Maintenance, method, and use of DNA fragment in eucaryotic cell

Abstract

To provide a method for reducing enzymatic destruction of a linear DNA fragment transfected into a cell.SOLUTION: A method for enhancing a function of a DNA construct once the construct is introduced into an eucaryotic cell includes: (a) providing an eukaryotic expression vector which encodes expression of protein that can protect an introduced DNA construct from enzymatic digestion; (b) transfecting the protein expression vector into an eucaryotic cell; and (c) introducing a DNA construct to which DNA protection protein binds into the eucaryotic cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the fields of biotechnology, molecular biology and cell biology, more particularly to the protection of DNA constructs introduced into eukaryotic cells from the activity of cellular DNases; the modification of eukaryotic cells to enhance the production of viral vectors; more particularly to the modification of packaging cells and their use to generate batches of recombinant viral vectors. [Background technology]

[0002] Among the most commonly used vectors for transporting genetic material into human cells are adenoviruses (Ad).

[0003] Adenoviruses have been isolated from a number of different species, and over 100 different serotypes have been reported. The overall organization of the adenovirus genome is conserved among serotypes, such that certain functions are similarly arranged. The adenovirus genome is a linear, nonsegmented, double-stranded DNA, approximately 34-43 kilobase pairs (kbp) in length (size varies among groups). The adenovirus genome is flanked on both sides by (left and right) inverted terminal repeats (LITR and RITR), which are essential for adenovirus replication. The viral infection cycle is divided into early and late stages. In the early stage, the virus is uncoated, and transcription of the early gene regions E1-E4 is activated after the genome is transported to the nucleus.

[0004] Early region 1 (E1) contains two transcriptional regions designated E1A and E1B. The E1A region (sometimes called the immediate early region) encodes two major proteins involved in the modification of the host cell cycle and the activation of other viral transcriptional regions. The E1B region encodes two major proteins, 19K and 55K, which, through different pathways, prevent the induction of apoptosis caused by the activity of the E1A protein. In addition, the E1B-55K protein is required for selective transport of viral mRNA at late stages and inhibit host protein expression. Early region 2 (E2) is also divided into the E2A and E2B regions, which together encode three proteins, the DNA-binding protein, the viral DNA polymerase (Pol), and the pre-terminal protein (Tp), all of which are involved in the replication of the viral genome. The E3 region encodes several proteins that are not required for replication in vitro but that disrupt the host's defense mechanisms against viral infection. The E4 region encodes at least six proteins involved in several different functions in viral mRNA splicing and transport, host cell mRNA transport, viral and cellular transcription and transformation.

[0005] All late proteins required for viral capsid formation and viral genome packaging are generated from the major late transcription unit (MLTU), which becomes fully active after the onset of viral DNA replication. A complex method of differential splicing and polyadenylation gives rise to more than 15 mRNA species that share a tripartite leader sequence. The early proteins E1B-55K and E4-0rf3 and Orf6 play pivotal roles in regulating the processing and export of late viral mRNAs from the nucleus.

[0006] Packaging of the newly formed viral genome into a preformed capsid is mediated by at least two adenoviral proteins, the late protein 52 / 55K, and the intermediate protein IVa2, through interaction with the viral packaging signal (Ψ) located at the left end of the adenovirus 5 genome. The second intermediate protein, piX, is part of the capsid and is known to stabilize hexon-hexon interactions. In addition, piX has been described to transactivate TATA-containing promoters such as the E1A promoter and the major late promoter (MLP).

[0007] Adenovirus-based vectors and adenovirus packaging cell lines Adenovirus-based vectors have been used as a means to achieve high-level gene transfer into various cell types, as vaccine delivery vehicles, to transfer genes into allogeneic tissue grafts for gene therapy, and to express recombinant proteins in cell lines and tissues that are otherwise difficult to transfect with high efficiency.Currently known systems for packaging adenovirus-based vectors consist of a host cell and a source of adenovirus late genes.

[0008] Currently known host cell lines, such as human embryonic kidney 293 (HEK293), QBI, and PERC6 cells, express only the early (nonstructural) adenovirus (Ad) genes, and not the late (structural) adenovirus genes required for packaging, which are already provided by either the adenovirus vector itself, a helper adenovirus, or an expression plasmid.

[0009] "Gutless" adenoviral vectors have been developed, i.e., vectors that lack all DNA sequences encoding viral proteins. Gutless adenoviral vectors contain only the ends of the viral genome (LITR and RITR), the therapeutic gene sequence, and the normal packaging recognition signal (Ψ), so that this genome can be selectively packaged and released from the cell.

[0010] Helper virus-dependent adenovirus vectors In order to propagate gutless adenoviral vectors, helper adenoviruses (helpers) containing the adenoviral gene packaging recognition signal (Ψ) are required. This helper-dependent system allows the introduction of foreign DNA up to about 32 kb, but the helper virus contaminates the preparation of gutless adenoviral vectors. This replicative contaminating helper virus poses serious problems for gene therapy, vaccine, and transplantation applications because of the replicative virus and because of the host immune response to the adenoviral genes in the helper virus. One approach to reduce helper contamination in this helper-dependent vector system has been to introduce a conditional genetic defect in the packaging recognition signal (Ψ) to make the DNA less likely to be packaged into virions. Gutless adenoviral vectors produced in such systems are still significantly contaminated with the helper virus. If gutless adenoviral gene transfer vectors could be produced without helper virus contamination, toxicity would be further reduced and gene expression in animals would be extended.

[0011] Helper virus-independent adenovirus vector In a more recent approach to the production of gutless adenovirus vectors, the adenovirus genes required for replication and packaging of the adenovirus-derived vector genome are provided by a replication-deficient circular expression plasmid that is deleted for at least the packaging recognition signal (Ψ) and one of the ITRs. This fully deleted helper virus-independent adenovirus (fdAd) vector platform consists of two genetic constructs, the fdAd vector genome module and the fdAd packaging expression plasmid, and packaging cells.

[0012] fdAd vector modules are designed to accommodate transgene constructs up to 33 kb in size. They contain the selected transgene, i.e., the left and right ITRs and packaging signal (Ψ) along with the transgene or transgene construct, and a size-compensating stuffer sequence if necessary to provide vector genome size for optimal packaging.

[0013] The fdAd packaging plasmids are designed to provide the necessary late and early adenoviral genes of different serotypes, species, or animal types of adenovirus. They contain the adenoviral late genes L1, L2, L3, L4, L5 in trans along with the early genes E2 and E4, and are deleted of the packaging signal (Ψ) and at least one ITR.

[0014] Packaging of helper virus-independent adenovirus vectors The fdAd vector module is packaged into adenovirus capsid with the aid of fdAd packaging plasmid. To encapsidate the fdhiAd vector module, the fdhiAd vector genome is released from the plasmid by enzymatic cleavage. As shown in Figure 1, a linear vector genome is generated. The transgene, i.e., the transgene or transgene construct, is flanked by a packaging recognition signal (Ψ) and both ITR, LITR and RITR, optionally together with an inactive stuffer DNA sequence.

[0015] The released linear fdAd vector genome module DNA is transfected into eukaryotic packaging cells together with the DNA of the packaging expression plasmid (Figure 2). The fdhiAd vector module DNA is then replicated in the packaging cells and packaged into the adenovirus capsid encoded by the packaging expression plasmid. The assembled adenovirus vector with the fdAd vector genome can be collected and used. The packaging expression plasmid is not packaged.

[0016] Packaging cells for helper virus-independent adenovirus vectors Packaging of the fdAd vector genome by this method involves the introduction of linear DNA into packaging cells, such as HEK293-derived cells or other cells engineered to express early adenoviral genes, such as the adenoviral E1 gene. In the packaging cells, the linear DNA of the fdhiAd vector genome is exposed to cellular DNases. These DNases can be cellular nucleases that can digest the free ends of linear DNA fragments. This enzymatic digestion reduces the amount of packageable fdAd vector genome, thereby reducing the efficiency of vector packaging and production. Furthermore, linear DNA fragments can be toxic to mammalian cells, causing cell cycle arrest and / or cell death.

[0017] It has been demonstrated that certain terminal sequences of linear DNA fragments can protect linear double-stranded DNA from the activity of certain bacterial and eukaryotic exonucleases. The terminal telomeric sequences of chromosomes are protected by nucleoprotein caps that mask the ends from constitutive exposure to the DNA damage response.

[0018] Other proteins known to bind to certain linear proteins. In the case of adenoviruses, the terminal protein (Tp) precursor is covalently bound to nucleotides in the ITRs of the DNA strand. It remains covalently bound to the 5' end of the viral DNA and is cleaved into mature Tp during virion maturation. Together with the adenovirus DNA polymerase, Tp is responsible for the replication of the adenovirus genome. Phage Tp can also be covalently bound to the viral genome stimulating DNA replication. In both cases, Tp prevents the DNA exonuclease from enzymatically destroying the linear DNA.

[0019] To protect the linear fdAd vector genome during vector packaging, HEK293 cells harboring the adenovirus E1 gene for transcriptional activation were modified by stably transfecting with a plasmid expressing the adenovirus genes encoding terminal protein (Tp) and DNA polymerase (Pol). For this purpose, a eukaryotic expression vector was constructed harboring the Tp and Pol genes from adenovirus 5 under the control of the bidirectional promoter Surfeit1 (Surf1). The design and sequence of the vector are shown in Figure 3.

[0020] Adenoviral Vectors for Gene Therapy and Protein Expression Gene delivery or gene therapy is a promising method for the treatment of acquired and inherited diseases. Numerous genes are being continually cloned and identified whose aberrant expression is associated with life-threatening human diseases. The ability to express such cloned genes in humans will ultimately enable the prevention and / or treatment of many important human diseases, diseases for which treatments are currently inadequate or non-existent.

[0021] Two advances that attempt to overcome the problem of immunity to adenovirus are the use of "gutless" (completely deleted) adenovirus vectors and the use of rare adenovirus serotypes, as well as pseudotyping of the adenovirus hexon. The use of "gutless" adenovirus vectors removes the L3 gene from the therapeutic vector, but the propagation of these "gutless" viruses may still require the presence of a helper adenovirus that contains the L3 gene. Moreover, these helper viruses are significant contaminants in therapeutic formulations of "gutless" adenovirus vectors.

[0022] Using rare adenovirus serotypes or animal adenoviruses can avoid the existing immune problems in some patients who have never been exposed to a given adenovirus.However, because adenovirus hexon protein is highly immunogenic, treatment with minimally modified adenovirus gene delivery vectors based on rare serotypes induces immune responses such as neutralizing antibodies that interfere with the subsequent use of human or animal serotypes or species of adenovirus vectors.This immune response can induce inflammatory responses and interfere with the therapeutic function of the vector.

[0023] Adenoviruses as vaccine vectors Adenoviruses are moving from a means of gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors because they induce both innate and adaptive immune responses in the mammalian host. Currently, adenovirus vectors are being tested as subunit vaccine systems for a number of infectious pathogens, ranging from malaria to HIV-1. In addition, they are being investigated as vaccines against a number of tumor-associated antigens. Thus far, most efforts have focused on vectors derived from human or simian serotypes of adenoviruses for eventual use as human vaccines, while bovine, porcine, and ovine adenoviruses have been investigated for use in veterinary medicine.

[0024] The kinetics of adenovirus gene expression has made the production of true adenovirus packaging cell lines difficult. Expression of the adenovirus early functional transcription region (E1A) gene induces expression of the adenovirus late genes (structural, immunogenic genes) and results in cell killing. Thus, host cells that constitutively express the adenovirus early genes cannot have a "wild-type" adenovirus late cistron. Previous host cells that propagate adenovirus vectors are not "packaging" cells. Specifically, 293, QBI, and PER.C6 cells express only the early (nonstructural) adenovirus genes, not the adenovirus late genes required for packaging. The adenovirus late genes are already provided by either the adenovirus vector itself or by a helper adenovirus. These adenovirus late genes in adenovirus vectors or helper adenoviruses are involved in inflammatory reactions to adenovirus vectors; interfere with immune responses to adenovirus-based vaccines; induce immune non-responsiveness to adenovirus in allogeneic transplant applications; and are involved in contamination of adenovirus-based protein expression. In addition, they are occupying. Summary of the Invention

[0025] The present invention addresses the problem of DNA stability in eukaryotic cells and provides engineered cell lines and their use for the protection of linear DNA fragments of DNA in eukaryotic cells and the production of adenovirus-based gene transfer vectors.

[0026] According to embodiments exemplified herein, eukaryotic packaging cell lines are provided that have been engineered to be permissive for the replication of adenoviral vectors, harboring the adenoviral early region 1 (E1) coding sequence as well as the adenoviral Tp and Pol, both of which are either stably integrated into the cellular genome or transiently expressed within the cell with the aid of a eukaryotic expression vector.

[0027] According to other embodiments exemplified herein, there are provided eukaryotic expression vector systems that drive expression of adenovirus Tp and / or adenovirus Pol genes, which are stably integrated into the cellular genome or transiently expressed in eukaryotic cells.

[0028] According to embodiments exemplified herein, designs are provided for engineering linear DNA fragments to which adenoviral Tp and / or Pol can bind, such that the linear DNA fragments are protected from intracellular DNases found in eukaryotic cells.

[0029] According to embodiments exemplified herein, a system is provided that includes: (a) an adenovirus packaging cell engineered to express adenovirus E1, Tp and / or Pol; (b) an fdAd vector genome model; and (c) a packaging expression plasmid, wherein the fdAd vector genome and packaging construct are transfected into the adenovirus packaging cell to induce encapsidation of the fdAd vector genome in a helper adenovirus-independent manner.

[0030] According to embodiments exemplified herein, adenovirus packaging cells are provided that are engineered to express adenovirus E1, Tp and / Pol and enhance packaging of minimally modified, such as E1 deleted, non-fully deleted adenovirus vectors.

[0031] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows the design of the fdAd vector construct, along with restriction enzyme sites that contain multiple cloning sites (MCS) allowing accommodation of various genes of interest. [Diagram 2] FIG. 1 describes the method of packaging the fdAd vector genome (GreVac module) into adenovirus capsids. The linearized fdAd vector genome is co-transfected with a packaging expression plasmid (pPaCh) into packaging cells (host cells), where the fdAd vector genome is replicated and packaged into the adenovirus capsid encoded by the packaging expression plasmid. [Diagram 3] 1 shows a eukaryotic expression vector designed to drive expression of adenovirus terminal protein and DNA polymerase. Expression of the Tp and Pol genes is driven from the human bidirectional Surfeit1 promoter. [Figure 4] Figure 1 shows transduction of eukaryotic cells with fdAd-derived adenoviral vectors carrying green fluorescent protein (GFP). The degree of green fluorescence was detected by fluorescence microscopy. fdAdGFP vectors were encapsidated by cotransfection of the fdAdGFP vector genome and a packaging expression plasmid into unmodified HEK293 cells. [Diagram 5]The packaging efficiency of unmodified parent HEK293 cells was compared to that of a cloned line of Q7 cells transfected to stably express adenovirus terminal protein and DNA polymerase. The collected packaging fdAdGFP vector was used to transduce eukaryotic cells. The degree of green fluorescence was detected by fluorescence microscopy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present disclosure provides, inter alia, improved packaging cells for encapsidation of adenoviral vector genomes, including but not limited to, the fdAd vector genome, with or without the involvement of a packaging expression plasmid.

[0034] The adenoviral vectors encapsidated by the improved packaging cells are used as gene transfer vectors for gene and protein expression, vaccine development, and immunosuppressive therapy.

[0035] In one embodiment, the adenovirus Tp and Pol genes are obtained from an adenovirus of human serotype 5. In other embodiments, the adenovirus Tp and Pol genes are obtained from adenoviruses of other human serotypes or simian or other animal adenovirus types.

[0036] In other embodiments, the Tp and Pol genes are obtained from other viruses.

[0037] In one embodiment, the fdAd vector genome is derived from an adenovirus of human serotype 5. In other embodiments, the fdAd vector genome is derived from an adenovirus of other human serotypes or simian or other animal adenovirus types.

[0038] In one embodiment, a linear DNA fragment is defined as a double-stranded DNA fragment that is not linked to a closed circular structure. In another embodiment, a linear DNA fragment is defined as a single-stranded DNA fragment that is not linked to a closed circular structure.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are well known and commonly used in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). In general, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0040] Techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout this document (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Units, prefixes, and symbols may be indicated in SI approved format. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes. Unless otherwise defined, software, electrical, and electronic terms used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms, 5th ed., 1993. As used throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings, and are more fully defined by reference to the specification in its entirety:

[0041] The terms "adenovirus" and "adenovirus particle" as used herein include any and all viruses that can be classified as adenovirus, including any adenovirus that infects humans or animals, including all groups, subgroups, and serotypes. Thus, as used herein, "adenovirus" and "adenovirus particle" refer to the virus itself or its derivatives, covering all serotypes and subtypes, and both naturally occurring and recombinant forms. In one embodiment, such adenovirus infects human cells. Such adenoviruses can be wild type or modified in various ways known in the art or disclosed herein. Such modifications include modifications to the adenovirus genome packaged in the particle to generate infectious virus. Such modifications include deletions known in the art, such as deletions in one or more of E1a, E1b, E2a, E2b, E3, or E4 coding regions.

[0042] "Adenovirus packaging cell" or "packaging cell" or "host cell" as understood herein is a cell that can package adenovirus genome or modified genome to produce viral particles. It can provide missing gene products or their equivalents. Thus, the packaging cell can provide the function of complementing the gene deleted in the adenovirus genome, and can package the adenovirus genome into adenovirus particles. The production of such particles requires the production of proteins necessary for genome replication and infectious virus assembly.

[0043] Particles may also require certain proteins that are necessary for viral particle maturation. Such proteins can be provided by vector, packaging construct, or packaging cell. Exemplary packaging cells that can be used to generate packaging cell lines according to the present invention include, but are not limited to, A549, HeLa, MRC5, W138, CHO cells, Vera cells, human embryonic retina cells, or any eukaryotic cells that can be modified to express Tp and / or Pol.

[0044] Some cell lines that can be modified by expression of the adenoviral genes Tp and / or Pol include adipocytes, chondrocytes, epithelial, fibroblasts, glioblastomas, hepatocytes, keratinocytes, leukemia, lymphoblastoid cells, monocytes, macrophages, myoblasts, and neurons. Other cell types include, but are not limited to, cells from primary cell cultures, such as human primary prostate cells, human embryonic retina cells, and human stem cells. Eukaryotic diploid and aneuploid cell lines are included within the scope of the present invention. To generate high-titer stocks of recombinant gene transfer vectors, as adenovirus packaging cells, the cells must be capable of expressing the products of the fdAd vector genome and / or packaging expression plasmids into those products at appropriate levels.

[0045] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence. Transcription and translation of the coding sequence are typically regulated by "control elements" including, but not limited to, transcriptional promoters, transcriptional enhancer elements, Shine and Delagamo sequences, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimizing translation initiation (located 5' to the coding sequence), and translation termination sequences.

[0046] The term "construct" refers to at least one of a fully deleted adenoviral vector construct, a packaging expression plasmid, or any other plasmid and linear DNA designed to drive expression of a particular gene.

[0047] The term "delete" or "deleted" as used herein means to remove, erase, or eliminate.

[0048] As used herein, the term "E1 region" refers to a group of genes present in the adenovirus genome. These genes, including but not limited to E1A and E1B, are expressed during the early stages of viral replication and activate the expression of other viral genes. In one embodiment, the adenovirus packaging cell line of the present disclosure contains the entire coding sequence that constitutes the E1 region.

[0049] The term "terminal protein" as used herein means a protein for which the gene is present in the genome of an adenovirus or another virus.

[0050] As used herein, the term "DNA polymerase" refers to a protein whose function is the replication of DNA sequences. In one embodiment, the DNA polymerase is encoded by an adenovirus.

[0051] In one embodiment, the adenovirus packaging cell line of the present disclosure contains several coding sequences that make up the E1 region (eg, E1A or E1B).

[0052] In one embodiment, the adenoviral packaging cell line of the present disclosure contains coding sequences constituting the terminus and / or polymerase genes.

[0053] The term "expression" refers to the transcription and / or translation of an endogenous gene, transgene, or coding region in a cell.

[0054] The terms "fully deleted adenoviral vector," "fdAd," "gutless," "gut," "mini," "fully deleted," "D," or "pseudo" vector as used herein refer to a linear, double-stranded DNA molecule having inverted terminal repeats (ITRs) spaced approximately 28 to 37 kb apart, a viral packaging signal (\), and at least one DNA insert (all or a fragment of at least one gene of interest).

[0055] Regulation of gene expression can be achieved by one of the following: 1) alteration of gene structure: site-specific recombinases (e.g., Cre based on the Cre-loxP system) can activate gene expression by removing sequences inserted between the promoter and the gene; 2) alteration of transcription: by induction (protection) or relief of inhibition; 3) alteration of mRNA stability by specific sequences or siRNA incorporated into the mRNA; and 4) alteration of translation by sequences of the mRNA.

[0056] fdAd does not contain any viral coding genes. fdAd is also called a "large-capacity" adenovirus because it can accommodate up to 36 kilobases of DNA. Because vector capsids efficiently package only 75-105% of the DNA of the entire adenovirus genome, and therapeutic expression cassettes are usually no larger than 36 kb, a "stuffer" DNA must be used to complete the genome size for encapsidation.

[0057] Certain fdAd require a helper adenovirus that contains the essential adenoviral coding regions and are therefore referred to as "helper-dependent" adenoviruses.

[0058] As used herein, the term "gene expression construct" refers to a promoter, at least a fragment of a gene of interest, and a polyadenylation signal sequence.

[0059] A "gene of interest" may be one that exerts its effect at the RNA or protein level. Examples of genes of interest include, but are not limited to, therapeutic genes, immunomodulatory genes, viral genes, bacterial genes, protein-producing genes, inhibitory RNAs or proteins, and regulatory proteins. For example, a protein encoded by a therapeutic gene can be used to treat a genetic disease, e.g., in the treatment of cystic fibrosis, eDNA encoding the cystic fibrosis transmembrane conductance regulator can be used. Furthermore, a therapeutic gene can exert its effect at the RNA level, e.g., by encoding an antisense message or a ribozyme, an siRNA as known in the art, an alternative RNA splice acceptor or donor, a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the expression level of another gene in a cell (i.e., where gene expression is broadly considered to include all steps from the initiation of transcription to the production of a processed protein), perhaps by mediating, among other things, changes in the rate of mRNA accumulation, changes in mRNA transport, and / or changes in post-transcriptional regulation.

[0060] As used herein, the phrase "gene therapy" refers to the delivery of genetic material of interest (e.g., DNA or RNA) to a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (e.g., a protein polypeptide, peptide, or functional RNA) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, or (poly)peptide of therapeutic value. Examples of genetic material of interest include DNA encoding cystic fibrosis transmembrane conductance regulator (CFTR), factor VIII, low density lipoprotein receptor, beta galactosidase, alpha galactosidase, beta glucocerebrosidase, insulin, parathyroid hormone, and alpha-1-antitrypsin.

[0061] By "gene transfer vector" is meant a composition of DNA constructs capable of transporting genetic material into cells and tissues, including the encapsidated fully deleted adenovirus-based vectors of the present disclosure packaged without helper adenovirus.

[0062] The term "helper-independent" as used herein refers to a method of generating an encapsidated, fully deleted adenovirus-based gene transfer vector that does not require the presence of a helper virus for its replication.

[0063] Adenoviral vectors include "first generation" and "second generation" adenoviral vectors.First generation adenoviral vectors refer to adenoviruses in which exogenous DNA replaces E1 region, or in some cases E3 region, or in some cases both E1 and E3 regions.Second generation adenoviral vectors refer to first generation adenoviral vectors that contain additional deletions in E2 region, E4 region, or any other region, or combination thereof, of the adenoviral genome in addition to E1 and E3 regions.

[0064] As used herein, the term "helper virus" refers to a virus that is used to generate copies of a helper-dependent viral vector that does not have the ability to replicate by itself. The helper virus is used to co-infect cells with the gutless virus, providing the enzymes required for replication of the gutless virus genome and the structural proteins required for assembly of the gutless virus capsid.

[0065] By "immune response" is preferably meant an acquired immune response, such as a cellular or humoral immune response.

[0066] The term "inverted terminal repeat" (ITR) as used herein refers to DNA sequences located at the left and right ends of the adenoviral genome. These sequences are identical to each other but are arranged in opposite directions. The length of the adenoviral inverted terminal repeats varies from about 50 bp to about 170 bp depending on the type of adenovirus or virus or different families or genera. The ITRs may contain several different cis-acting elements necessary for viral growth, such as the core origin of viral DNA replication and enhancer elements for activation of the E1 region. The ITRs may also contain sequences and structures that allow covalent and / or non-covalent binding of viral and / or cellular proteins, such as but not limited to Tp and Pol proteins.

[0067] "In vivo gene therapy" and "in vitro gene therapy" are intended to encompass all past, present, and future variations and modifications of therapies that are commonly known and referred to by those of skill in the art as "gene therapy," including ex vivo applications.

[0068] The term "introduce" as used herein refers to the stable incorporation of gene sequences into cells or the delivery of expression vectors for transient maintenance. A vector can be introduced into a cell by transfection, which typically refers to the insertion of heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which typically refers to the introduction by an infectious agent, i.e., a virus; or transduction, which typically refers to the stable infection of a cell by a virus or the transfer of genetic material from one microorganism to another by a viral agent (e.g., a bacteriophage). As mentioned above, a vector can be a plasmid, a virus, or other vehicle.

[0069] As used herein, the term "linear DNA" refers to a DNA molecule that is not circularized.

[0070] The term "naturally" as used herein means found in nature; in wild form; essentially or inherently.

[0071] The term "nucleic acid" refers to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) that have the essential property of natural nucleotides to hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0072] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous. Enhancers, however, do not have to be contiguous. Linking is accomplished by ligation at convenient restriction enzyme recognition sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0073] The term "packaging expression plasmid" or "packaging expression construct" refers to an engineered plasmid construct of a circular double-stranded DNA molecule, in which the DNA molecule contains at least a subset of the adenoviral late genes (e.g., L1, L2, L3, L4, L5, E2A, and E4) under the control of a promoter. Packaging Expression Plasmids Packaging expression plasmids do not contain a packaging signal sequence (Ψ) and multiple ITRs.

[0074] The packaging expression plasmid is "replicate-incompetent"; the viral genome does not contain sufficient genetic information to permit independent replication alone to produce infectious viral particles within a cell.

[0075] Any subtype, mixture of subtypes, or chimeric adenovirus can be used as the DNA source for generation of the fdAd vector genome and packaging expression plasmids.

[0076] As used herein, the term "packaging signal" refers to a nucleotide sequence present in the viral genome and necessary for the incorporation of the viral genome into the viral capsid during viral assembly. The adenoviral packaging signal is naturally located at the left end downstream of the left inverted terminal repeat and can be represented as "\".

[0077] A "permissive" cell supports viral gene expression and / or viral replication.

[0078] As used herein, the term "plasmid" refers to an extrachromosomal DNA molecule that is separate from chromosomal DNA, often circular and double-stranded.

[0079] The term "polylinker" is used for a short, artificially synthesized strand of DNA that has many unique restriction sites that allow for easy insertion of any promoter or DNA segment. The term "heterologous" is used for any combination of DNA sequences that are not normally found closely related in nature.

[0080] The term "promoter" refers to a regulatory region of DNA that facilitates transcription of a particular gene. Promoters usually contain a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site of a particular coding sequence. Promoters may further contain other recognition sequences, generally located upstream or 5' of the TATA box, called upstream promoter elements, that affect the rate of transcription initiation. "Constitutive promoter" refers to a promoter that allows for continuous transcription of the associated gene in many cell types. "Inducible promoter system" refers to a system that uses regulators (including small molecules such as tetracycline, peptides, and steroid hormones, neurotransmitters, and environmental factors such as heat and osmolarity) to induce or silence genes. Such systems are "analogs" in the sense that the response changes stepwise depending on the concentration of the regulator. Also, such systems are reversible upon removal of the regulator. The activity of these promoters is induced by the presence or absence of biotic or abiotic factors. Inducible promoters are powerful tools in genetic engineering because they can turn on or off the expression of genes operably linked to them at certain stages of development or in particular tissues of an organism.

[0081] The term "proliferate" or "proliferated" as used herein means to reproduce, increase, or increase in number, amount, or degree by any means.

[0082] As used herein, the term "purified" refers to a process by which a substance is purified or made free of foreign, extraneous, or undesirable elements.

[0083] As used herein, the term "regulatory sequence" (also called "regulatory region" or "regulatory element") refers to a promoter, enhancer, or other segment of DNA to which regulatory proteins, such as transcription factors, preferentially bind.

[0084] Regulatory sequences control gene expression and therefore protein expression.

[0085] The term "recombinase" as used herein refers to an enzyme that catalyzes genetic recombination, such as, but not limited to, Cre, Hin, Tre, or FLP recombinase or the CRISPR / Cas-based DNA recombinase system. Recombinase enzymes catalyze the exchange of short pieces of DNA between two long strands of DNA, in particular the exchange of homologous regions between paired maternal and paternal chromosomes.

[0086] The term "restriction enzyme" (or "restriction endonuclease") refers to an enzyme that cuts double-stranded DNA. The term "restriction enzyme recognition site" or "restriction recognition site" refers to a specific sequence of nucleotides recognized by a restriction enzyme as a site for cutting a DNA molecule. These sites are commonly palindromic (because restriction enzymes usually bind as homodimers), but they do not have to be, and a particular enzyme can cut between two nucleotides somewhere within or near its recognition site.

[0087] The term "replication" or "replicating" as used herein means making identical copies of an entity, such as, for example, but not limited to, a virus particle.

[0088] The term "replication defective" as used herein refers to the characteristic of a virus that cannot replicate in natural environment.Replication defective virus is a virus that is deleted one or more of the genes essential for its replication, such as but not limited to the E1 gene of adenovirus.Replication defective virus can be propagated in laboratory in cell line that expresses deleted gene.

[0089] As used herein, the term "stuffer" refers to a DNA sequence that is inserted into another DNA sequence to increase its size. For example, a stuffer fragment can be inserted into the adenovirus genome to increase its size to about 36 kb. A stuffer fragment usually does not code for any protein, nor does it contain regulatory elements for gene expression, such as transcription enhancers or promoters.

[0090] As used herein, the term "target" or "targeted" refers to a biological entity, such as, but not limited to, a protein, a cell, an organ, or a nucleic acid, whose activity can be modified by an external stimulus. Depending on the nature of the stimulus, there may be no direct change in the target or a conformational change in the target may be induced.

[0091] As used herein, a "target cell" can exist as a single entity or may be part of a larger collection of cells. Such a "larger collection of cells" may include, for example, a cell culture (mixed or single), a tissue (e.g., epithelial or other tissue), an organ (e.g., heart, lung, liver, gallbladder, bladder, eye, or other organ), an organ system (e.g., circulatory system, respiratory system, digestive system, urinary system, nervous system, integumentary system, or other organ system), or an organism (e.g., a bird, a mammal, particularly a human, etc.). Preferably, the targeted organs / tissues / cells are of the circulatory system (including but not limited to, e.g., heart, blood vessels, and blood), respiratory system (e.g., nose, pharynx, larynx, trachea, bronchi, bronchioles, lungs, etc.), digestive system (e.g., mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, liver, gallbladder, etc.), urinary system (e.g., kidneys, ureters, bladder, urethra, etc.), nervous system (including but not limited to, e.g., brain and spinal cord, specialized sensory organs such as eyes), and integumentary system (e.g., skin). Even more preferably, the cells are selected from the group consisting of heart, blood vessels, lungs, liver, gallbladder, bladder, eye cells, and stem cells. In one embodiment, the targeted cells are natural stem or progenitor cells, including but not limited to, oocytes or spermatocytes. In one embodiment, the targeted cells are induced stem or progenitor cells.

[0092] The term "transfection" as used herein means introduction into cellular DNA as DNA (e.g., introduction of an isolated nucleic acid molecule or construct of the present disclosure). The packaging cell lines disclosed herein are transfected with a genetic construct that causes expression of a specific viral gene together with at least one of the adenoviral genome or packaging expression vectors. The term "transduction" as used herein means introduction into cellular DNA as DNA or by a genetic transfer vector. The genetic transfer vector can be introduced into the target cell.

[0093] The term "vector" refers to a nucleic acid that is used to infect a host cell and into which a polynucleotide can be inserted. An expression vector allows for transcription of a nucleic acid inserted therein. Some common vectors include, but are not limited to, plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. The term "vector" can also refer to an element that aids in the transport of genes from one location to another.

[0094] As used herein, the term "viral DNA" refers to the sequences of DNA found in virus particles.

[0095] As used herein, the term "viral genome" refers to the entirety of DNA found in a virus particle that contains all the elements necessary for viral replication. The genome is replicated during each cycle of viral replication and transmitted to viral progeny.

[0096] The term "virion" as used herein means a virus particle. Each virion consists of genetic material within a protective protein capsid.

[0097] The term "wild type" as used herein means the typical form of an organism, strain, gene, protein, nucleic acid, or characteristic occurring in nature. Wild type refers to the most common phenotype in a natural population. The terms "wild type" and "naturally occurring" are used interchangeably.

[0098] The following examples illustrate various aspects of the present invention. Of course, it should be understood that the examples merely illustrate certain embodiments of the invention and do not limit the scope of the invention, which is defined by the claims appended at the end of this specification.

[0099] Although the disclosure has been described in detail, it will be apparent that modifications and variations are possible without departing from the scope of the appended claims. EXAMPLES

[0100] The following non-limiting examples are provided to further illustrate the present disclosure.

[0101] Example 1 Engineering of terminal protein and DNA polymerase expression vectors An expression vector was generated that drives expression of the Tp and Pol genes of human serotype 5 adenovirus (Figure 3 and SEQ ID NO:1). A bidirectional expression cassette carrying both the adenovirus terminal protein (Tp) and DNA polymerase (Pol) genes along with a puromycin selection marker was engineered by moving them into the multiple cloning site of pUC57 (GeneScript).

[0102] The resulting Tp / Pol vector is engineered using a forced cloning strategy to incorporate defined restriction enzyme recognition sites. The Tp / Pol expression vector, pUTp / Pol, is constructed as follows: 1. Vector DNA (1-431) and (7576-9860) belong to the cloning vector pUC57 (complete sequence Y14837). 2. A DNA cassette was synthesized and transferred into the pUC57 cloning vector. It was composed of the following components: (i) The polyadenylation site of the Tp gene (vector DNA 440 to 505) corresponds to the “artificial” sequence. (ii) The adenovirus Tp gene (vector DNA506 to 2,521) corresponds to nucleotides 8,583 to 10,590 of wild-type adenovirus human serotype 5 (AC000008). (iii) The Surfeit bidirectional promoter (vector DNA 2,522 to 2,689) corresponds to nucleotides 37,174 to 37,293 of the genomic sequence of human chromosome 9q34 (AC002107). (iv) The DNA polymerase (Pol) gene (vector DNA 2,690 to 6,287) corresponds to nucleotides 8,785 to 5,197 of wild-type adenovirus human serotype 5 (AC000008). (v) The polyadenylation site of the Pol gene (vector DNA 6,296 to 6,414) corresponds to the “artificial” sequence. and (vi) The puromycin selection marker (vector DNA 6,423 to 7,575) corresponds to nucleotides 231,893 to 231,290 of human herpesvirus 5 (AD169-BAC isolate, complete sequence AC146999).

[0103] In other embodiments, expression vectors are produced that have the Tp and / or Pol genes behind a constitutive or inducible eukaryotic promoter, such as, but not limited to, the cytomegalovirus (CMV) immediate early promoter, the simian vacuolating virus 40 (SV40), the elongation factor 1a (EFla) promoter, the human ubiquitin C gene (Ubc) promoter, the human b-actin promoter, or the tetracyline response element (TRE) promoter.

[0104] In other embodiments, expression vectors are generated that have both Tp and Pol genes separated by an internal ribosome entry site (IRES), such as, but not limited to, those obtained from poliovirus, rhinovirus, hepatitis virus, or human immunodeficiency virus.

[0105] In other embodiments, the expression vectors carrying the Tp and / or Pol genes also carry a positive or negative eukaryotic selection marker, such as, but not limited to, the following drugs: puromycin, hygromycin, G418 / neomycin, or bleomycin.

[0106] In other embodiments, expression vectors carrying the Tp and / or Pol genes are used to transfect cells with the aid of transfectant reagents such as, but not limited to, calcium phosphate, lipids, or polyethyleneimine.

[0107] In other embodiments, expression vectors carrying the Tp and / or Pol genes are used to transfect cells such that the genes are transiently maintained within the cells.

[0108] In other embodiments, expression vectors carrying the Tp and / or Pol genes are used to establish stable expression of Tp and / or Pol in transfected cells.

[0109] Example 2 Engineering enhanced viral vector packaging cells A helper virus-free adenovirus vector can be prepared by the following method, as summarized in FIG. (1) packaging cells, such as HEK293 derived cells, are transfected with a mixture of linearized adenoviral vector genomic DNA, such as, but not limited to, an fdAd vector genome having ITRs at or near the 5' and 3' ends, and a packaging expression plasmid; (2) maintaining the transfected packaging cells in culture for a period of time during which the linearized adenoviral vector genome is replicated and packaged into adenoviral capsids; (3) The transfected packaging cells are harvested to recover the encapsidated adenoviral vector.

[0110] The functionality of the harvested encapsidated fdAd vectors was tested as exemplified herein using an fdAd vector carrying green fluorescent protein behind the CMV immediate early / promoter enhancer, fdAdGFP (Figure 4). The encapsidated fdAdGFP vector was used to transduce HEK293-derived cells. After 24 hours of culture, the cells are analyzed for expression of GFP.

[0111] Introduction of linear viral genomes, such as but not limited to adenovirus vectors, into eukaryotic cells can cause destruction by cellular nucleases, such as exonucleases. To increase the activity level of linear genomes in cells, it is necessary to protect the genome from nucleases. New packaging cells were engineered based on HEK293 cells to increase the rate of encapsidation of fdAd vectors. HEK293 cells were originally immortalized by transfection with truncated adenovirus type 5 DNA (Graham, 1977). Sequencing of the adenovirus 5 insert showed that a contiguous segment from nucleotides 1 to 4,344 is integrated into chromosome 19 (19ql3.2) (Louis, 1997). HEK293 cells are known to express the adenovirus E1A and E1B genes, and possibly other adenovirus genes.

[0112] HEK293 cells were modified by stable transfection of a eukaryotic expression vector (Figure 3, and SEQ ID NO: 1) carrying the genes for adenovirus 5-terminal protein (Tp) and DNA polymerase (Pol) under the control of the bidirectional promoter Surfeit1 (Surf1). The Tp / Pol vector was linearized by restriction enzyme digestion with NdeI and then used to transfect HEK293 cells. Transfectant cells were selected for puromycin resistance. Resistant cells were cloned. Cloned transduced packaging cells, designated Q7, were selected for high vector production efficiency. The stability of the Tp / Pol vector integration was investigated by measuring the expression of Tp and Pol over time in HTP7 cells by quantitative reverse transcriptase PCR (RT-PCR).

[0113] The encapsidation efficiency of Q7 cells was compared with that of the unmodified HEK293 parental cell line. For this purpose, the fdAdGFP linearized vector genome was co-transfected with a packaging expression plasmid into two packaging cells, Q7 cells and HEK293 parental cells. Encapsidated fdAdGFP vectors were collected from both cell lines and used for transfection of human cells. After 24 h of culture, the cells are analyzed for the expression of GFP. As illustrated in Figure 5, the packaging efficiency of Q7 for fdAdGFP was approximately 50-100 times higher than that of HEK293 parental cells.

[0114] Example 3 The adenovirus terminal protein (Tp) precursor is covalently bound to nucleotides within the ITRs of the linear adenovirus genome. The Tp precursor remains covalently bound to the 5' end of the viral DNA and is cleaved into mature Tp during virion maturation. Tp, together with adenovirus DNA polymerase (Pol), is responsible for the replication of the adenovirus genome. Tp, by itself or in combination with adenovirus Pol, prevents DNA exonuclease from enzymatically destroying linear DNA.

[0115] In one embodiment, the linear DNA fragment is modified by linking adenovirus ITR to the end of the linear fragment. When adenovirus Tp is provided by itself or together with adenovirus Pol to eukaryotic cells that are transfected or have been transfected with the ITR-containing linear DNA fragment, the enzymatic destruction of the ITR-containing linear DNA fragment will be limited. As a result, the ITR-containing linear DNA fragment is maintained at a higher level in the cell, leading to the enhancement of the function of the ITR-containing linear DNA fragment.

[0116] The adenoviral Tp can be provided to the cell by itself or together with the adenoviral Pol, by transient or stable transfection with a eukaryotic expression vector, or as a protein or modified protein that is introduced into the cell.

[0117] Example 4 In one embodiment, the linear DNA fragment is modified at the 5' and 3' ends so that phage Tp can covalently bind to the linear DNA fragment. Providing Tp to eukaryotic and / or prokaryotic cells that are or have been transfected with the modified linear DNA fragment will limit the enzymatic destruction of the modified linear DNA fragment. As a result, the modified linear DNA fragment is maintained at a higher level in the cell, resulting in enhanced function of the modified linear DNA fragment.

[0118] Phage Tp can be provided to cells by transient or stable transfection with a eukaryotic expression vector, or as a protein or modified protein that is introduced into the cells.

[0119] Example 5 In one embodiment, linear DNA fragments are modified at 5' and 3' ends so that certain proteins can bind to linear DNA fragments.Providing such proteins (such as, but not limited to, proteins obtained from eukaryotic cells and / or prokaryotic cells and capable of binding to linear DNA) to cells that are transfected or have been transfected with the modified linear DNA fragments will limit the enzymatic destruction of the modified linear DNA fragments.As a result, the modified linear DNA fragments are maintained at higher levels in cells, leading to enhanced function of the modified linear DNA fragments.

[0120] In one embodiment, proteins that can bind to linear and / or circular DNA fragments can be collected or engineered so that enzymatic destruction in cells is limited.Providing such proteins (such as, but not limited to, proteins obtained from eukaryotic and prokaryotic cells and capable of binding to linear DNA) to cells that are transfected or have been transfected with said DNA fragments will limit enzymatic destruction of said DNA fragments.As a result, said DNA fragments are maintained at higher levels in cells, leading to enhanced function of said DNA fragments.

[0121] Such protective proteins can be provided to cells by transient or stable transfection with eukaryotic expression vectors, or as proteins or modified proteins introduced into the cells.

[0122] Example 6 Linear and / or circular DNA fragments protected from destruction in eukaryotic and / or prokaryotic cells by protective proteins can be used to deliver genetic constructs, including but not limited to: - viral genome; - Genes for therapeutic purposes; - Protein-coding genes; - a set of genes that code for proteins and / or enzymes; - Genes for proteins and / or enzymes that modify cell functions; - genes for enzymes that modify DNA, such as, but not limited to, DNA recombinases, DNA integrases; CRISPR mechanisms, as well as DNA and / or RNA constructs; - DNA and RNA fragments that are integrated into the cell genome; and - DNA and RNA fragments that regulate cell functions.

[0123] When describing elements of this disclosure or its preferred embodiments, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0124] In view of the above, it will be seen that the several objects of the disclosure are achieved and other advantageous results attained.

[0125] It is intended that all matter contained in the above description be interpreted in an illustrative and not a limiting sense, as various changes can be made in the products and methods described above without departing from the scope of the disclosure.

Claims

1. A method for enhancing the function of a packaging cell that propagates an adenovirus and an adenovirus-based gene transfer vector, comprising: (a) providing a eukaryotic expression vector encoding the expression of a terminal protein (Tp); (b) transfecting the terminal protein expression vector into a packaging cell; and (c) introducing into the packaging cell an adenovirus-derived genome having terminal sequences at both the 5' and 3' ends of a linear fragment of the adenovirus-derived genome to which the terminal protein can bind.

2. The method of claim 1 , wherein the Tp eukaryotic expression vector is transiently maintained in the packaging cell.

3. The method of claim 1 , wherein the Tp eukaryotic expression vector is stably integrated into the genome of the packaging cell.

4. The method of claim 1 , wherein the Tp eukaryotic expression vector codes for expression of the adenoviral terminal protein.

5. 2. The method of claim 1, wherein the eukaryotic expression vector that codes for the expression of the adenoviral terminal protein also codes for the expression of the adenoviral DNA polymerase.

6. The method of claim 1, wherein the adenovirus-derived genome has left and right adenovirus inverted terminal repeats (ITRs) and an adenovirus packaging signal (Ψ).

7. The method of claim 1, wherein the adenovirus-derived genome is partially deleted of endogenous adenovirus genes.

8. The method of claim 1, wherein the adenovirus-derived genome is completely deleted of all endogenous adenovirus genes.

9. 2. The method of claim 1, wherein the adenovirus-derived genome is completely deleted of endogenous adenovirus gene structures other than the adenovirus left and right inverted terminal repeats (ITRs) and the adenovirus packaging signal (Ψ).

10. The method of claim 1, wherein the adenovirus-derived genome is linear.

11. The method of claim 1 , wherein the adenovirus-derived genome has modified and / or non-adenovirus genes.

12. The method of claim 1, wherein the adenovirus-derived genome is used for genetic medical purposes, including but not limited to gene therapy, vaccination, tissue engineering, and genome modification.

13. 2. The method of claim 1, wherein the virus or expression vector is co-transfected into a packaging cell that provides the genetic information necessary for packaging the adenovirus-derived genome into an adenovirus capsid.

14. 1. A method for enhancing the function of a DNA construct once introduced into a eukaryotic cell, comprising: (a) providing a eukaryotic expression vector that codes for the expression of a protein capable of protecting an introduced DNA construct from enzymatic digestion; (b) transfecting the protein expression vector into a eukaryotic cell; (c) A method comprising introducing into a eukaryotic cell a DNA construct to which the DNA protection protein binds.

15. The method of claim 14, wherein the eukaryotic expression vector for the protection protein is transiently maintained in the packaging cell.

16. The method of claim 14, wherein the eukaryotic expression vector for the protection protein is stably integrated into the genome of the packaging cell.

17. 15. The method of claim 14, wherein the eukaryotic expression vector for the protection protein codes for the expression of a bacteriophage or bacterial terminal protein.

18. The method of claim 14 , wherein the DNA construct is circular.

19. The method of claim 14 , wherein the DNA construct is linear.

20. The method of claim 14, wherein the DNA construct is modified to enhance binding of a bacteriophage or bacterial terminal protein.

21. The method of claim 14, wherein the DNA construct is used for genetic medical purposes, including but not limited to gene therapy, vaccination, tissue engineering, and genome modification.

22. 15. The method of claim 14, wherein the eukaryotic expression vector for the protective protein encodes a protein of eukaryotic or prokaryotic origin.

23. The method of claim 14, wherein the DNA construct is modified to enhance binding of the protective protein.

24. A method of enhancing the function of a DNA construct once introduced into a eukaryotic cell by altering the DNA sequence of the DNA such that enzymatic destruction of the DNA construct is reduced.

25. 25. The method of claim 24, wherein the DNA construct is linear and its 5' and 3' ends are modified to reduce digestion by nucleases.

26. 25. The method of claim 24, wherein the linear DNA construct is chemically altered.

27. 25. The method of claim 24, wherein the 5' and 3' terminal sequences are modified to limit DNA exonuclease function.

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