yield DNAs capable of encoding recombinant proteins such as single chain antibodies, antibodies with enhanced affinity, or other antibody-based polypeptides (see, for example, Larrick et al., Biotechnology 7:934-938, 1989; Reichmann et al., Nature 332:323-327, 1988; Roberts et al., Nature 328:731-734, 1987; Verhoeyen et al., Science 239:1534-1536, 1988; and Chaudhary et al., Nature 339:394-397, 1989).

[0112] The invention also provides for coexpression of more than one polypeptide. Coexpressed polypeptides may interact with one another. For example, co-expressed light chain and heavy chain polypeptides of an antibody may interact with each other. Co-expressed polypeptides may also modulate the level of expression of another. Also by way of example, one may modulate the processing, such as the folding, of another. Ahn et al., Toxicol. Lett. 153:267-272 (2004), have reported a high yield of cytochrome P450 1 A2 (CYP 1 A2) expression in E. coli by coexpressing CYP 1 A2 with Hsp70, a molecular chaperone known to assist proteins to fold correctly.

[0113] In vivo Modulation

[0114] In vivo expression of the nucleic acids of the invention leads to the production of encoded polypeptides or antisense molecules that modulate a physiological process. This modulation may encompass an increase or a decrease, a stimulation, inhibition, or a blockage in a measurable cellular activity, when compared to a suitable control. This modulation may also encompass an increase or a decrease in the level or activity of an endogenous mRNA or polypeptide of interest, when compared to a suitable control.

[0115] Moreover, the expressed modulators of the invention may act as agonists or antagonists, interfering with the binding or activity of endogenous polypeptides or polynucleotides. Such modulators, include, for example, polypeptide variants, whether agonist or antagonist; antibodies, whether agonist or antagonist; soluble receptors, usually antagonists; and antisense nucleic acids, usually antagonists. In some embodiments, an expressed modulator is an antibody specific for a subject "target" polypeptide. Modulation may include the recruitment of other molecules that directly effect the modulation. For example, an expressed antibody that modulates the activity of a cell surface receptor may bind to the receptor and fix complement, thereby activating the complement cascade and resulting in lysis of the cell. An expressed molecule which modulates a biological activity of a target polypeptide or polynucleotide increases or decreases the activity or binding at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 80%, or at least about 2-fold, at least about 5-fold, or at least about 10-fold or more, when compared to a suitable control. [0116] Expression of Variant and Mutant Polypeptides

[0117] The invention provides nucleic acids for the expression of proteins that were engineered to improve or alter the characteristics of any polypeptide. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can show desirable properties, such as enhanced activity or increased stability. [0118] For many proteins, including the extracellular domain of a membrane- associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993), reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino-terminal amino acid residues were missing. [0119] However, even if deletion of one or more amino acids from the N- terminus or C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature from of the protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- terminal residues of a complete protein retains such immunologic activities can be determined by routine methods known in the art.

[0120] Many examples of biologically functional, terminal deletion muteins are known. For instance, interferon gamma increases in activity as much as ten fold when 8-10 amino acid residues are deleted from the carboxy terminus of the protein, see, for example, Dobeli et al, J Biotechnology, 7:199-216 (1988). [0121] hi addition to terminal deletion forms of a protein, it also will be recognized by one of ordinary skill in the art that some amino acid sequences of a polypeptide can be varied without significant effect on the structure or function of the protein. Such mutants include internal deletions, insertions, inversions, repeats, and type substitutions. These can be selected according to general rules known in the art, so as to have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-10 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections, or screens, to identify sequences that maintain functionality.

[0122] The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, et al., supra, and the references cited therein. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and Gm, exchange of the basic residues Lys and Arg, and replacements between the aromatic residues Phe and Tyr.

[0123] In contrast, replacing amino acids can also change the functionality of a protein in a desired way. For example, Ostade et al., Nature 361 :266-8 (1993), describe mutations resulting in a change of selectivity of the binding of a ligand, TNF-α, to cell surface receptors, such that mutated TNF-α selectively binds to only one of the two known types of TNF receptors. Sites that are critical for ligand- receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffmity labeling, for example, Smith et al., J MoI. Biol, 224:899-904 (1992), and de Vos et al., Science 255:306-12 (1992). [0124] Use of Secretory Leader Sequences

[0125] The invention provides nucleic acids for the expression of secreted proteins, which are capable of being directed to the endoplasmic reticulum (ER), secretory vesicles, or the extracellular space as a result of a secretory leader, signal peptide, or leader sequence, as well as proteins released into the extracellular space without necessarily containing a signal sequence. If a secreted protein is released into the extracellular space, it may undergo extracellular processing to a mature polypeptide. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

[0126] Typically, a secretory leader sequence used in the invention directs certain proteins to the ER. The ER separates the membrane-bound proteins from other proteins. Once localized to the ER, proteins can be further directed to the Golgi apparatus for distribution to vesicles, including secretory vesicles; the plasma membrane; lysosomes; and other organelles.

[0127] Proteins targeted to the ER by a secretory leader sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space via exocytosis. Exocytosis can occur constitutively or upon receipt of a triggering signal. In the latter case, the proteins may be stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a linker holding the protein to the membrane.

[0128] As demonstrated in U.S. Patent No. 60/647,013, in order for some secreted proteins to express and secrete in larger quantities, a secretory leader sequence from another, different, secreted protein is desirable. Employing heterologous secretory leader sequences may be advantageous and a resulting mature amino acid sequence of the secreted polypeptide is not altered as the secretory leader sequence is removed in the ER during the secretion process.

[0129] Identified secretory leader sequences that can be used in the practice of the invention include, for example, those derived from interleukin-9 precursor, T cell growth factor P40, P40 cytokine, triacylglycerol lipase, pancreatic precursor, somatoliberin precursor, vasopressin-neurophysin 2-copeptin precursor, beta- enoendorphin-dynorphin precursor, complement C2 precursor, small inducible cytokine A14 precursor, elastase 2A precursor, plasma serine protease inhibitor precursor, granulocyte-macrophage colony-stimulating factor precursor, interleukin-2 precursor, interleukin-3 precursor, alpha-fetoprotein precursor, alpha-2-HS- glycoprotein precursor, serum albumin precursor, inter-alpha-trypsin inhibitor light chain, serum amyloid P-component precursor, apolipoprotein A-II precursor, apolipoprotein D precursor, colipase precursor, carboxypeptidase Al precursor, alpha-sl casein precursor, beta casein precursor, cystatin SA precursor, follitropin beta chain precursor, glucagon precursor, complement factor H precursor, histidine- rich glycoprotein precursor, interleukin-5 precursor, alpha-lactalbumin precursor, Von Ebner's gland protein precursor, matrix Gla-protein precursor, alpha- 1 -acid glycoprotein 2 precursor, phospholipase A2 precursor, dendritic cell chemokine 1, statherin precursor, transthyretin precursor, apolipoprotein A-I precursor, apolipoprotein C-III precursor, apolipoprotein E precursor, complement component C8 gamma chain precursor, serotransferrin precursor, beta-2-microglobulin precursor, neutrophils defensins 1 precursor, triacylglycerol lipase gastric precursor, haptoglobin precursor, neutrophils defensins 3 precursor, neuroblastoma suppressor of tumorigenicity 1 precursor, small inducible cytokine Al 3 precursor, CD5 antigen-like precursor, phospholipids transfer protein precursor, dickkopf related protein-4 precursor, elastase 2B precursor, alpha- 1 -acid glycoprotein 1 precursor, beta-2- glycoprotein 1 precursor, neutrophil gelatinase-associated lipocalin precursor, C- reactive protein precursor, interferon gamma precursor, kappa casein precursor, plasma retinol-binding protein precursor, and interleukin-13 precursor. [0130] Expression of Fusion Molecules

[0131] The invention provides nucleic acids for the expression of fusion proteins that combine heterologous polypeptide moieties, resulting in chimeric polypeptides. Such fusion proteins may facilitate purification or detection and show an increased half-life in vivo. Suitable moieties for derivatization of a heterologous polypeptide include, for example, the constant domain of immunoglobulins, all or part of human serum albumin (HSA); fetuin A; fetuin B; a leucine zipper domain; a tetranectin trimerization domain; mannose binding protein (also known as mannose binding lectin), for example, mannose binding protein 1; and an Fc region, as described herein and further described in U.S. Patent No. 6,686,179, and U.S. Patent Application Nos. 60/589,788 and 60/654,229. Methods of making fusion proteins are well-known to the skilled artisan.

[0132] Examples of heterologous proteins with increased half-life are provided by chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP 0 394 827; Traunecker et al., Nature 331 :84-6 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric proteins or protein fragments, for example, as described by Fountoulakis et al., J. Biochem. 270:3958-64 (1995).

[0133] In another example, the short plasma half-life of unmodified interferon alpha makes frequent dosing necessary over an extended period of time, in order to treat viral and proliferative disorders. Interferon alpha fused with HSA has a longer half life and requires less frequent dosing than unmodified interferon alpha; the half- life was 18-fold longer and the clearance rate was approximately 140 times slower (Osbom et al., J. Pharmacol. Exp. Ther. 303:540-8 (2002)). Interferon beta fused with HSA also has favorable pharmacokinetic properties; its half life was reported to be 36-40 hours, compared to 8 hours for unmodified interferon beta (Sung et al., J. Interferon Cytokine Res. 23:25-36 (2003)). A HS A-interleukin-2 fusion protein has been reported to have both a longer half-life and favorable biodistribution compared to unmodified interleukin-2. This fusion protein was observed to target tissues where lymphocytes reside to a greater extent than unmodified interleukin 2, suggesting that it exerts greater efficacy (Yao et al., Cancer Immunol. Immunother. 53:404-10 (2004)). [0134] In yet another example, the Fc receptor of human immunoglobulin G subclass 1 has been recombinantly linked to two soluble p75 tumor necrosis factor (TNF) receptor molecules. This fusion protein has been reported to have a longer circulating half-life than monomeric soluble receptors, and to inhibit TNFα-induced proinflammatory activity in the joints of patients with rheumatoid arthritis (Goldenberg, Clin. Ther. 21 :75-87 (1999)). This fusion protein has been used clinically to treat rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis (Nanda and Bathon, Expert Opin. Pharmacother. 5:1175- 86 (2004)).

[0135] Moreover, peptide moieties and/or purification tags may be added to the polypeptides to facilitate purification or detection, engender secretion or excretion, improve stability, or for other reasons. Such moieties are added by familiar and routine techniques in the art. Suitable tags include, for example, V5, HISX6, HISX8, avidin, and biotin. As described in Gentz et al., Proc. Natl. Acad. Sd. USA 86:821-4 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the hemagglutinin HA tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767-78 (1984)). [0136] Expression of Antibodies

[0137] The invention provides nucleic acids for the expression of antibodies, including antibodies corresponding to isolated natural polyclonal or monoclonal antibodies, altered antibodies, chimeric antibodies, and hybrid antibodies (see, for example, Winter et al., Nature 349:293-9 (1991), and U.S. Patent No. 4,816,567); F(ab')₂ and F(ab) fragments; Fv molecules (noncovalent heterodimers; see, for example, Inbar et al., Proc. Natl. Acad. Sd. USA 69:2659-62 (1972), and Ehrlich et al., Biochem. 19:4091-6 (1980)); single chain Fv molecules (sFv) (see, for example, Huston et al., Proc. Natl. Acad. Sd. USA 85:5879-83 (1980)); dimeric and trimeric antibody fragment constructs; minibodies (see, for example, Pack et al., Biochem. 31:1579-84 (1992), and Cumber et al., J. Immunology 149B:120-6 (1992)); humanized antibody molecules (see, for example, Riechmann et al., Nature 332:323-7 (1988) and Verhoeyan et al., Science 239:1534-6 (1988)); bispecific antibodies (see, for example, U.S. Patent No. 6,010,902 and U.S. Patent Application No. 2002/0155604); and any functional fragments obtained from such molecules, wherein such fragments retain specific binding.

[0138] Techniques developed for the production of chimeric antibodies

(Morrison et al., Proc. Natl. Acad. ScL USA 81:851-5 (1984); Neuberger et al., Nature 312:604-8 (1984); Takeda et al., Nature 314:452-4 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity have been described. Sequences encoding chimeric antibodies, which are antibodies in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, for example, humanized antibodies, and insertion/deletions relating to cdr and framework regions, are suitable for use in the invention. [0139] The invention includes the expression of humanized antibodies, i.e., those with mostly human immunoglobulin sequences. Humanized antibodies generally refer to non-human immunoglobulins that have been modified to incorporate portions of human sequences. A humanized antibody may include a human antibody that contains entirely human immunoglobulin sequences. [0140] Antibodies expressed according to the invention specifically bind to their respective antigen(s); they may display high avidity and/or high affinity to a specific polypeptide, or more accurately, to an epitope of an antigen. Antibodies expressed according to the invention may bind to one epitope, or to more than one epitope. They may display different affinities and/or avidities to different epitopes on one or more molecules.

[0141] It will be appreciated that Fab and F(ab')₂ and other fragments of antibodies may be expressed by the nucleic acids of the invention according to the methods disclosed herein. Such fragments correspond to those produced by proteolytic cleavage of natural antibodies, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). Sequences encoding such fragments can be produced through the application of recombinant DNA technology. Humanized chimeric monoclonal antibodies are suitable for in vivo use in humans. Such humanized antibodies can be produced using genetic constructs derived from hybridoma cells producing monoclonal antibodies of interest. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 0 171 496; Morrison et al., EP O 173 494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature, 314:268 (1985). Methods for producing single chain antibodies are also known in the art. See, for review, Smith et al., J. Clin. Pathol. 57:912 (2004); Pastan et al., U.S. Pat. No. 6,099,842; Solomon et al., U.S. Pat. No. 6,630,584.

[0142] In an example for the application of antibody expression, the expressed antibodies are neutralizing antibodies that provide therapy for diseases such as cancer and proliferative disorders. Neutralizing antibodies can specifically recognize and bind a protein involved in disease progression, for example, in a bodily fluid or the extracellular space, thereby modulating the biological activity of the protein. For example, neutralizing antibodies specific for proteins that play a role in stimulating the growth of cancer cells can be useful in modulating the growth of cancer cells. Similarly, neutralizing antibodies specific for proteins that play a role in the differentiation of cancer cells can be useful in modulating the differentiation of cancer cells. It is apparent to one skilled in the art that there are many other applications of expressing antibodies via the nucleic acids of the invention. [0143] In Vivo Delivery Methods

[0144] A nucleic acid can be delivered to a cell to express an exogenous nucleotide sequence, to modulate expression of an endogenous nucleotide sequence, and/or to express a specific physiological characteristic not naturally associated with the cell. In vivo gene expression depends on introduction of foreign DNA into a cell, for example by transfection. Transfection in vivo can be achieved by both viral delivery techniques and non- viral delivery techniques. A non- viral delivery technique provided by the invention is the introduction of naked DNA into an animal. The DNA molecules of the invention are free of sequences derived from viruses that infect eukaryotic cells. The DNA molecules of the invention can also be free of transfection agents. Transfection agents bind to or complex with oligonucleotides or polynucleotides, and mediate their entry into cells. Examples of transfection agents include cationic liposomes and lipids, polyamines, polyethylenimine, and polylysine complexes.

[0145] In some embodiments, the invention provides for transfection of nucleic acids via a hydrodynamics-based procedure, such as, for example, the hydrodynamic tail vein injection method, as described, for example, by Zhang et al., Hum. Gene Ther. 10:1735-7 (1999), and U.S. Pat. No. 6,627,616. This method has been successfully used to transfect cells in vivo with a gene of interest. The invention also provides for the manipulation of the level of gene expression by controlling the amount and frequency of intravascular DNA administration. The term "intravascular" refers to a route of administration in which a nucleic acid is placed within a vessel that is connected to a tissue or organ within the body of an animal. Within the cavity of the vessel, a bodily fluid flows to or from a body part. Examples of bodily fluids include blood, lymphatic fluid, and bile. Examples of vessels include arteries, veins, lymphatics, and bile ducts. The intravascular route includes delivery of nucleic acids through the tail vein or iliac artery of a rodent, for example, a mouse or a rat (Kameda et al., Biochem. Biophys. Res. Commun. 309:929-36 (2003); Jiang et al., Biochem. Biophys. Res. Commun. 289: 1088-92 (2001)).

[0146] In an embodiment, the invention provides a method of intravascular injection of naked DNA, wherein the permeability of the blood vessel is increased. Permeability of the blood vessel can be increased by increasing the pressure against the vessel wall, whereby increasing the pressure is achieved by increasing the volume of the fluid within the vessel. Hence, the permeability of the blood vessel is increased by injecting a relatively large volume within a relatively short time period. [0147] The injection volume depends on the size of the injected animal (U.S.

Pat. No. 6,627,616). Generally, injection volumes are approximately 0.03 ml/g - 0.1 ml/g or more. Suitable volumes for injecting DNA molecules of the invention into the tail rein of mice are about 1.0, 1.5, and 2.0 ml. Suitable volumes for injecting DNA molecules of the invention into the iliac arteries of rats are about 6-35 ml or more. Suitable volumes for injecting DNA molecules of the invention into the blood vessels of primates, including humans, are about 70-200 ml or more. [0148] Similarly, the speed of injection depends in part on the volume to be injected, the size of the injected vessel, and the size of the injected animal (U.S. Pat. No. 6,627,616). For example, a volume of 1-3 ml can be injected into mice within 5- 15 seconds. In another example, a volume of 6-35 ml can be injected into rats within 7-20 seconds. In another example, a volume of 7-200 ml can be injected into monkeys within 120 seconds or less.

[0149] Permeability of the blood vessels can also be increased, for example, by biologically active molecules. Suitable biologically-active molecules include papaverine, histamine, and vascular endothelial growth factor (U.S. Pat. No. 6,627,616). Intravascular pressure can also be increased, for example, by increasing the osmotic pressure in the vessel. Compositions suitable for increasing the intravascular pressure include hypertonic salts, sugars, and polyols (U.S. Pat. No. 6,627,616).

[0150] The hydrodynamic injection of nucleic acids of the invention provides a method in which the composition is injected intravascularly and under pressure. Such hydrodynamic injection of nucleic acids of the invention provides a method of inducing sustained expression of a protein in an animal by providing a composition, injecting the composition into the animal, and allowing expression of the protein. This method can be used to obtain expression and protein activity that are detectable on 5 to 15 days, 16 to 25 days, 26 to 35 days, or 36 to 45 days post-injection. [0151] In an embodiment, DNA molecules of the invention are delivered to an animal using commercially available products (Mirus Bio Corp., Madison, WI). [0152] Therapeutic compositions and formulations

[0153] The nucleic acids of the present invention may be employed in combination with a suitable pharmaceutical carrier or excipient to comprise a pharmaceutical composition for administration by injection. Such compositions comprise a therapeutically effective amount of the nucleic acids and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient can be saline, e.g., phosphate buffered saline, or a buffer. In an embodiment, the carrier or excipient is neither a liposome nor a DNA complexing agent. The composition may also comprise a nucleotide sequence encoding a protein that enhances expression and/or folding of the protein of interest encoded by the second sequence element of a nucleic acid of the invention. The formulation should suit the mode of administration. [0154] The compositions will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual subject, the site of delivery of the nucleic acid composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The effective amount of the nucleic acids of the invention for purposes herein is thus determined by such considerations.

[0155] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the nucleic acids of the present invention may be employed in conjunction with other therapeutic compounds.

[0156] The pharmaceutical compositions may be administered by hydrodynamic injection in a manner deemed most appropriate for the specific purpose. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. [0157] Alternatively, nucleic acids of the invention may also be administered in aerosol formulations via inhalation, or in powder form intranasally or via inhalation, as conventional in the art. The nucleic acids of the invention may also be administered by intramuscular jet injection as described (Furth et al., Anal. Biochem. 205:365-8 (1992)). Furthermore, the nucleic acids of the invention can be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (Tang et al., Nature 356:152-4 (1992)), where gold microprojectiles are coated with the DNA, then bombarded into skin cells. [0158] A wide variety of pharmaceutically acceptable excipients are known in the art (Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3^rd ed., Pharmaceutical Press (2000)) and available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, and the like, are available to the public.

[0159] Animals

[0160] In another aspect, the invention provides an animal injected with one or more of the compositions described above. Animals of the invention include, but are not limited to, humans, mice, rats, guinea pigs and other rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, avians, mammalian farm animals, mammalian sport animals, and mammalian pets. Animals of the invention may be referred to as "subjects," "individuals," and "patients," terms used interchangeably herein.

[0161] Therapeutic Applications

[0162] The invention provides a method of delivering a nucleic acid of the invention to hepatocytes in vivo by intravascular injection. Hepatocytes divide slowly, thus the DNA molecules persist extrachromosomally for extended periods of time. Methods of the invention do not require the expression and purification of the expressed gene products from bacteria or cultured cells for in vivo application and do not require continuous administration. Proteins expressed by methods of the invention have native post-translational modifications, which can be important for their biological activity.

[0163] The invention may be used in diagnosing, prognosing, preventing, treating, and developing treatments for many different disorders in an animal, including a human. This encompasses preventing a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, or preventing a disease from recurring in a subject who has been diagnosed as having had the disease previously. This also encompasses treatment methods that inhibit a disease, i.e., arrest its development; or relieve a disease, i.e., cause its regression; restore or repair a lost, missing, or defective function; and/or stimulate an inefficient process. The methods of the invention are also suitable for identifying and validating drug targets.

[0164] In an embodiment, a nucleic acid of the invention is delivered to hepatocytes in vivo to express an intracellular, transmembrane or secreted polypeptide which may induce, inhibit, or otherwise affect liver disorders, including, but not limited to hepatitis, alcohol toxicity, bile duct disorders, dyslipoproteinemias, diabetes, obesity, sepsis, inflammation, and other liver disorders. [0165] In another embodiment, a nucleic acid of the invention is delivered to hepatocytes in vivo to express a secreted polypeptide which may act locally or systemically to affect a disorder in any organ or anatomical site that is accessible via the vascular system. Such disorders include, but are not limited to, cancer, immune diseases, such as an autoimmune disease or an inflammatory disease, ischemic diseases, infectious diseases, bone diseases, cardiovascular diseases, and neural diseases. As an example, nucleic acids of the invention can direct the expression of polypeptides that are useful in a variety of settings for the treatment of animal cancer by inhibiting the multiplication of tumor cells or cancer cells. [0166] An effective amount of a nucleic acid of the invention is administered to the host. In an embodiment, the nucleic acid is administered at a dosage sufficient to produce a desired result. The dosage will, of course, vary depending upon the polypeptide expressed and the disease targeted. Administration is generally by injection and often by injection to a localized area. The frequency of administration will be determined by the duration of exogenous polypeptide expression and by the care given based on patient responsiveness.

[0167] Effective dosages can be readily determined by one of ordinary skill in the art through trials establishing dose response curves. Those of skill will readily appreciate that dose levels of the administered nucleic acid can vary as a function of the specific polypeptide expressed, the severity of the symptoms, and the susceptibility of the subject to side effects. [0168] Vaccine Therapy

[0169] The invention provides a method for prophylactic or therapeutic treatment of a subject needing or desiring such treatment by providing a vaccine, that can be administered to the subject. A vaccine may comprise one or more nucleic acids of the invention, in form of a nucleic acid vaccine composition, expressing one or more polypeptides whereby the polypeptides correspond to antigens specific for, for example, cancer, other proliferative disorders, inflammatory, immune, metabolic, bacterial, or viral disorders. Administration of a vaccine comprising a minicircle DNA described herein, leads to persistent expression and release of the therapeutic immunogen over a period of time.

[0170] In some embodiments, a nucleic acid-based vaccine expresses a molecule that is involved in the control of cell proliferation. A resulting immune response can cause the inhibition of undesirable cell proliferation. Therefore, expression of such molecules can be useful for treating disorders that involve abnormal cell proliferation, including, but not limited to, cancer, psoriasis, and scleroderma.

[0171] In some embodiments, the vaccine can be a cancer vaccine, and the expressed polypeptide can be a tumor antigen. Over 1770 tumor antigens have been identified to date (Yu and Restifo, J. CHn. Invest. 110:289-294, 2002). The expressed tumor antigen can be, for example, an extracellular fragment of a polypeptide that is expressed on the surface of cancer cells. The expressed tumor antigens may be altered such that the antigens are more highly antigenic than in their native state. These alterations address the need in the art to overcome the poor in vivo immunogenicity of most tumor antigens by enhancing tumor antigen immunogenicity via modification of epitope sequences (Yu et al., J. CHn. Invest. 110:289-294 (2002)). [0172] In other embodiments, antibodies themselves can be expressed as antigens by anti-idiotype nucleic acid-based vaccines. That is, expressing an antibody to a tumor antigen stimulates B cells to make antibodies to that antibody, which in turn recognize the tumor cells.

[0173] Besides stimulating anti-tumor immune responses by inducing humoral responses, vaccines of the invention can also induce cellular responses, including stimulating T-cells that recognize and kill tumor cells directly. For example, nucleic acid-based vaccines of the invention encoding tumor antigens can be used to activate the CD8⁺ cytotoxic T lymphocyte arm of the immune system. [0174] In some embodiments, the vaccines activate T-cells directly, and in others they enlist antigen-presenting cells to activate T-cells. Killer T-cells are primed, in part, by interacting with antigen-presenting cells, for example, dendritic cells. In some embodiments, the nucleic acid molecules of the invention enter antigen-presenting cells, which in turn display the encoded tumor-antigens that contribute to killer T-cell activation. [0175] Whether a particular molecule and/or therapeutic regimen of the invention is effective in reducing unwanted cellular proliferation, for example, in the context of treating cancer, can be determined using standard methods. For example, the number of cancer cells in a biological sample such as blood, a biopsy sample, and the like, can be determined. The tumor mass can be determined using standard radiological or biochemical methods.

[0176] Vaccines comprising genetic material, such as nucleic acids of the invention, can be given directly, either alone, in conjunction with other molecules, or in combination with other conventional or unconventional therapies. For example, nucleic acids expressing immunogenic molecules can be combined with other molecules that have a variety of antiproliferative effects, or with additional substances that help stimulate the immune response, such as adjuvants or cytokines.

MODES FOR CARRYING OUT THE INVENTION

[0177] The invention provides a recombinant DNA molecule comprising a first sequence which comprises a promoter of a liver-expressed gene operably linked to a second sequence which encodes a protein other than a reporter gene, wherein the DNA molecule does not comprise sequences of a virus that infects eukaryotic cells and wherein the DNA molecule can be expressed in vivo in an animal to produce a protein which is functionally active in the animal. This DNA molecule may further comprise a third sequence operably linked to the first and second sequences, wherein the third sequence comprises an intron sequence. This intron may be heterologous and/or may comprise a nucleotide sequence selected from SEQ. ID. NO.:245 to SEQ. ID. NO.:394.

[0178] In an embodiment, DNA molecules of the invention may encode a secreted protein, for example, an extracellular fragment of a transmembrane protein or a naturally secreted protein, such as a growth factor. In another embodiment, DNA molecules of the invention may encode a transmembrane protein, for example, a growth factor receptor. In another embodiment, DNA molecules of the invention may encode an intracellular protein, for example, an intracellular fragment of a transmembrane protein or a naturally intracellular protein, such as a signal transduction molecule or transcription factor. [0179] The invention provides DNA molecules which are expressed in vivo in a human individual or in an animal, for example a mouse or another rodent. The invention also provides DNA molecules which encode a human protein or a mouse protein. The invention further provides DNA molecules which encode a protein that is not alpha- 1 -antitrypsin.

[0180] In another embodiment, the invention provides a composition comprising at least one DNA molecule as described above and a pharmaceutically acceptable carrier. This carrier, in an embodiment, is not a liposome. This carrier, in another embodiment, is not a DNA complexing agent. Carriers of the invention may be saline or a buffer, for example, phosphate buffered saline. [0181] In another embodiment, these compositions may further comprise a nucleotide sequence that encodes a protein that enhances expression and/or folding of the protein encoded by the second sequence element of the DNA molecule. [0182] In another embodiment, DNA molecules of the invention comprise a promoter which includes a transcription start site, wherein the transcription start site is a start sequence selected from SEQ. ID. NO.:1 to SEQ. DD. NO.:122 or a fragment of any of these.

[0183] The invention also provides a DNA molecule as described above that further comprises an origin of replication. This DNA molecule may further comprise a nucleotide sequence encoding a reporter gene and/or an antibiotic resistance gene. The invention also provides a recombinant host cell comprising a DNA molecule as described above. These may be prokaryotic or eukaryotic. The invention further provides an animal injected with a composition comprising at least one DNA molecule of the invention and a pharmaceutically acceptable carrier. This animal may be a laboratory animal, for example, a mouse.

[0184] m another aspect, the invention provides a method of inducing sustained and/or high-level expression of a protein in an animal comprising (a) providing a composition comprising at least one DNA molecule of the invention and a pharmaceutically acceptable carrier; (b) injecting the composition into the animal; and (c) allowing expression of the protein. This method may be practiced by injecting the composition under pressure. It may also be practiced by injecting the composition intravascularly, for example, intravenously. In an embodiment, the duration of the injecting is about five seconds. In an embodiment, the injected animal is a mouse and the composition has a volume of about 1, 1.5, or 2 ml. The invention provides that expression and protein activity are detectable on 5 to 15 days, 16 to 25 days, 26 to 35 days, or 36 to 45 days post-injection.

[0185] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. AU such modifications are intended to be within the scope of the claims appended hereto. [0186] With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values. Moreover, the invention also encompasses ranges excluding either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

[0187] Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art to which this invention belongs. One of ordinary skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. Further, all publications mentioned herein are incorporated by reference.

[0188] It must be noted that, as used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a subject polypeptide" includes a plurality of such polypeptides and reference to "the agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. [0189] Further, all numbers expressing quantities of ingredients, reaction conditions, % purity, polypeptide and polynucleotide lengths, and so forth, used in the specification and claims, are modified by the term "about," unless otherwise indicated. Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits, applying ordinary rounding techniques. Nonetheless, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors from the standard deviation of its experimental measurement.

[0190] The specification is most thoroughly understood in light of the references provided herein, all of which are hereby incorporated by reference in their entireties. The disclosures of the patents and other references cited above are also hereby incorporated by reference.

[0191] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

INDUSTRIAL APPLICABILITY

[0192] The invention provides a safe, reproducible, and easy-to-use in vivo gene delivery and expression system for testing genes in a high-throughput manner in animal models, including disease models. It facilitates the functional evaluation of proteins, including secreted and engineered proteins, in vivo and aids in the identification of novel therapeutic molecules. The invention also provides a safe in vivo gene delivery system for therapeutic gene therapy.

EXAMPLES

[0193] The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. [0194] Example 1: Constructs Containing the Human Cytochrome P450

3A4 Promoter

[0195] Plasmid constructs containing the human cytochrome P450 3A4 promoter were made using pBlueScript (Stratagene; La Jolla, CA) as a backbone and with standard recombinant DNA methodology. The resulting constructs are represented schematically in Figure 1. The top construct contained the cytochrome P450 3A4 promoter operably linked to a monkey erythropoietin (EPO) gene. The bottom construct contained the same promoter operably linked to lacZ. Both constructs also contained poly A tails. Similar constructs may be made with an intron, for example, a heterologous intron, located between the promoter and the functional gene of interest. Suitable introns include those shown in Tables 2 and 3. The insertion of such an intron can enhance in vivo expression of a sequence of interest. ^r

[0196] Example 2. Promoter-Driven EPO Expression Following Tail

Vein Injection

[0197] The DNA constructs described in Example 1 were purified using a

Qiagen Plasmid Maxi Kit (Qiagen, Inc.; Valencia, CA) according to manufacturer's instructions and resuspended at a concentration of 25 μg/ml in saline. Two groups of mice were injected in the tail veins with the naked DNA constructs. Each mouse in the first group was injected with 2 ml of the EPO construct, and each mouse from the second group was injected with 2 ml of the lacZ construct (Figure 1). The duration of each injection was approximately 5-8 seconds.

[0198] On days 1, 3, and 7 following injection of the constructs described in

Example 1, serum EPO levels of three anesthetized mice from each group were determined by ELISA according to manufacturer's directions (R&D Systems; Minneapolis, MN). Figure 2 shows the EPO values of each mouse at days 1, 3, and 7 following injection. All of the mice injected with the lacZ construct had undetectable levels of EPO in their blood serum (bgal#l, bgal#2, and bgal#3). All of the mice injected with the EPO construct had detectable levels of EPO on day 1, which continued to increase through days 3 and 7 (EPO#1, EPO#2, and EPO#3), demonstrating overexpression of the introduced EPO construct. [0199] In a separate experiment, serum EPO levels were measured by ELISA in mice injected with the EPO or lacZ constructs, as described above, on days 8, 11, and 13 post-injection. Serum EPO levels of individual mice were determined by ELISA. Mice injected with the EPO construct had high levels of EPO, and overexpressed levels were sustained at least until post-injection day 13. Penetrance was observed to be 100%, since all injected mice had elevated EPO levels. [0200] Example 3: Cytochrome P450 3A4 Promoter-Driven LacZ

Expression

[0201] On day 11 after injection, the livers of mice injected with either the

EPO construct or the lacZ construct were harvested. As negative controls, the livers of wild-type (WT), uninjected, mice were also harvested. The livers were fixed in paraformaldehyde, then whole-mount stained with 1 mg/ml X-gal using a kit from Specialty Media (Phillipsburg, NJ). The results are shown in Figure 3. The dark punctate staining shows the expression of lacZ protein in the livers of the mice injected with the lacZ construct. No X-gal staining was observed in either the mice injected with the EPO construct or in wild-type uninjected mice. [0202] Example 4: Cytochrome P450 3A4 Promoter-Driven EPO expression

[0203] On day 13 after injection, blood was drawn from mice injected as described in Example 2 with either the lacZ or the EPO construct, and sent to Quality Clinical Lab (Glendale, CA) for determination of hematocrit levels. Figure 4a shows that, consistent with the overexpression of EPO, the average hematocrit levels of three mice injected with the EPO constructs was significantly higher than that of the three mice injected with the lacZ constructs.

[0204] Three mice injected as described above with either the lacZ or the EPO construct were sacrificed on day 14 and their spleens were isolated. EPO over- expression caused obvious splenomegaly in mice that had been injected with EPO constructs, as shown in Figure 4b. Extramedullary hematopoeisis was also observed in these animals. [0205] Example 5: Production of Mini-circle DNA Vectors [0206] A master construct for the production of mini-circle DNA molecules was prepared in accordance with standard recombinant DNA methodology (Figure 5). The ApoE locus control region (LCR), the alpha-antitrypsin promoter, the human Factor JX intron, a multiple cloning site, and a bovine polyadenylation sequence were amplified by polymerase chain reaction (PCR) and ligated head-to-tail into a cloning vector in the above order. The combination of ApoE LCR and alpha-antitrypsin promoter was designed to drive the expression of genes of interest in liver cells. The human Factor DC intron was positioned downstream of the promoter to enhance the expression of genes of interest. The intron was positioned in a way that it is flanked on each side by a unique cloning site that can be used to insert genes of interest. The unique cloning sites are Nhel and Sfil restriction sites. The entire expression cassette was excised from the cloning vector and transferred into a mini-circle producer plasmid described by Chen et al., Hum. Gene Ther. 16:126-31 (2005). [0207] Alternative master constructs are prepared in similar fashion, substituting promoters of other liver-expressed genes, such as the cytochrome P450 genes, for the alpha-antitrypsin promoter. These constructs include the transcription start site and may also include 5' untranslated regions. They may optionally include intron and/or enhancer sequences. Minicircles can be purified using a restriction enzyme digestive step followed by ultracentrifugation or by a one-step procedure with commercially available affinity columns, as described by Chen et al., Hum. Gene Ther. 16:126-31 (2005).

[0208] Example 6: Protein Expression Following Tail Vein Injection of

Minicircle DNA

[0209] The cDNA encoding a secreted protein of interest, FGFRl -IHc-Fc

(described in an U.S. Provisional Patent Application filed on January 10, 2006), was inserted into the Sfil unique cloning site of the master minicircle producer construct described in Example 5 by standard recombinant DNA methodology. The minicircle DNA molecules were produced according to the procedure described in Chen et al., Hum. Gene Ther. 16:126-31 (2005). Minicircle DNA molecules were then injected into the tail veins of three mice using the hydrodynamic injection method described above at a DNA concentration of 15 μg/ml in saline, injecting two ml per mouse in 5- 8 seconds. [0210] Serum samples were collected from the mice at days 2, 9, 16, 24, 30,

37, and 44 days post-injection and assayed for FGFRl -lHc-Fc by ELISA, as shown in Figure 6. The injected minicircle DNA molecules expressed high levels (10-50 μg/ml) of FGFRl -IIIc-Fc. The mice demonstrated sustained protein expression for at least 44 days post-injection.

[0211] Example 7: Evaluating Protein Function with Sequences of the

Invention

[0212] Minicircle constructs containing sequences encoding two or more proteins, or two or more constructs each containing sequences encoding a protein, can also be injected intravascularly into animals. Interactions between molecules can then be studied in vivo. In addition, differences in the function of a protein injected alone and that of a protein injected along with other proteins can be determined using methods of invention.

[0213 ] Gene expression and biodistribution of constructs containing sequences encoding animal proteins of interest may be monitored by, for example, according to the methods of Kobayashi et al., J Pharmacol. Exp. Ther. 297:853-60 (2001), following intravascular injection. After allowing the protein to be expressed, the function of a protein, for example, a secreted protein, can be determined by any method known in the art. Changes in physiological markers or changes manifest in organ histology can be examined. The function of a protein that remains in liver cells can be evaluated by observing changes in liver structure and/or function. For example, the function of single-chain antibodies can be determined by studying immune markers. Methods of the invention can be used to inject mice with various constructs, as described herein, and to quickly determine the function of many, varied types of proteins.

[0214] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. Table 1. Annotation of Liver-Expressed Genes

Table 2. Sequence Identification Numbers

Table 3. Intron Coordinates

Claims

1. A recombinant DNA molecule comprising a first sequence which comprises a promoter of a liver-expressed gene operably linked to a second sequence which encodes a protein other than a reporter gene, wherein the DNA molecule does not comprise sequences of a virus that infects eukaryotic cells and wherein the DNA molecule can be expressed in vivo in an animal to produce a protein which is functionally active in the animal.

2. The DNA molecule of claim 1, further comprising a third sequence operably linked to the first and second sequences, wherein the third sequence comprises an intron.

3. The DNA molecule of claim 2, wherein the intron is heterologous.

4. The DNA molecule of claim 2, wherein the intron comprises a nucleotide sequence, or a fragment thereof, selected from the group consisting of SEQ. ID. NO.:245 to SEQ. ID. NO.:394.

5. The DNA molecule of claim 2, wherein the intron is positioned downstream of the promoter and upstream of a cloning site, so as to enhance the expression of genes inserted into the cloning site.

6. The DNA molecule of claim 1 , wherein the protein is secreted.

7. The DNA molecule of claim 1, wherein the protein is a transmembrane protein or an extracellular fragment of a transmembrane protein.

8. The DNA molecule of claim 1 , wherein the protein is an intracellular protein or an intracellular fragment of a transmembrane protein.

9. The DNA molecule of claim 1 , wherein the animal is a human.

10. The DNA molecule of claim 1, wherein the animal is a mouse.

11. The DNA molecule of claim 1 , wherein the protein is a human protein.

12. The DNA molecule of claim 1, wherein the protein is a mouse protein.

13. The DNA molecule of claim 1 , wherein the protein is not alpha- 1 -antitrypsin.

14. A composition comprising at least one DNA molecule of claim 1 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein the carrier is not a liposome or DNA complexing agent.

16. The composition of claim 14, wherein the carrier is saline or a buffer.

17. The composition of claim 16, wherein the carrier is phosphate buffered saline.

18. The composition of claim 14, further comprising a nucleotide sequence that encodes a protein that enhances expression and/or folding of the protein encoded by the second sequence of the DNA molecule.

19. The DNA molecule of claim 1 , wherein the promoter comprises a transcription start site, and the transcription start site is selected from those included in SEQ. ID. NO.:1 to SEQ. DD. NO.: 122.

20. The DNA molecule of claim 1, wherein the promoter comprises a promoter sequence, or a fragment thereof, selected from the group consisting of SEQ. ID. NO.:1 to SEQ. ID. NO.: 122.

21. The DNA molecule of claim 1, further comprising an origin of replication.

22. The DNA molecule of claim 21 , further comprising a nucleotide sequence encoding a reporter gene.

23. The DNA molecule of claim 21, further comprising a nucleotide sequence encoding an antibiotic resistance gene.

24. A recombinant host cell comprising the DNA molecule of claim 1.

25. The host cell of claim 24, wherein the cell is a prokaryotic cell.

26. The host cell of claim 24, wherein the cell is a eukaryotic cell.

27. An animal injected with the composition of claim 14.

28. The animal of claim 27, wherein the animal is a laboratory animal.

29. The animal of claim 27, wherein the animal is a mouse.

30. A method of inducing sustained and/or high-level expression of a protein in an animal comprising:

(a) providing the composition of claim 14;

(b) injecting the composition into the animal; and

(c) allowing expression of the protein.

31. The method of claim 30, wherein the composition is injected under pressure.

32. The method of claim 30, wherein the composition is injected intravascularly.

33. The method of claim 32, wherein the composition is injected intravenously.

34. The method of claim 33, wherein the composition is injected into the tail vein of a rodent.

35. The method of claim 30, wherein the duration of the injecting is about five seconds.

36. The method of claim 30, wherein the animal is a mouse and the composition has a volume of about 1, 1.5, or 2 ml.

37. The method of claim 30, wherein the animal is a human and the composition has a volume of about 7, 20, 60, or 200 ml.

38. The method of claim 30, wherein expression and protein activity are detectable at least 4 days, at least 5-15 days, at least 16-25 days, at least 26- 35 days, or at least 36-45 days post-injection.