EP1045919A1 - Promotorelemente für andauernde genexpression - Google Patents

Promotorelemente für andauernde genexpression

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Publication number
EP1045919A1
EP1045919A1 EP99902292A EP99902292A EP1045919A1 EP 1045919 A1 EP1045919 A1 EP 1045919A1 EP 99902292 A EP99902292 A EP 99902292A EP 99902292 A EP99902292 A EP 99902292A EP 1045919 A1 EP1045919 A1 EP 1045919A1
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Prior art keywords
transgene
cmv
expression cassette
seq
expression
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French (fr)
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Donna Armentano
Nelson Yew
John Marshall
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Genzyme Corp
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Genzyme Corp
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • the invention is directed to novel promoter elements for the persistent expression of a transgene which is delivered to a target cell.
  • the promoter elements are derived from the cytomegalovirus intermediate early promoter (CMV).
  • CMV cytomegalovirus intermediate early promoter
  • the invention is also directed to enhancer elements which, when placed upstream (or 5') to the novel promoter elements of the invention operably linked to a transgene, increase the levels of expression of the transgene.
  • adenoviral vectors or plasmids comprising the CMV-derived promoter elements, operatively linked to a transgene, are used to achieve persistent expression of the transgene.
  • RNA, antisense R . NA, protein, hormone, enzyme, etc. a biologically active and useful product
  • a biologically active and useful product e.g. RNA, antisense R . NA, protein, hormone, enzyme, etc.
  • Transgene transfer has involved the use and development of viral vectors, such as those derived from retroviruses, adenoviruses, he es viruses, vaccinia and adeno-associated virus, among others. Also, transgene transfer has been effectuated using plasmids, naked DNA, lipids and combinations of all of these. For in vivo transgene delivery, much attention has focused on the use of viral vectors, particularly those derived from adenovirus.
  • Adenovirus is a non-enveloped, nuclear DNA virus with a genome size of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M.S., "Adenoviridae and Their Replication,” in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990).
  • the viral genes are classified into early (k. nown as E1-E4) and late (known as L1-L5) transcriptional units, representing two temporal classes of viral proteins.
  • the demarcation between early and late viral protein expression is viral DNA replication.
  • the human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 subgroups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, nucleic acid and amino acid compositions and relatedness, and antigenic relationships.
  • Recombinant adenoviruses have several advantages for use as transgene transfer vectors, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berl ner, K.L., CUIT. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1 :51-64, 1994).
  • the cloning capacity of an adenovirus vector is proportional to the size of the adenovirus genome present in the vector.
  • a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as El whose function may be restored in trans from 293 cells (Graham, F.L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Therapy 3:75-84, 1996).
  • El- deleted vectors are rendered replication-defective.
  • the upper limit of vector DNA capacity for optimal carrying capacity is about 105%- 108% of the length of the wild-type genome.
  • adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (.Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Allowed U.S. Patent Application Serial No. 08/895 J 94, incorporated herein by reference; Caravokyri et al., J. Virol. 69:6627-6633, 1995; .Krougliak et al., Hum. Gene Ther.
  • Transgenes that have been expressed to date by adenoviral vectors include, ter alia, p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5: 130-134, 1993; erythropoietin (Descamps et al., Human Gene Therapy 5:979-985, 1994; ornithine transcarbamylase (Stratford-Perricaudet et al., Human Gene Therapy 1 :241-256, 1990; We et al., J. Biol. Chem.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Adenovirus vectors engineered to carry the CFTR gene have been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75 :207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994).
  • tissue specific expression can be obtained from adenovirus vectors by the use of tissue specific promoter sequences.
  • liver specific adenovirus expression vector characterized in that a therapeutic transgene which is coupled to a liver-specifrc promoter consisting of enhancer elements of the hepatitis B virus and an enhancer-less minimal promoter and is optionally surrounded by SAR elements is inserted in the adenovirus genome.
  • adenovirus vectors in transgene transfer studies to date indicates that persistence of transgene expression is often transient. At least some of the limitation is due to the generation of a cellular immune response to the viral proteins which are expressed even from a replication-defective vector, triggering a pathological inflammatory response which may destroy or adversely affect the adenovirus-infected cells (Yang et al., J. Virol. 69:2004-2015, 1995; Yang et al., Proc. Natl. Acad. Sci. USA 91 :4407-4411, 1994; Zsengeller et al., Hum Gene Ther. 6:457- 467, 1995; Worgall et al., Hum. Gene Ther.
  • adenovirus does not integrate into the cell genome, host immune responses that destroy virions or infected cells have the potential to limit adenovirus-based transgene transfer.
  • Long-term expression can be achieved if the recombinant adenoviral vector is introduced into nude mice or mice that are given both the adenoviral vector and immunosupressing agents. Dai, Y. et al., 1995, Proc. Natl. Acad. Sci. USA 92:1401-1405. These results bolster the notion that the immune response is behind the short-term expression that is usually obtained from adenoviral vectors.
  • the adenovirus E3 gpl9K protein can complex with MHC Class I antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol. 437-443, 1994), suggesting that its presence in a recombinant adenoviral vector may be beneficial.
  • CTLs cytotoxic T-lymphocytes
  • adenovirus vector- delivered transgenes may also be due to limitations imposed by the choice of promoter or transgene contained in an expression cassette or a transcription unit (Guo et al., Gene Therapy 3:802-801, 1996; Tripathy et al., Nature Med. 2:545-550, 1996).
  • plasmids comprising a transgene may be used to deliver the transgene to a target cell. It is desirable that plasmids for use as gene transfer vehicles also be able to replicate in the recipient cells or tissues of individuals, since continued presence of the plasmid could provide correction of the genetic defect (in the case of cystic fibrosis, a vector may provide a functioning CFTR protein in the cell membrane of lung epithelial cells or other cells to replace the non-functioning or missing endogenous protein) over an extended period of time. There has been some concern that plasmids that cannot replicate in the targeted cells or tissues may be degraded after only a relatively short period of maintenance therein, thus requiring excessive repeat administrations.
  • one strategy has been to provide for continued maintenance of plasmids in target cells by other means.
  • One such strategy is to construct a plasmid capable of being maintained separately in the nucleus of a target (i.e. in an episome).
  • C. McWhinney et al. Nucleic Acids Research. 18, 1233-1242, 1990 have determined that the 2.4 kb Hindlll-Xhol fragment that is present immediately 5' to exon 1 of the human c-myc gene contains an origin of replication which when cloned into a plasmid and transfected into HeLa cells was shown to allow the plasmid to persist in the cells for more than 300 generations under drug selection. Replication was shown to be semiconservative (C.
  • pCFl-CAT PCT publication WO 96/18372 Figure 18A
  • pCFl-CAT PCT publication WO 96/18372 Figure 18A
  • the increase in plasmid size that results from insertion of the 2.4 kb fragment (or multiple copies thereof) is predicted to provide an additional benefit, that is, to facilitate plasmid unwinding, thus facilitating the activity of DNA polymerase. See PCT publication WO 96/18372, incorporated herein by reference.
  • Use of this origin of replication, or multiple copies thereof, allows the resultant plasmid to replicate efficiently in human cells.
  • Other DNAs comprising other origins of replication may also be used (for example, as found in the human ⁇ -globin gene, or the mouse DHFR gene).
  • a plasmid containing the cytomegalovirus promoter and enhancer, an intron, the CFTR cDNA, the bovine growth hormone polyadenylation signal, the kanamycin resistance transposon Tn903, and 4 copies of the 2.4 kb 5' flanking region of the human c-myc gene is shown in Figure 20 of WO 96/18372.
  • adenoviral vectors and plasmids for persistent transgene expression in target cells and tissues may also involve the design of expression control elements, such as promoters, which confer persistent expression to an operably linked transgene.
  • promoter elements which function independently of particular viral genes to confer persistent expression of a transgene may allow the use of vectors which contain reduced viral genomes, increasing the carrying capacity of the vector while decreasing the potential for host immune reaction or the generation of replication-competent viruses.
  • the invention is directed to a novel promoter element for persistent expression of an operably linked transgene.
  • the element is derived from the cytomegalovirus intermediate early promoter (CMV).
  • CMV cytomegalovirus intermediate early promoter
  • an adenoviral vector comprising a CMV-derived promoter element operably linked to a transgene is administered to recipient cells.
  • a plasmid comprising a CMV-derived promoter element of the invention operably linked to a transgene is administered to recipient cells.
  • the plasmid may also be delivered to a cell in conjunction with a lipid, such as those disclosed in WO 96/18372 or U.S. Patent No. 5,650,096.
  • enhancer elements derived from the human albumin gene which when operably linked to the CMV-derived promoter elements of the invention increase the expression of a transgene operably linked to the promoter elements.
  • the invention is also directed the use of such adenoviral vectors and plasmids comprising the enhancer and promoter elements of the invention in transgene transfer.
  • Figure 1 Sequence of the CMV intermediate early promoter, showing nucleotides -523 to -14 (SEQ ID NO:l).
  • FIG. 1 Sequence of a CMV-derived promoter element of the invention, showing nucleotides -295 to -14 (SEQ ID NO:2).
  • FIG. 3 Sequence of a CMV-derived promoter element of the invention, showing nucleotides -299 to -19 (SEQ ID NO:3).
  • FIG. 4 Sequence of a CMV-derived promoter element of the invention, showing nucleotides -242 to -14 (SEQ ID NO:4) Figure 5. Sequence of a human albumin gene-derived enhancer element of the invention showing a 65 nucleotide sequence found 1.7 kilobases upstream from the transcription initiation start site of the human albumin gene (SEQ ID NO:4) Figure 6. Schematic representation of transcriptional repressor binding sites in the CMV promoter.
  • Figure 7. Expression of ⁇ -galactosidase in rat hepatocytes using a promoter of the invention.
  • Figure 8. Expression of ⁇ -galactosidase in human hepatocytes using a promoter of the invention.
  • Figure 9 Expression of ⁇ -galactosidase in Balb/c lungs using a promoter of the invention.
  • FIG. 10 Expression of CAT in mice using a promoter of the invention.
  • Figure 1 Increased expression of a transgene operably linked to a CMV-derived promoter element of the invention through the use of enhancer elements derived from the human albumin gene placed 5' to the CMV-derived promoter element in 293 cells and Hep3B cells.
  • the present invention is directed to a novel promoter element for the persistent expression of an operably linked transgene.
  • the element is derived from the cytomegalovirus intermediate early promoter (CMV).
  • CMV cytomegalovirus intermediate early promoter
  • an adenoviral vector comprising a CMV-derived promoter element of the invention operatively linked to a transgene is used to achieve persistent expression of a transgene when administered to target cell.
  • a plasmid comprising CMV-derived promoter element operably linked to a transgene is used to achieve persistent expression of a transgene when administered to a target cell.
  • enhancer elements which effectuate increased expression of a transgene operably linked to CMV-derived promoter elements.
  • the enhancer elements are derived from the human albumin gene.
  • the invention is also directed to an expression cassette or transcription unit comprising at least a CMV-derived promoter element of the present invention and a transgene.
  • the expression cassette or transcription unit may also comprise an enhancer element.
  • a transgene is defined as a nucleic acid molecule coding for, inter alia, a protein (e.g. an enzyme, a hormone, a cell-surface molecule), ribozyme, RNA, and antisense RNA heterologous to the carrier vector.
  • a transgene may be delivered to a cell or tissue for example, but not by way of limitation , by a viral vector, a plasmid, a lipid, including a liposome, naked DNA, combinations thereof or other means known to those of skill in the art for delivery of transgenes.
  • Persistent expression is defined as generating and maintaining a sustained level of expression of a transgene over time.
  • a CMV-derived promoter element of the invention is defined as a promoter element which contains a nucleotide sequence derived from the wild-type cytomegalovirus (CMV) immediate early promoter (Boshart et al., Cell 41 :521-530, 1985, incorporated herein by reference) ( Figure 1) (SEQ ID NOJ), and provides for persistent expression of a transgene operably linked thereto.
  • CMV cytomegalovirus
  • Particular embodiments of the invention include a CMV-derived promoter element containing nucleotides -295 to -14 ( Figure 2) (SEQ ID NO:2), a CMV-derived promoter element containing nucleotides 299 to -19 of the CMV promoter ( Figure 3) (SEQ ID NO: 3), and a CMV-derived promoter element containing nucleotides -242 to -14 of the wild-type CMV promoter ( Figure 4) (SEQ ID NO:4) (referred to as ⁇ CMV promoter elements).
  • promoter elements which are within the scope of the invention, are also derived from the nucleotide sequence of the CMV promoter and confer persistent expression to an operably linked transgene in a target cell.
  • a promoter element of the invention may be identified by its ability to confer persistent expression of a transgene when delivered to a cell in an adenoviral vector lacking the E4 region.
  • promoter elements which are capable of conferring persistent expression may be constructed, for example, by deletion of sites within the CMV promoter sequence to which transcription repressor proteins can bind.
  • repressor proteins include YY1 (Liu et al., Nucleic Acids Research 22:2453- 2459, 1994; Gualberto et al., Mol. Cell Biol. 12:4209-4214, 1992; Galvin et al., Mol. Cell Biol. 17:3723-3732, 1997); MDBP (Zhang et al., Nucleic Acids Res. 18:6253- 6260; Zhang et al., Virology 182: 865-869; Supekar et al., Nucleic Acids Res.
  • Three YY1 binding sites are located in the wild-type CMV promoter between -300 and -522 relative to the transcriptional start site. Also, there are at least five potential binding sites for CREB and three binding sites for methylation-dependent binding protein. In addition, repressors such as Drl can also act on the core promoter complex. One skilled in the art can readily remove any of these sites by standard techniques of recombinant DNA technology.
  • CMV-derived promoter elements that are within the scope of the invention can retain or add in any nucleotides that correspond to transcriptional activator sites in order to achieve persistent expression.
  • activators include, for example, NFkappa ⁇ (Boshart et al., Cell 41 :521-530, 1985; Chang et al., J.Virol. 64:264-277, 1990; Neller et al., Nucleic Acids Res. 19:3715-3721, 1991).
  • Nucleotide sequences in the native CMV promoter to which transcriptional repressor and activator proteins bind are .known to those skilled in the art.
  • CMV-derived promoter elements of the invention can be engineered using standard techniques of molecular biology, such as restriction enzyme digestion, polymerase chain reaction (PCR), and site-directed mutagenesis.
  • a CMV-derived promoter element can be operably linked to a particular transgene by standard techniques .known in molecular biology for ligating DNA fragments.
  • nucleotide substitutions within the CMV-derived promoter elements of the invention that allow the promoter elements to retain the capability for persistent expression of a transgene are within the scope of the invention.
  • Such nucleotide substitutions can include those that, for example, alter the binding sites for the transcriptional repressor proteins discussed above (e.g. YYl), such that the repressors can no longer bind.
  • CMV-derived promoter elements of the invention which have capability to confer persistent expression of a transgene, include those which contain nucleotides -295 to -14, -299 to -19 and -213 to -14.
  • Other truncations of the wild-type CMV promoter to create CMV-derived promoter elements which are within the scope of the invention include, but are not limited to, those containing nucleotides -406 to -19; -299 to -10; -299 to +1; and -299 to +31; -277 to -19; -277 to -14; and - 213 to -19.
  • CMV-derived promoter elements of the invention can also comprise transcription factor binding sites which can be added, for example, to the 5' end of a CMV-derived promoter element of the invention. Such sites are .known to those skilled in the art.
  • CMV-derived promoter elements of the invention may include cellular promoter sequences which contribute to persistent expression of the operably linked transgene.
  • Such sequences can be derived from, for example but not by way of limitation, actin, mucin, and other constitutive cellular promoters.
  • promoter elements derived from wild-type promoters other than CMV which exhibit dependence on the adenovirus E4 region for persistent transgene expression, such as the Rous sarcoma virus (RSV).
  • RSV Rous sarcoma virus
  • an RSV-derived promoter element can be constructed to delete or alter the serum response elements (SRE) to which the transcriptional repressor protein YYl can bind, so as to create a promoter element which can confer persistent expression to an operably linked transgene (Gualberto et al., Mol. Cell Biol. 12:4209-4214, 1992).
  • SRE serum response elements
  • Transgenes which can be delivered and expressed from a promoter element of the invention include, but are not limited to, those encoding enzymes, blood derivatives, hormones, lymphokines such as the interleukins and interferons, coagulants, growth factors, neurotransmitters, tumor suppressors, apoliproteins, antigens, and antibodies, and other biologically active proteins.
  • transgenes which may be operably linked to the promoter elements of the invention include, but are not limited to, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin, glucocerebrosidase, tumor necrosis factor, p53, retinoblastoma (Rb), von- hippel lindau (VHL), pten tumor suppressor, pi 6, Glut4, .and adenosine deaminase (ADA).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Dytrophin glucocerebrosidase
  • tumor necrosis factor p53
  • Rb retinoblastoma
  • VHL von- hippel lindau
  • pten tumor suppressor pi 6, Glut4, .and adenosine deaminase (ADA).
  • Transgenes encoding antisense molecules and ribozymes are also within the scope of the invention.
  • an adenoviral vector or plasmid for gene transfer not only comprises the promoter element of the invention operably linked to a DNA encoding a transgene but may also comprise any other expression control sequences such as another promoter or enhancer , a polyadenylation element and any other regulatory elements that may be used to modulate or increase expression or a transgene when operably linked thereto.
  • enhancer elements include apoE enhancer elements (Shachter N.S., etal., 1993, J Lipid Res. 34:1699-707; Allan CM. et al., 1995, J. Biol. Chem.
  • porcine alpha-skeletal actin gene enhancer elements (Reecy, J.M. et al., A im.. Biotechnol. 9:101-120, 1998) and human albumin gene enhancer elements positioned at -1.7 and -6 kb upstream from the transcriptional start site of the wild- type human albumin gene (Hayashi, Y. et al., J. Biol. Chem. 267:14580-14585, 1992; incorporated herein by reference).
  • an enhancer element of the invention is derived from human albumin gene enhancer sequences and, when placed 5' to the CMV-derived promoter elements of the invention operably linked to a transgene, increases the expression levels of the transgene ( Figure 11).
  • the use of any other expression control sequences, or regulatory elements, which facilitate persistent expression of the transgene is also within the scope of the invention. Such sequences or elements may be capable of generating tissue-specific expression or be susceptible to induction by exogenous agents or stimuli.
  • Polyadenylation signals which may be positioned at the 3' end of the transgene in a transcription unit or expression cassette include, but are not limited to, those derived from bovine growth hormone (BGH) and SV40.
  • a human albumin gene-derived enhancer element of the invention is defined as an enhancer element which contains a nucleotide sequence derived from enhancer sequences found 1.7 kilobases (TTGTCAATTAGTAACAA; SEQ ID NO:5) and 6.0 kilobases (GCCAAACA; SEQ ID NO:6) upstream from the transcriptional initiation site of the wild-type human albumin gene (Hayashi, Y. et al., J. Biol. Chem. 267:14580-14585, 1992), and provides for increased expression of a transgene operably linked to a CMV-derived promoter element of the invention.
  • enhancer element which contains a nucleotide sequence derived from enhancer sequences found 1.7 kilobases (TTGTCAATTAGTAACAA; SEQ ID NO:5) and 6.0 kilobases (GCCAAACA; SEQ ID NO:6) upstream from the transcriptional initiation site of the wild-type human albumin gene (Hayashi, Y.
  • Preferred human albumin gene-derived enhancer elements of the invention which have the ability to increase the expression of a transgene operably linked to the CMV-derived promoter elements of the invention include a 65 nucleotide sequence located - 1797 to - 1737 bases upstream from the transcriptional initiation site of the wild-type human albumin gene comprising a 17 nucleotide enhancer element (-1.7kb enhancer element) ( Figure 5) (SEQ ID NO:7).
  • Another enhancer element within the scope of the invention is located -6 kilobases from the human albumin gene transcriptional start site (-6kb enhancer element) (SEQ ID NO:6).
  • adenoviral vectors can be used to deliver a transgene which is operably linked to a CMV-derived promoter element of the invention to target cells in order to achieve persistent expression of a desired protein.
  • the promoter elements of the invention may also be used with other viral vectors useful for gene transfer, including, but not limited to, those 9/36557
  • adenoviral vectors which can be used in the invention include, for example, Ad2/CFTR-1 and Ad2/CFTR-2 and others described in U. S. Patent No. 5,670,488, issued September 23, 1997 (incorporated herein by reference).
  • Adenoviral vectors may include deletion of the El region, partial or complete deletion of the E4 region, and deletions within, for example, the E2 and E3 regions.
  • the vectors can contain all, part or none of the E4 region of the adenoviral genome because the CMV-derived promoter elements of the present invention confer persistent expression in the absence of the E4 region.
  • Such vectors therefore, may include, if desired, the ORF3, ORF4 or ORF6 open reading frames from the E4 region.
  • the vectors are preferably replication-defective, that is, they are incapable of generating a productive infection in the host cell.
  • chimeric viral vectors which contain an Ad 2 backbone with one or more heterologous capsid proteins or fragments thereof (see PCT publication No. WO 98/22609, incorporated herein by reference, and allowed U.S. application Serial No. 08/752,760, filed November 20, 1996, allowed October 16, 1998 incorporated herein by reference).
  • Other adenoviral vectors include those derived from U.S. Patent No. 5,707,618 and U.S. Patent No. 5,824,544 (both incorporated herein by reference).
  • the CMV-derived promoter elements of the invention can be used to confer persistent expression of a transgene in E4-deleted adenoviral vectors, allowing for the design of such vectors with increased carrying capacity, and reduced potential for the generation of a host immune response or replication-competent viruses, all of which are desirable features for a vector used for gene transfer in vivo.
  • adenoviral vectors can also be constructed using adenovirus serotypes from the well-studied group C adenoviruses, especially Ad2 and Ad5.
  • Adl7 is also a preferred serotype.
  • adenoviral vectors for use in the invention derived from other group C or non-group C adenovir. uses are also within the scope of the invention, including chimeric adenoviral vectors which contain nucleotide sequences from one or more serotypes.
  • an adenoviral vector for use in the invention, reference may be made to the substantial body of literature on how such vectors may be designed, constructed and propagated using techniques from molecular biology and microbiology that are well-known to the skilled artisan.
  • the skilled artisan can use the standard techniques of molecular biology to engineer a transgene operably linked to a promoter element, preferably a CMV-derived promoter element, of the invention into a backbone vector genome (Berkner, K.L., Curr. Top. Micro. Immunol. 158:39-66, 1992).
  • an adenoviral genomic fragment may be co-transfected with a linearized viral genome derived from an adenoviral vector of interest into a recipient cell under conditions whereby homologous recombination occurs between the genomic fragment and the virus.
  • the transgene and the operably linked CMV- derived promoter element of the invention are inserted into the site of an El deletion.
  • the transgene is inserted into the adenoviral genome at the site in which it was cloned into the plasmid, creating a recombinant adenoviral vector.
  • the adenoviral vectors may also be constructed using standard ligation techniques. Construction of the adenoviral vectors may be based on adenovirus
  • DNA sequence information widely available in the field e.g., nucleic acid sequence databases such as GenBank.
  • Preparation of replication-defective adenoviral vector stocks may be accomplished using cell lines that complement viral genes deleted from the vector, e.g., 293 or A549 cells containing the deleted adenovirus El genomic sequences.
  • HER3 cells human embryonic retinoblasts transformed by Ad 12
  • the viruses may be recovered by freeze-thawing and may subsequently be purified using cesium chloride centrifugation.
  • virus purification may be performed using chromatographic teclmiques, e.g., as disclosed in PCT Publication No. WO 97/08298, incorporated herein by reference.
  • Titers of replication-defective adenoviral vector stocks may be determined by plaque formation in a complementing cell line, e.g., 293 cells. End- point dilution using an antibody to the adenoviral hexon protein may be used to quantitate virus production or infection efficiency of target cells (Armentano et al., Hum. Gene Ther. 6:1343-1353, 1995, incorporated herein by reference).
  • An example of an adenoviral vector containing a CMV-derived promoter element of the invention is ⁇ CMV- ⁇ gal-1, which comprises a CMV-derived promoter element comprising nucleotides -295 to -14, operably linked to a ⁇ - galactosidase gene, and the SV40 polyadenylation signal, in an El deletion that is further deleted for the E4 region.
  • Plasmids which may be used to deliver a transgene operably linked to a CMV-derived promoter element of the invention can be may be engineered using standard recombinant DNA technology. Large scale production and purification of such plasmids may be performed using techniques known to those skilled in the art (see, e ⁇ g., Current Protocols in Molecular Biology. Ausubel et al., eds., Jolin Wiley & Sons, Inc., New York, 1995). Plasmids may be delivered to target cells using such techniques as transfection, electroporation, microinjection, and other DNA transfer methods known to those skilled in the art. Plasmids may also be delivered in conjunction with a lipid, e.g.
  • a cationic lipid such as N 4 -spermine cholesteryl carbamate and N 4 -spermidine cholesteryl carbamate as disclosed in U.S. Patent No. 5,650,096 and PCT publication WO 96/18372, both incorporated herein by reference.
  • the delivery of a transgene operably linked to a promoter element of the invention to a target cell in the form of naked DNA is also within the scope of the invention.
  • the transgene is a marker or reporter gene
  • it may be used as to determine the persistence of expression using a CMV-derived promoter element of the invention.
  • a nonlimiting example is a plasmid such as pCFl-CAT (PCT publication WO 96/18372 Figure 18A), containing the chloramphenicol acetyltransferase (CAT) gene operatively linked to the wild-type CMV promoter which may be truncated to generate the CMV-derived promoter elements of the invention operably linked to CAT.
  • Other marker genes within the scope of the invention include, but are not limited to, the genes encoding ⁇ -galactosidase and luciferase. Proteins expressed from marker genes may be readily detected by standard techniques.
  • the plasmid pCFA-299/- 19 CAT (Exa preferred embodiment, the plasmid pCFA-299/- 19 CAT (Example
  • plasmid backbone 4 below is used as a plasmid backbone to construct a plasmid for transgene transfer to a target cell, in which the CAT marker gene is replaced by a transgene of interest.
  • Infection of target cells by adenoviral vectors or plasmids comprising a transgene operably linked to a CMV-derived promoter element of the invention may also be facilitated by the use of cationic molecules, such as cationic lipids disclosed in U.S. Patent No. 5,650,096 and PCT Publication No. WO 96/18372, published June 20, 1996, both incorporated herein by reference.
  • cationic molecules such as cationic lipids disclosed in U.S. Patent No. 5,650,096 and PCT Publication No. WO 96/18372, published June 20, 1996, both incorporated herein by reference.
  • Adenoviral vectors complexed with cationic molecules are also described in U.S. Application Serial No. 08/755,035, filed November 22, 1996 and PCT Publication No. WO 98/22144, incorporated herein by reference.
  • Cationic amphiphiles have a chemical structure which encompasses both polar and non-polar domains so that the molecule can simultaneously facilitate entry across a lipid membrane with its non-polar domain and attach to a biologically useful molecule to be transported across the membrane with its cationic polar domain.
  • Cationic amphiphiles which may be used to form complexes with the adenoviral vectors or plasmids of the invention include, but are not limited to, cationic lipids such as those disclosed in U.S. Patent No. 5,650,096, PCT publication No. WO 96/18372, and PCT publication No. WO 98/43994; DOTMA (Feigner et al., Proc. Natl. Acad. Sci.
  • the cationic amphiphiles useful to complex with and facilitate transfer of the vectors and plasmids of the invention are those lipids disclosed in U.S. Patent No. 5,650,096 and in PCT
  • cationic amphiphiles described herein to be used in the delivery of the plasmids and/or viruses include, inter alia, GL-53, GL-67, GL-75, GL-87 and GL-89, including protonated, partially protonated, and deprotonated forms thereof as set forth Figures 1 , 7 and 9 of WO 96/18372. Further embodiments include the use of non-T-shaped amphiphiles as disclosed in the aforementioned patent publications, including protonated, partially protonated and deprotonated forms thereof.
  • the cationic amphiphile which can be used to deliver the vectors and plasmids of the invention is either N 4 -spermine cholesteryl carbamate (GL-67) having the following formula (I)
  • N 4 -spermidine cholesteiyl carbamate having the following formula (II)
  • one or more cationic amphiphiles may be fo ⁇ nulated with neutral co-lipids such as dileoylphosphatidylethanolamine (DOPE) to facilitate delivery of the vectors into a cell.
  • neutral co-lipids such as dileoylphosphatidylethanolamine (DOPE)
  • DOPE dileoylphosphatidylethanolamine
  • Other co-lipids which may be used in 9/36557
  • these complexes include, but are not limited to, diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines, other phosphatidylethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and cholesterol.
  • a preferred molar ratio of cationic amphiphile to colipid is 1 : 1. However, it is within the scope of the invention to vary this ratio, including also over a considerable range.
  • the cationic amphiphile N4-spermine cholesterol carbamate (GL-67) having the formula (I)
  • the neutral co-lipid DOPE are combined in a 1 :2 molar ratio, respectively, before complexing with an adenoviral vector for delivery to a cell.
  • a preferred range of 10 7 - 10 10 infectious units of vims may be combined with a range of 10 4 - 10 6 cationic amphiphile molecules/viral particle.
  • a preferred range of from A mM - 1 mM of cationic amphiphile may be combined with a range of 3 mM - 8 mM of plasmid to form the complexes.
  • Infection efficiency from adenoviral vectors containing the CMV- derived promoter elements of the invention may be assayed by standard techniques. Such methods include, but are not limited to, plaque formation, end-point dilution using, for example, an antibody to the adenoviral hexon protein, and cell binding assays using radiolabelled virus. Improved infection efficiency may be characterized as an increase in infection of at least one order of magnitude with reference to a control virus.
  • Persistent expression of a transgene from adenoviral vectors comprising the promoter elements of the invention following the infection of target cells or persistent expression from plasmids comprising the promoter elements of the invention following transfection, electroporation or other method of plasmid transfer to target cells may be assayed by standard techniques.
  • an adenoviral vector or plasmid comprising the promoter element of the invention encodes a marker or other transgene
  • relevant molecular assays to determine expression include the measurement of transgene mRNA, by, for example, but not by way of limitation, Northern blot, SI analysis or reverse transcription-polymerase chain reaction (RT-PCR).
  • the presence of a protein encoded by a transgene may be detected by Western blot, immunoprecipitation, immunocytochemistry, or other techniques known to those skilled in the art. Marker-specific assays can also be used, such as X-gal staining of cells infected with an adenoviral vector encoding ⁇ -galactosidase.
  • Preferred target cells which can be used in tissue culture to determine persistence of transgene expression from an adenoviral vector comprising a transgene operably linked to a promoter element of the invention include, but are not limited to, primary cells such as hepatocytes, airway epithelial cells, muscle cells and endothelial cells.
  • Preferred target cells for determining the persistence of transgene expression from a plasmid containing a transgene operably linked to a promoter element of the invention include established cell lines, such as HeLa or COS cells, or primary cells.
  • Any cells or cell lines which may be transfected with the plasmids or infected with the viruses comprising a transgene operably linked to a promoter element of the invention are suitable for assays which measure the level and duration of expression of such a transgene.
  • Demonstration of persistent expression of a transgene from adenoviral vector or plasmid comprising a transgene operably linked to a promoter element of the invention in, for example, animal and/or human hepatocytes can be predictive of the ability of such a plasmid or virus to achieve persistent expression of the transgene in the liver of an animal or human.
  • animal models may be particularly relevant in order to assess transgene expression persistence against a background of potential host immune response.
  • a model may be chosen with reference to certain parameters such as ease of delivery, identity of transgene, relevant molecular assays, and assessment of clinical status.
  • an animal model which is representative of the disease state may optimally be used in order to assess a specific phenotypic result and clinical improvement through the persistent expression of the transgene.
  • knockout mice e.g. Fabry knockout mice (Ohshima et al., 1997, Proc. Natl. Acad. Sci. USA 94:2540-2544) and CFTR .knockout mice (Zeiher, B.G et al., 1995, J. Clin. Invest. 98:2051-2064)
  • vectors comprising the expression cassettes of the present invention which comprise at least a CMV-derived promoter element and a transgene.
  • Such .knockout mice may be used to assess the biological activity and persistent expression of a transgene of interest.
  • an expression cassette of the present invention comprising at least a CMV-derived promoter element and ⁇ -galactosidase as the transgene, may be administered to Fabry knockout mice in order to assess persistent transgene expression of the gene, biological activity of the expressed transgene and clinical improvement of the knockout mice (see U.S. Patent Application Serial No. 09/182,245, filed October 29, 1998 and PCT
  • an expression cassette of the present invention comprising at least a CMV derived promoter element and the CFTR as the transgene may be administered to CFTR .knockout mice to assess persistent transgene expression, biological activity of the expressed transgene and clinical improvement of the knockout mice. See Scaria, A. et al., 1998, Journal of Virology 72:7302-7309, U.S. Patent Application Serial No. 08/839,553, filed April 14, 1997 and PCT Publication No. WO 98/46780, incorporated herein by reference).
  • adenoviral vectors may display enhanced infection efficiency only in human model systems, e.g., using primary cell cultures, tissue explants, or permanent cell lines.
  • human model systems e.g., using primary cell cultures, tissue explants, or permanent cell lines.
  • reference to art-recognized human cell culture models may be relevant and definitive.
  • Relevant animals in which the adenoviral vectors or plasmids may be assayed include, but are not limited to, mice, rats, monkeys, and rabbits.
  • Suitable mouse strains in which the vectors may be tested include, but are not limited to, C3H, C57BL/6 (wild-type and nude) and Balb/c (available from Taconic Farms, Germantown, New York).
  • testing in immunocompetent and immunodeficient animals may be compared in order to define specific adverse responses generated by the immune system.
  • the use of immunodeficient animals, e.g., nude mice may be used to characterize vector or plasmid performance and persistence of transgene expression, independent of an acquired host response.
  • transgene is the gene encoding cystic fibrosis transmembrane conductance regulator protein (CFTR) which is administered to the respiratory epithelium of test animals
  • expression of CFTR may be assayed in the lungs of relevant animal models, for example, C57BL/6 or Balb/c mice, cotton rats, or Rhesus monkeys.
  • CFTR mRNA Molecular markers which may used to determine expression include the measurement of CFTR mRNA, by, for example, Northern blot, SI analysis or RT- PCR.
  • the presence of the CFTR protein may be detected by Western blot, immunoprecipitation, immunocytochemistry, or other techniques known to those skilled in the art.
  • Such assays may also be used in tissue culture where cells deficient in a functional CFTR protein which have been infected with the adenoviral vectors may be assessed to determine the presence of functional chloride ion channels - indicative of the presence of a functional CFTR molecule. See, for example, Zabner et al., J. Clin. Invest. 97:1504-1511 (1996).
  • the adenoviral vectors and plasmids comprising the promoter elements of the invention have a number of in vivo and in vitro utilities.
  • the vectors and plasmids can be used to transfer a normal copy of a transgene encoding a biologically active protein to target cells in order to remedy a deficient or dysfunctional protein.
  • the vectors and plasmids can be used to transfer marked transgenes (e.g. , containing nucleotide alterations) which allow for distinguishing expression levels of a transduced transgene from the levels of the corresponding endogenous gene.
  • the adenoviral vectors can also be used to define the mechanism of specific viral protein- cellular protein interactions that are mediated by specific virus surface protein sequences.
  • the adenoviral vectors can also be used to optimize infection efficiency of specific target cells by adenoviral vectors, for example, but not by way of limitation, using a chimeric adenoviral vector containing Ad 17 fiber protein to infect human nasal polyp cells (e.g. PCT Publication No. WO 98/22609 incorporated herein by reference). Where it is desirable to use an adenoviral vector for transgene transfer to cancer cells in an individual, an adenoviral vector can be chosen which selectively infects the specific type of target cancer cell and avoids promiscuous infection.
  • the cells may be tested against a panel of adenoviral vectors and plasmids to select a vector or plasmid with optimal infection efficiency for transgene delivery.
  • the vectors can further be used to transfer transgenes encoding tumor antigens to dendritic cells which can then be delivered to an individual to elicit an anti-tumor immune response.
  • the adenoviral vectors can also be used to evade undesirable immune responses to particular adenovirus serotypes which compromise the gene transfer capability of adenoviral vectors.
  • compositions which comprise the adenoviral vectors and plasmids comprising the promoter elements of the invention which can be administered to cells or tissues in an amount effective to deliver one or more desired transgenes to the cells of an individual in need of such molecules and cause expression of a transgene encoding a biologically active protein to achieve a specific phenotypic result or to produce the biologically active protein.
  • the cationic amphiphile-plasmid complexes or cationic amphiphile-virus complexes similarly may be formulated into compositions for administration to an individual in need of the delivery of the transgenes.
  • compositions can include physiologically acceptable carriers, including any relevant solvents.
  • physiologically acceptable carriers including any relevant solvents.
  • physiologically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the compositions is contemplated.
  • Routes of administration for the compositions comprising the adenoviral vectors or plasmids of the invention include conventional and physiologically acceptable routes such as, but not limited to, direct delivery to a target organ or tissue, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parenteral routes of administration.
  • the invention is further directed to methods for using the compositions of the invention in in vivo or ex vivo applications in which it is desirable to deliver one or more transgenes into cells such that the transgene produces a biologically active protein for a normal biological or phenotypic effect.
  • In vivo applications involve the direct administration of one ore more adenoviral vectors or plasmids formulated into a composition and delivered to the cells of an individual.
  • Ex vivo applications involve the direct transfer of compositions comprising the vector or plasmid to autologous cells which are maintained in vitro, followed by readministration of the transduced cells to a recipient.
  • Dosage of the adenoviral vector or plasmid to be administered to an individual for expression of a transgene encoding a biologically active protein and to achieve a specific phenotypic result is determined with reference to various parameters, including the condition to be treated, the age, weight and biological or clinical status of the individual, and the particular molecular defect requiring the furnishing of a biologically active protein.
  • the dosage is preferably chosen so that administration causes a specific phenotypic result, as measured by molecular assays or clinical markers.
  • determination of the infection efficiency of an adenoviral vector or plasmid containing the CFTR transgene which is administered to an individual can be performed by molecular assays including the measurement of CFTR mRNA, by, for example, Northern blot, SI or RT-PCR analysis or the measurement of the CFTR protein as detected by Western blot, immunoprecipitation, immunocytochemistry, or other techniques .known to those skilled in the art.
  • Relevant clinical studies which could be used to assess phenotypic results from delivery of the CFTR transgene include, but are not limited to, PFT assessment of lung function and radiological evaluation of the lung.
  • Transgene expression and phenotypic alteration associated with transgene expression can be assayed analogously, using the specific biological parameters most relevant to the condition.
  • Dosages of an adenoviral vector comprising the promoter elements of the invention which are effective to provide expression of a transgene encoding a biologically active protein and achieve a specific phenotypic result range from approximately 10 8 infectious units ( U.) to 10" I.U. for humans.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active ingredient calculated to produce the specific phenotypic effect in association with the required physiologically acceptable carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependant on the unique characteristics of the adenoviral vector or plasmid and the limitations inherent in the art of compounding.
  • the principal active ingredient (the adenoviral vector or plasmid) is compounded for convenient and effective administration in effective amounts with the physiologically acceptable carrier in dosage unit form as discussed above.
  • adenoviral vectors and plasmids of the invention may require repeated administration.
  • Such repeated administration may involve the use of the same adenoviral vector or plasmid, or, alternatively, may involve the use of different adenoviral vectors which are rotated in order to alter viral antigen expression and decrease host immune response.
  • ⁇ CMV ⁇ gal-1 is based on Ad2/ ⁇ gal-5 (complete E4 deletion, Armentano et al. 1997, J. Virol. 71:2408-2416, 1997) and contains a promoter element which is a truncated CMV promoter, containing nucleotide sequences -295 to -14 (see Figure 2; SEQ ID NO. 2).
  • a pre-virus plasmid, pAdCMV ⁇ gal (Armentano et al. 1997, J. Virol. 71 :2408-2416, 1997) was cut with restriction endonucleases Clal and SnaBI which removes all sequences of the CMV promoter upstream of the SnaBI site (-242).
  • the removed sequences were replaced with a Clal - SnaBI oligonucleotide adapter (containing CMV promoter sequences -295 to -242; see Figure 4, SEQ ID NO:4) to extend promoter sequences to the -295 position.
  • the resulting plasmid, pAd ⁇ CMV ⁇ gal-1 was cut with BstBI and recombined with PshAI digested Ad2/ ⁇ gal-5 DNA in VK2-20 cells to generate ⁇ CMV ⁇ gal.
  • Rat hepatocytes were isolated from Sprague-Dawley rats by perfusion with .05% collagenase, washed with Hepato-Stim media (Beckton-Dickinson) several times and plated in a hepatocyte differentiation environment (Becton-Dickinson). The following day hepatocytes were infected with Ad2/ ⁇ gal-4, Ad2/ ⁇ gal-2 or ⁇ CMV ⁇ gal- 1 at an moi of 50. The media was changed every other day throughout the course of the experiment. At the indicated time points cultures were treated with dispase to remove cells from the extracellular matrix. See Figure 7. Cells were pelleted, washed with PBS, pelleted again and resuspended in lysis buffer.
  • Human hepatocytes were obtained from Clonetics and were maintained in Hepatocyte Maintenance media (Clonetics). Cultures were infected at an moi of 50 with Ad2/ ⁇ gal-4, Ad2/ ⁇ gal-2 or ⁇ CMV ⁇ gal- 1 alone or were co-infected with Ad2/CMVAAT, a vector that could supply E4 function in trans. Cells were harvested at the indicated time points ( Figure 8) by incubation with dispase and analyzed as in Example 1 for ⁇ -galactosidase activity. The results in Figure 8 indicate that expression from Ad2/ ⁇ gal-4 could persist to day 11 and was not further enhanced by the co-infection of a virus that supplies E4 functions in trans.
  • ⁇ CMV ⁇ gal- 1 on days 3 and 11 was in the range of levels seen with Ad2/ ⁇ gal-4 and was not further enhanced by co-infection with Ad2/CMVAAT. The results indicate that the ⁇ CMV promoter element does not require E4 for maintained elevated levels of expression and is no longer influenced by supplying E4 in trans.
  • EXAMPLE 4 Effect of a CMV-derived promoter element on plasmid-provided transgene expression in mice.
  • Plasmid pCFl-299/-19-CAT was constructed by first digesting pCFl- SEAP (pCFl plasmid containing the gene for secreted alkaline phosphatase (SEAP) and an additional upstream polylinker called PCFA) (Yew et al., Hum Gene Ther. 8:575-584, 1997) with Pme I and Bgl I, blunting the ends with the Klenow fragment of DNA polymerase I, then religating.
  • This vector was digested with Not I to excise SEAP and the CAT cDNA was ligated into the Not I site to form pCFl-299-CAT. Tliis vector was then digested with Sac I and Xba I blunted with Klenow, then relegated.
  • the promoter element in the plasmid comprises the sequence of Figure 3 (SEQ ID NO. 3).
  • Cationic Hpid:DOPE:pDNA complexes were prepared as described previously (Lee et al., Hum. Gene Ther. 7:1701-1717, 1996; U.S. Patent No. 5,650,096 and PCT Publication No. WO 96/18372, all incorporated herein by reference ). Briefly, equal volumes of liposomes and plasmid DNA were mixed to a final concentration of 0.6 mM GL-67: 1.2 mM DOPE: 3.6 mM pCF A-299 9-CAT and allowed to sit 15 minutes at room temperature. Nude B ALB/c mice were instilled intranasally with 100 ⁇ l of lipid:pDNA complex as described previously (Lee et al., Hum. Gene Ther.
  • mice were instilled within 15 minutes of complex formation. At different days post- instillation, lungs were harvested and frozen at -80 °C for later processing. CAT activity was assayed as described in the afore-mentioned references.
  • EXAMPLE 5 Effect of a human albumin gene-derived enhancer element operatively linked to
  • Plasmids pBsl.7-2HI-AGA. L, pBsl.7-3HI-AG.AL, and pBsl.7-5HI- AGAL were constructed as follows: A double stranded 65bp fragment comprising the - 1.7kb human albumin-derived enhancer element (SEQ ID NO:7) was generated by annealing two complimentary oligos (sythesized by Operon, Alameda, CA) by standard techniques. See, eg., Current Protocols in Molecular Biology. Ausubel et al., eds., John Wiley & Sons, Inc., New York, 1995.
  • the annealed double stranded 65bp fragment comprising the -1.7kb human albumin-derived enhancer element has 5' overhangs that can be ligated into a Clal restriction site. Multiple copies were ligated into pBluescriptIIsk+ (Strategene, La Jolla, CA) which was digested with Clal restriction enzyme. PbluescriptIIsk+ vectors containing 2, 3 or 5 copies were isolated and were digested with EcoRV and Xbal.
  • the digested vectors were ligated to a SnaBI-Xbal digested fragment of the wild-type CMV promoter (-242 to -14) (SEQ ID NO:4), a hybrid intron (from pAD ⁇ , Clonetech), wild-type ⁇ -galactosidase cDNA and the SV40 polyadenylation signal.
  • Plasmids pBsl .7-2HI-AGAL, pBsl .7-3HI-AGAL, .and pBsl.7-5HI-AGAL contain 2, 3 and 5 copies of the 65bp -1.7 human albumin- derived enhancer element respectively.
  • 293 cells were obtained from Frank Graham and were maintained in DMEM supplemented with 1 mM L-glutamine and 10% fetal bovine serum.
  • Hep3B cells hepatocellular cell line; ATCC
  • ATCC hepatocellular cell line
  • Eagle's minimum essential medium supplemented with 2 mM L-glutamine, Earle's BSS (balanced salt solution) to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyiuvate and 10% fetal bovine serum.
  • Cell lines were transfected with the indicated plasmids ( Figure 11) by the CaPO 4 precipitation method. See Graham, F.L. and van der Eb, A.J., 1973, Virology 52:456-467.

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