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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• a variety of different gene transfer approaches are available to deliver recombinant genes into cells and tissues.
• these are several non-viral vectors, including DNA/liposome complexes, DNA, and targeted viral protein DNA complexes.
• viral vectors including adenoviruses, adeno-associated viruses, retroviruses, and others have previously been well-described.
• Most viral vectors have several limitations, including possible biohazard from possible recombination with wild-type vectors, low viral titer and low expression levels.
• Adenoviral vectors in contrast, are an effective means for introducing genes into tissues in vivo because of their high level of expression and efficient transformation of cells both in vi tro and in vivo, see Davidson, et al.
• This invention provides a novel expression vector useful for inserting and expressing foreign nucleic acid molecules in a host cell.
• the expression vector of this invention is derived from an adenoviral vector and has as its components the adenoviral Inverted Terminal Repeat, an adenoviral packaging sequence, and the DNA molecule to be inserted.
• This invention also provides an adenoviral expression vector having a foreign or heterologous DNA molecule inserted within adenoviral capsid proteins. These vectors are useful for gene therapy.
• Figure 1 graphically depicts a strategy for introducing plasmid DNA into adenoviral particle.
• the inverted terminal repeat (ITR) packaging sequence of the virus is introduced into a plasmid in such a fashion that the plasmid can be linearized and co-transfected with a mutant full-length virus.
• the production of viral proteins occurs and allows the plasmid DNA to be packaged in the particle.
• Figure 2 shows a segment of adenoviral DNA subcloned into a cosmid vector and linearized before co- transfection into the packaging cell line.
• Figure 3 shows the use of a packaging plasmid with the packaging site deleted, but the ITR sequence maintained viral genomic DNA.
• Figure 4 schematically depicts purification and cloning of adenoviral type 5, wild-type and sub 360 genomic DNA.
• Figure 5 is a restriction map of plasmid Psi RSV beta-gal
• Figure 6 is a restriction map of RSV beta-gal.
• Figure 7 is a restriction map of plasmid Psi RSV beta-gal-2.
• Figure 8 is a restriction map of plasmid Psi RSV beta-gal after partial digestion with Aatll, treated with Klenow fragment and created a unique Xba I site.
• Figure 9 is a restriction map of the cosmid vector Cos Psi RSV beta-gal.
• Figure 10 is a restriction map of packaging plasmid Psi RSV beta-gal LS.
• Figures 11A through 11C are restriction maps of cosmid vectors.
• Figure 11A is the cosmid Psi RSV beta-gal A2.
• Figure 11B is the cosmid Psi RSV beta-gal S2 and
• Figure 11C is the cosmid Psi RSV beta-gal AS2.
• Figures 12A through 12C are the maps of the adenoviral expression vectors of this invention.
• FIG. 12A is the map of Psi RSV beta-gal LSA2.
• Figure 12B is the restriction map of Psi RSV beta-gal LSS2 and
• Figure 12C is the restriction map of Psi RSV beta-gal LSAS2.
• An object of this invention is to provide adenoviral vectors which can be grown to high titer and infect cells efficiently. These vectors also are useful for gene therapy because the probability of recombination with wild-type virus is extremely low and they express no adenoviral gene products.
• another object of this invention is to provide an alternate method for introducing recombinant genes into cells for the purposes of treating disease. This is accomplished through the development of a unique adenoviral vector that contains a plasmid DNA rather than adenoviral DNA.
• This invention offers an advantage over retroviral vectors and conventional prior art adenoviral vectors because it can be grown to high titer stocks, can infect cells efficiently, and is extremely unlikely to recombine in the population.
• This invention provides a pseudo-adenovirus vector comprising, from the 5' end to the 3' end, a DNA molecule corresponding to a first adenovirus Inverted Terminal Repeat, a DNA molecule encoding adenovirus packaging sequence, a heterologous DNA, and a DNA molecule corresponding to a second adenovirus Inverted Terminal Repeat.
• a pseudo-adenovirus vector is intended to include DNA molecules that can be transferred into the host cell in adenovirus cap'sids to express a recombinant gene.
• expression vector is intended to mean a vehicle that promotes the expression of a gene inserted into it; typically, a restriction fragment that carries a regulatory sequence for the particular gene and sequences that provide for RNA polyadenylation and processing.
• heterologous DNA is intended to encompass a DNA polymer.
• the heterologous DNA comprises plasmid vector DNA or cosmid vector DNA.
• the heterologous DNA Prior to insertion into the pseudo-adenoviral vector, the heterologous DNA is in the form of a separate fragment, or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
• Recombinant is intended to mean that a particular DNA sequence is the product of various combination of cloning, restriction, and ligation steps resulting in a construct having a sequence distinguishable from homologous sequences found in natural systems.
• Recombinant sequences can be assembled from cloned fragments and short oligonucleotides linkers, or from a series of oligonucleotides.
• the pseudo- adenovirus expression vector and the adenovirus capsids are derived from adenovirus type 5 virus.
• Other suitable adenoviral subtypes are human types 1-41 or murine strains.
• the vector further contains a DNA molecule containing adenovirus packaging sequence which allows the genetic material to be assembled and packaged into the adenoviral particle.
• This sequence is comprised of multiple, (6-20) oligonucleotide repeats derived from sequence 3' to the left ITR (Grable et al.(1990) infra. ) .
• the heterologous DNA also can contain additional
• DNA molecules which comprise a transcriptional initiation region so that DNA molecules downstream from the initiation region can be transcribed to a sequence of interest, usually mRNA, whose transcription and, as appropriate, translation will result in the expression of a polypeptide, a protein, a ribozyme and/or the regulation of other genes, e.g. antisense, expression of transcriptional factors, etc.
• adenoviral DNA there are technical considerations in introducing adenoviral DNA into adenoviral complexes.
• the cis- acting DNA sequences required for packaging are the inverted terminal repeats (ITR) , which are required for replication of the DNA in cells that contain adenoviral gene products.
• ITR inverted terminal repeats
• the packaging sequence is required. These sequences have been defined, in part, by deletion analysis of minimal regions required for packaging, and have been previously described (Grable et al., J. Virol. 64:2047-2056 (1990) incorporated herein by reference) .
• the length of the DNA to be packaged within the adenoviral sequence needs to be considered.
• several means to introduce the recombinant DNA into the adenoviral particle have been set forth.
• adenoviral packaging is accomplished using a plasmid containing the left end of the adenoviral genome which is replication defective and co- transfecting with wild type adenoviral DNA inactivated to prevent its replication.
• Figure 1 the ITR packaging sequence of the virus is introduced into a plasmid in such a fashion that the plasmid can be linearized and co-transfected with the virus DNA.
• the production of viral proteins occurs and allows the plasmid DNA to be packaged in the particle.
• a segment of adenoviral DNA is subcloned into a cosmid vector and linearized before co-transfection into the packaging cell line, thus also allowing for packaging of the recombinant DNA in the transfected cell line.
• the advantage of this approach is that an artificial form of the truncated virus is used, thus minimizing the possibility that uncut viral DNA will be present in the cell culture and will allow for the replication of wild-type adenovirus.
• the packaging plasmid is used, together with an adenovirus in which the packaging site has been deleted but the ITR sequence is maintained, thus allowing for the replication of defective virus and viral proteins at the same time that the plasmid DNA is replicated within cells, allowing for the higher titer virus.
• a further development of this technology is a permanent packaging cell line which provides the viral packaging proteins in trans, and thus require only the transfection of the plasmid DNA with the packaging sequence within.
• the present studies demonstrate the feasibility of using a packaging sequence and ITR anti-plasmid to allow incorporation of the DNA into the antiviral particle.
• the addition of nonviral DNA sequences to further improve efficiency are within the scope of this invention.
• adenoviral sequences to further define the other cis-acting regulatory elements required for packaging, and finally, to introduce additional consensus packaging sequences into the background of irrelevant DNA (phage DNA) to further improve the efficiency of packaging of the plasmid vector.
• the transformed human embryonic kidney cell line The transformed human embryonic kidney cell line,
• D-MEM Dulbecco's Modified Eagle Medium
• Serum FBS, Gibco
• penicillin 50 U/ml penicillin
• 50 ⁇ g/ml streptomycin 50 ⁇ g/ml streptomycin
• 2 mM L-Glutamine 50 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-Glutamine.
• the sub360 DNA was treated with T4 polynucleotide kinase and Klenow fragment to repair the ends of the genomic DNA. Following the ligation of Xba I linkers (Promega) to each end, the genomic DNA was digested with Xba I. The right hand fragment of sub360 was cloned into the Xba I site of cosmid vector p E15 (Strategene) which was modified by creating a new Xba I site into the BamHI site according to the manufacturer's instructions.
• pAd-Bgl II plasmid (Davidson, et al. , Nature Genetics, 3:219-223 (1993) incorporated herein by reference) was digested with Eco RI and repaired by Klenow fragment of E. coli DNA polymerase. After ligation of BamHI linkers (Boehringer) to the blunted Eco RI sites, the plasmid was digested with BamHI and Bgl II. A DNA fragment containing the terminal sequence and packaging signal sequence (370 bp) was introduced into the BamHI site of RSV ⁇ Gal (Stewart, et al .
• 5' -ACAGAATTCGCTAGCATCATCAATAATATACC-3' (Seq. I .D. No. 1) and anti-sense primer (200-173) containing a BamHI site, 5' -ACAGGATCCGGCGCACACCAAAAACGTCACTTTTGCC-3' (Seq. I .D. No. 2) .
• the PCR conditions were 94°C 30 seconds; 65°C 30 seconds; and 72°C 30 seconds for the first 5 cycles, then 94°C 30 seconds; and 72°C 30 seconds for 30 cycles.
• the amplified terminal sequence (212 bp) was digested with Eco RI and BamHI and subcloned into pBluescript (Strategene) .
• the terminal sequence fragment was purified by BamHI digestion, and introduced into the BamHI site of Pack+RSV jSGal plasmid to generate an Inverted Terminal Repeat (ITR) .
• ITR Inverted Terminal Repeat
• the ⁇ RSV j ⁇ Gal plasmid was propagated in E. coli , SURE Cells (Strategene) .
• the terminal sequence in the above pBluescript plasmid was purified by digestion with Nhe I and BamHI, and cloned into the Nhe I and Bgl II sites of pAd Bgl II.
• Co-transfection was performed by the calcium phosphate method (Sambrook, et al. , Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference) in 100 mm diameter petri dishes, 293 cells were transfected with 10 ⁇ g Eco RI digested pAd ⁇ i', lO ⁇ g Nhe I digested ⁇ RSV /SGal, and varying amounts of Xba I and Cla I digested sub 360 genome, or Xba I and Klenow fragment-treated pWEsub360. In control experiments, lO ⁇ g of BamHI digested RSV / SGal was used in place of ⁇ RSV / SGal. Eight days post-transfection, cells were harvested, suspended in 1.5 mis of medium and freeze- thawed 3 times in dry ice-ethanol. Supernatants were used as viral lysates in the subsequent experiments. Titration of Virus
• Confluent 293 cells in 60 mm diameter dishes were infected with 0.5 ml of viral lysate for 1 hr. After infection, 4.5 mis of medium were overlaid, and cells were cultured for 24 hours at 37°C. The infected cells were harvested, washed with PBS twice, and fixed with 1.25% glutaraldehyde-PBS solution for 5 min. at room temperature. Fixed cells were washed with PBS twice and stained with Solution X [50 mM Tris HC1, pH 7.5, 2.5 mM Ferriferrocyanide, 15 mM NaCl, ImM MgCl 2 and 0.5 mg/ml X- gal] overnight in 6 well culture plates. The number of blue stained cells and total cells in each well were counted (Table 1) .
• Adenovirus packaging sequence induces incorporation of linearized plasmid DNA into virus particles - evidence of transduction and expression.
• Psi RSV beta-gal plasmid ( Figure 7) was used as a parental plasmid to construct the large-size plasmids.
• Psi RSV beta-gal plasmid was partially digested with Aatll and treated with Klenow fragment, then an Xbal linker (Progega) was introduced (nucleotide position, 5,775) .
• This plasmid was tentatively named Psi RSV beta-galXbal ( Figure 8) .
• Cos Psi RSV beta-gal plasmid was digested NotI, treated with Calf intestinal alkaline phosphatase, then, additionally digested with Xbal .
• Yeast genomic DNA was completely digested with Nhel and treated with alkaline phosphatase. The DNA fragments were separated on 0.5% low melting agarose gel, the fragments ranging 20-30 kb were purified. These fragments were ligated to NotI, Xbal-digested Cos Psi RSV beta-gal plasmid, described above, then, packaged into lambda phage using the Gigapack II packaging kit (Stratagene) . The clones, whose total sizes ranged between 20-40kb were selected, and designated packaging plasmids Psi RSV beta-galLS ( Figure 10) .
• oligonucleotides which coded packaging signal element AV and AVI were designed as follows.
• Sense primer which had Apal restriction site at 3' end; 5' -GCGTAATATTTGTCTAGGGCCGCGGGGACTTTGGGGCC-3', (Seq. I.D. No. 3)
• anti-sense primer which had Apal site at 5' -end;
• All 5' -ends of sense and anti-sense oligo ⁇ nucleotides were phosphorylated by T4 polynucleotide kinase and annealed.
• the oligonucleotides which had either Apal site or Sapl site were introduced into Apal or Sapl site of cos Psi RSV beta-gal to create two (2) tandem copies and also to show the same direction as that of wild-type packaging signal in Cos Psi RSV beta-gal ( Figure 11) .
• the plasmid which contained oligonucleotides at Apal site was called Cos Psi RSV beta-galA2 and the Sap I site was termed Cos Psi RSV beta-galS2.
• the expression vectors of this invention can be inserted into host cells, for example, mammalian cells, particularly primate, more particularly human, but can be associated with any animal of interest, particularly domesticated animals, such as equine, bovine, murine, ovine, canine, feline, etc.
• host cells for example, mammalian cells, particularly primate, more particularly human, but can be associated with any animal of interest, particularly domesticated animals, such as equine, bovine, murine, ovine, canine, feline, etc.
• various types of cells may be involved, such as hematopoietic, neural, mesenchymal, cutaneous, mucosal, stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary, etc.
• hematopoietic cells which can include any of the nucleated cells which may be involved with the lymphoid or myelomonocytic lineages.
• members of the T- and B-cell lineages members of the T- and B-cell lineages, macrophages and monocytes.
• stem and progenitor cells such as hematopoietic neural, stromal, muscle, hepatic, pulmonary, gastrointestinal, etc.
• the heterologous DNA also can code for receptors which may include receptors for the ligands IL-2, IL-3, IL-4, IL-7 (interacts with p59fyn) ; erythropoietin (EPOR) , G-CSF, leukemia inhibitory factor (LIF) , ciliary neutryphic factor (CNTR) , growth hormone (GH) , herpesvirus thymidine kinase, histocompatibility genes, and prolactin (PRL) .
• receptors may include receptors for the ligands IL-2, IL-3, IL-4, IL-7 (interacts with p59fyn) ; erythropoietin (EPOR) , G-CSF, leukemia inhibitory factor (LIF) , ciliary neutryphic factor (CNTR) , growth hormone (GH) , herpesvirus thymidine kinase, histocompatibility genes
• the heterologous DNA also may contain DNA sequences which provides for the necessary transcriptional termination, and as appropriate, translational termination.
• the heterologous DNA can contain a wide variety of genes, where the gene encodes a protein of interest or an antisense sequence of interest.
• the gene can be any sequence of interest which provides a desired phenotype.
• the gene can express a surface membrane protein, a secreted protein, a cytoplasmic protein, or there may be a plurality of genes which may express different types of products.
• the gene also can encode an antisense sequence which may modulate a particular pathway by inhibiting a transcriptional regulation protein or turn on a particular pathway by inhibiting an inhibitor of the pathway.
• the proteins which are expressed, singly or in combination, may involve homing, cytotoxicity, proliferation, immune response, inflammatory response, clotting or dissolving of clots, hormonal regulation, or the like.
• the proteins expressed could be naturally-occurring, mutants of naturally-occurring proteins, unique sequences, or combinations thereof.
• the gene also can encode a product which is secreted by a cell, so that the encoded product may be made available at will, whenever desired or needed by the host.
• Various secreted products include hormones, such as insulin, human growth hormone, glucagon, pituitary releasing factor, ACTH, melanotropin, relaxin, etc.; growth factors, such as EGF, IGF-1, TGF- ⁇ , - ⁇ , PDGF, G-CSF, M-CSF, GM-CSF, FGF, erythropoietin, megakaryocytic stimulating and growth factors, etc.; interleukins, such as IL-1 to -11; TNF- ⁇ .
• tissue plasminogen activator members of the complement cascade, perforans, superoxide dismutase, coagulation factors, anti- thrombin-III, Factor VIIIc, Factor VIIIvW, ⁇ .-anti-trypsin, protein C, protein S, etc.
• enzymes such as tissue plasminogen activator, members of the complement cascade, perforans, superoxide dismutase, coagulation factors, anti- thrombin-III, Factor VIIIc, Factor VIIIvW, ⁇ .-anti-trypsin, protein C, protein S, etc.
• the gene also can encode a surface membrane protein.
• proteins may include homing receptors, e.g. L-selectin (Mel-14) , blood-related proteins, particularly having a kringle structure, e.g. , Factor VIIIc, Factor VIIIvW, hematopoietic cell markers, e.g.
• ions e.g., H + , Ca +2 , K + , Na + , Cl " , etc., and the like
• CFTR tyrosine activation motif
• zeta activation protein etc.
• intracellular proteins may be of interest, such as proteins in metabolic pathways, regulatory proteins, steroid receptors, transcription factors, etc., particularly depending upon the nature of the host cell. Some of the proteins indicated above may also serve as intracellular proteins.
• T-cells In T-cells, one may wish to introduce genes encoding one or both chains of a T-cell receptor.
• B-cells one could provide the heavy and light chains for an immunoglobulin for secretion.
• cutaneous cells e.g. keratinocytes
• the site may include anatomical sites, such as lymph nodes, mucosal tissue, skin, synovium, lung or other internal organs or functional sites, such as clots, injured sites, sites of surgical manipulation, inflammation, infection, etc.
• anatomical sites such as lymph nodes, mucosal tissue, skin, synovium, lung or other internal organs or functional sites, such as clots, injured sites, sites of surgical manipulation, inflammation, infection, etc.
• surface membrane proteins which will direct the host cell to the particular site by providing for binding at the host target site to a naturally-occurring epitope, localized concentrations of a secreted product may be achieved.
• Proteins of interest include homing receptors, e.g. L-selectin, GMP140, LCAM-1, etc., or addressins, e.g.
• ELAM-1, PNAd, LNAd, etc. clot binding proteins, or cell surface proteins that respond to localized gradients of chemotactic factors.
• a recombinant gene to malignant cells for the purpose of causing cell death or inducing immune recognition of tumors.
• autoimmune disease Cells of extended lifetime, e.g. endothelial cells could be employed.
• the heterologous DNA would provide for a homing receptor for homing to the site of autoimmune injury and for cytotoxic attack on cells causing the injury.
• the therapy would then be directed against cells causing the injury.
• Another alternative would be to secrete an anti-inflammatory product, which could serve to diminish the degenerative effects.
• the genes can be introduced in one or more DNA molecules or expression vectors, where there will be at least one marker and may be two or more markers, which will allow for selection of host cells which contain the gene(s) .
• the heterologous DNA, genes and expression vectors can be prepared in conventional ways, where the genes and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual DNA fragments including all or portions of a functional unit may be isolated, where one or more mutations may be introduced using "primer repair", ligation, etc. as appropriate. See Sambrook et al .
• Host cells can be grown and expanded in culture before introduction of the vector(s) followed by the appropriate treatment for introduction of the vectors and integration of the vector(s) . The cells will then be expanded and screened by virtue of a marker present in the vector.
• markers which may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
• the expression vectors can be introduced simultaneously or consecutively, each with the same or different markers.
• the cells may be administered in a wide variety of ways.
• Hematopoietic cells may be administered by injection into the vascular system, there being usually at least about 10 4 cells and generally not more than about 10 10 , more usually not more than about 10 8 cells.
• the number of cells which are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the therapeutic agent, the physiologic need for the therapeutic agent, and the like.
• skin cells which may be used as a graft, the number of cells would depend upon the size of the layer to be applied to the burn or other lesion.
• the number of cells will at least about 10 4 and not more than about 10 8 and may be applied as a dispersion, generally being injected at or near the site of interest.
• the cells will usually be in a physiologically-acceptable medium.
• the vectors of this invention can be used for the treatment of a wide variety of conditions and indications.
• B- and T-cells antigen-presenting cells or malignant cells themselves may be used in the treatment of cancer, infectious diseases, metabolic deficiencies, cardiovascular disease, hereditary coagulation deficiencies, autoimmune diseases, joint degenerative diseases, e.g. arthritis, pulmonary disease, kidney disease, nedocrine abnormalities, etc.
• Various cells involved with structure such as fibroblasts and myoblasts, may be used in the treatment of genetic deficiencies, such as connective tissue deficiencies, arthritis, hepatic disease, etc.
• Hepatocytes could be used in cases where large amounts of a protein must be made to complement a deficiency or to deliver a therapeutic product to the liver or portal circulation.
• This invention also provides a transgenic, non- human animal whose germ cells and somatic cells contain a heterologous DNA molecule that has been introduced into the animal, or an ancestor of the animal, at an embryonic stage.
• a heterologous DNA molecule encodes an product which produces a pathological condition in the animal
• these animals are useful to test materials suspected of treating the pathology.
• the heterologous DNA can be used to encode a therapeutic or prophylactic composition. These animals are useful to test the particular therapy.
• the transgenic animals can be produced.

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