AU2231099A - Novel promoter elements for persistent gene expression - Google Patents

Description

WO 99/36557 PCT/US99/00915 DESCRIPTION Novel Promoter Elements For Persistent Gene Expression Field of the Invention 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 5 are derived from the cytomegalovirus intermediate early promoter (CMV). 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. In particular embodiments of the invention, adenoviral vectors or plasmids comprising the CMV-derived promoter elements, 10 operatively linked to a transgene, are used to achieve persistent expression of the transgene. Background of the Invention The transfer of a transgene to recipient cells such that a biologically active and useful product (e.g. RNA, antisense RNA, protein, hormone, enzyme, etc.) 15 is produced in the recipient cell is an important aspect of effective gene transfer. There are two important parameters for consideration - the effective transfer of such transgenes to recipient cells and tissues and the persistent or continued expression of the transgene in the target cell. Transfer of transgenes to target cells has been the focus of much 20 inquiry and experimentation - leading to the development of various methods and modalities to accomplish such transfer. Transgene transfer has involved the use and development of viral vectors, such as those derived from retroviruses, adenoviruses, herpes viruses, vaccinia and adeno-associated virus, among others. Also, transgene transfer has been effectuated using plasmids, naked DNA, lipids and combinations of WO 99/36557 PCT/US99/00915 2 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 5 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 (known as E1-E4) and late (known as Ll-L5) transcriptional units, representing two temporal classes of viral proteins. The demarcation between early and late viral protein expression is viral DNA replication. 10 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 15 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 (Berkner, K.L., Curr. 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 20 of the adenovirus genome present in the vector. For example, 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). Such El 25 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. Further 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), 30 complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang WO 99/36557 PCT/US99/00915 3 et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Allowed U.S. Patent Application Serial No. 08/895,194, incorporated herein by reference; Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995). Maximal carrying capacity can be achieved using adenoviral 5 vectors deleted for all viral coding sequences (Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996). Transgenes that have been expressed to date by adenoviral vectors include, inter alia, p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin 10 (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. 271;3639-3646, 1996;); adenosine deaminase (Mitani et al., Human Gene Therapy 5:941-948, 1994); interleukin-2 (Haddada et al., Human Gene Therapy 4:703-711, 1993); and al-antitrypsin (Jaffe et al., Nature Genetics 1:372-378, 1992); 15 thrombopoietin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996). The particular tropism of adenoviruses for cells of the respiratory tract has relevance to the use of adenovirus in transgene transfer for cystic fibrosis (CF), which is the most common autosomal recessive disease in Caucasians. Mutations in 20 the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb the cAMP-regulated Cl- channel in airway epithelia result in pulmonary dysfunction (Zabner et al., Nature Genetics 6:75-83, 1994). 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 25 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). Recent studies have shown that administering an adenoviral vector containing a DNA encoding CFTR to airway epithelial cells of CF patients can restore a 30 functioning chloride ion channel in the treated epithelial cells (Zabner et al., J. Clin.

WO 99/36557 PCT/US99/00915 4 Invest. 97:1504-1511, 1996; U.S. Patent No. 5,670,488, issued September 23, 1997). Additionally, tissue specific expression can be obtained from adenovirus vectors by the use of tissue specific promoter sequences. See, for example, PCT publication WO 97/04117 which describes a liver specific adenovirus expression vector characterized 5 in that a therapeutic transgene which is coupled to a liver-specific 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. The use of adenovirus vectors in transgene transfer studies to date indicates that persistence of transgene expression is often transient. At least some of 10 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 15 467, 1995; Worgall et al., Hum. Gene Ther. 8:37-44, 1997; Kaplan et al., Hum. Gene Ther. 8:45-56, 1997). Because 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 20 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. Verma, I.N. and Somia, N., 1997, Science 389:239-242. An adverse immune response can pose a serious obstacle for high dose administration of an adenovirus 25 vector and for repeated administration (Crystal, R., Science 270:404-410, 1995). In order to circumvent the host immune response which can contribute to limiting the persistence of transgene expression, various strategies have been employed, generally involving either the modulation of the immune response itself or the engineering of a vector that decreases the immune response. The administration of 30 immunosuppressive agents together with vector administration has been shown to WO 99/36557 PCT/US99/00915 5 prolong transgene expression (Fang et al., Hum. Gene Ther. 6:1039-1044, 1995; Kay et al., Nature Genetics 11:191-197, 1995; Zsellenger et al., Hum. Gene Ther. 6:457 467, 1995) and allow for repeat administration of the vector. Engelhardt et al. (Proc. Natl. Acad. Sci. USA 91:6196-6200 (1994)) have demonstrated that the use of broad 5 innunosupressants can overcome the immunologic problems associated with repeat administration of Adenovirus vectors. Similarly, Dai et al. ((Proc. Natl. Acad Sci. USA 92:1401-1405 (1995)) have shown similar results using cytoablative agents to overcome the immune response of the host to first generation adenovirus vectors. However, although immunosuprressive drugs can extend the duration of expression 10 obtained from adenoviral vectors, it is more desirable to manipulate the vector to achieve prolonged expression rather than the patient. Verma, I.N. and Somia, N., 1997, Science 389:239-242. Modifications to genomic adenoviral sequences contained in the recombinant vector have been attempted in order to decrease the host immune 15 response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al., J. Virol. 70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996). The adenovirus E3 gp 19K protein can complex with MHC Class I antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and 20 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. The lack of persistence in the expression of adenovirus vector delivered transgenes may also be due to limitations imposed by the choice of promoter 25 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). Experiments in which adenovirus vectors were introduced into nude and immunocompetent mice demonstrated that the E4 region to the adenovirus genome contributed to persistent transgene expression, especially when the transgene 30 was operably linked to a wild-type CMV promoter which controlled the transgene WO 99/36557 PCT/US99/00915 6 (Kaplan et al., Hum. Gene Ther. 8:45-56, 1997; Armentano et al., J. Virol. 71:2408 2416, 1997; PCT publication WO 98/46781). In addition to the use of adenoviral vectors, plasmids comprising a transgene may be used to deliver the transgene to a target cell. It is desirable that 5 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 10 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. Long term correction of a missing or faulty gene product in a cell could perhaps be achieved using a vector designed to integrate into chromosomes in the 15 targeted cells (for example, vectors patterned on retrovirus). Such a strategy, however, involves risks including (1) that the vector will integrate into an essential region of a chromosome, (2) that the vector will integrate adjacent to an oncogene and activate it. Accordingly, one strategy has been to provide for continued 20 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 HindIII-XhoI fragment that is present immediately 5' to exon 1 of the human c-myc gene contains an origin of replication 25 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. McWhinney et al.). Approximately 5% of the plasmid population was lost per cell generation without drug selection demonstrating substantial stabilization which would be of benefit with 30 respect to the design of therapeutic plasmids for gene therapy.

WO 99/36557 PCT/US99/00915 7 One example of a replicating episomal vector, pCF 1-CAT (PCT publication WO 96/18372 Figure 18A), may be constructed in which a copy of the 2.4 kb HindIII-XhoI fragment is placed just 5' to the CMV enhancer/promoter region of the pCF 1 backbone. Alternatively, between 2 and about 4 - in tandem - copies of the 5 2.4 kb fragment may be similarly positioned. 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, 10 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 3-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, 15 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. Further optimization of 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 20 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. 25 WO 99/36557 PCT/US99/00915 8 Summary of the Invention The invention is directed to a novel promoter element for persistent expression of an operably linked transgene. In one aspect, the element is derived from the cytomegalovirus intermediate early promoter (CMV). In an embodiment of the 5 invention, an adenoviral vector comprising a CMV-derived promoter element operably linked to a transgene is administered to recipient cells. In another embodiment of the invention, 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 10 disclosed in WO 96/18372 or U.S. Patent No. 5,650,096. Also within the scope of the invention are 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 15 and promoter elements of the invention in transgene transfer. Brief Description of the Drawings Figure 1. Sequence of the CMV intermediate early promoter, showing nucleotides -523 to -14 (SEQ ID NO:1). Figure 2. Sequence of a CMV-derived promoter element of the 20 invention, showing nucleotides -295 to -14 (SEQ ID NO:2). Figure 3. Sequence of a CMV-derived promoter element of the invention, showing nucleotides -299 to -19 (SEQ ID NO:3). Figure 4. Sequence of a CMV-derived promoter element of the invention, showing nucleotides -242 to -14 (SEQ ID NO:4) 25 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) WO 99/36557 PCT/US99/00915 9 Figure 6. Schematic representation of transcriptional repressor binding sites in the CMV promoter. Figure 7. Expression of P-galactosidase in rat hepatocytes using a promoter of the invention. 5 Figure 8. Expression of P-galactosidase in human hepatocytes using a promoter of the invention. Figure 9. Expression of P-galactosidase in Balb/c lungs using a promoter of the invention. Figure 10. Expression of CAT in mice using a promoter of the 10 invention. Figure 11. 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. 15 Detailed Description of the Invention The present invention is directed to a novel promoter element for the persistent expression of an operably linked transgene. In one aspect, the element is derived from the cytomegalovirus intermediate early promoter (CMV). In an embodiment of the invention, an adenoviral vector comprising a CMV-derived 20 promoter element of the invention operatively linked to a transgene is used to achieve persistent expression of a transgene when administered to target cell. In another embodiment, 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. Also within the scope of the invention are enhancer 25 elements which effectuate increased expression of a transgene operably linked to CMV-derived promoter elements. In one aspect, 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 WO 99/36557 PCT/US99/00915 10 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 5 antisense RNA heterologous to the carrier vector. Such 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 10 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 NO: 1), and provides for 15 persistent expression of a transgene operably linked thereto. 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 20 containing nucleotides -242 to -14 of the wild-type CMV promoter (Figure 4) (SEQ ID NO:4) (referred to as ACMV promoter elements). Other 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. Where a wild 25 type CMV promoter is dependent upon adenoviral E4 sequences to confer persistent expression (see, e.g. WO 98/46781), 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. In another embodiment, promoter elements which are capable of conferring persistent expression may be 30 constructed, for example, by deletion of sites within the CMV promoter sequence to WO 99/36557 PCT/US99/00915 11 which transcription repressor proteins can bind. Removal of such sites from the wild type CMV promoter may lead to more sustained expression (see Figure 6). Examples of such 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. 5 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. 16:8029-8044, 1988); IE2 (Liu et al., J.Virol. 65:897-603, 1991), CREB/CREM (Foulkes et al., Cell 64:739-749, 1991; Karpinski et al., Proc.Natl. Acad. Sci. USA 89:4820-4824, 1992; Lamph et al., Proc.Natl.Acad.Sci.USA 87:4320-4324, 1990; 10 Lemaigre et al., Nucleic Acids Res. 21:2907-2911, 1993) and DrIl (Kim et al., Proc.Natl.Acad.Sci.USA 94:820-825, 1997; White et al., Science 266:448-450, 1994). 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 15 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. Alternatively, other 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 20 persistent expression. Such activators, include, for example, NFkappap (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. 25 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. Furthermore, 30 nucleotide substitutions within the CMV-derived promoter elements of the invention WO 99/36557 PCT/US99/00915 12 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. YY1), such that the repressors can no longer bind. 5 Preferred 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 10 -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 15 skilled in the art. Additionally, 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. 20 Also within the scope of the invention are 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). For example, an RSV-derived promoter element can be constructed to delete or alter the serum response elements (SRE) to which the transcriptional 25 repressor protein YY1 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). Transgenes which can be delivered and expressed from a promoter element of the invention include, but are not limited to, those encoding enzymes, 30 blood derivatives, hormones, lymphokines such as the interleukins and interferons, WO 99/36557 PCT/US99/00915 13 coagulants, growth factors, neurotransmitters, tumor suppressors, apoliproteins, antigens, and antibodies, and other biologically active proteins. Specific 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), 5 dystrophin, glucocerebrosidase, tumor necrosis factor, p53, retinoblastoma (Rb), von hippel lindau (VHL), pten tumor suppressor, p 16, Glut4, and adenosine deaminase (ADA). Transgenes encoding antisense molecules and ribozymes are also within the scope of the invention. Gene transfer vehicles of the invention, such as adenoviral vectors or plasmids, may contain one or more transgenes operably linked to a CMV 10 derived or other promoter element of the invention. In accordance with the present 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 15 polyadenylation element and any other regulatory elements that may be used to modulate or increase expression or a transgene when operably linked thereto. Non limiting examples of enhancer elements include apoE enhancer elements (Shachter N.S., etal., 1993, JLipidRes. 34:1699-707; Allan C.M. et al., 1995, J. Biol. Chem. 270:26278-81), al antitrypsin enhancer elements (Morgan K. et al., 1997, Biochim. 20 Biophys. Acta. 1362:67-76. , human fibrinogen enhancer elements (Hu C.H. et al., 1995 J Biol. Chem. 270:28342-9) the human cytochrome P4501A1 gene enhancer elements (Kress, S. et al., Eur. J. Biochem. 258:803-812, 1998), the human carboxyl ester lipase gene enhancer elements (Lidberg, U. et al., J. Biol. Chem. 273:31417 31426, 1998), porcine alpha-skeletal actin gene enhancer elements (Reecy, J.M. et al., 25 Anim. 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). In particular embodiments of the invention, an enhancer element of the 30 invention is derived from human albumin gene enhancer sequences and, when placed WO 99/36557 PCT/US99/00915 14 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 5 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. 10 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. 15 267:14580-14585, 1992), and provides for increased expression of a transgene operably linked to a CMV-derived promoter element of the invention. 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 20 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 25 NO:6). In a particular embodiment of the invention, 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. However, the promoter elements of the invention may also be used 30 with other viral vectors useful for gene transfer, including, but not limited to, those WO 99/36557 PCT/US99/00915 15 derived from retroviruses, herpesviruses, adeno-associated virus, and others known to those skilled in the art. Specific examples of adenoviral vectors which can be used in the invention include, for example, Ad2/CFTR-1 and Ad2/CFTR-2 and others described 5 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. For example, 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 10 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. Within the scope of the invention are also, for example, chimeric viral vectors which contain an Ad 2 15 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 20 incorporated herein by reference). In a particular embodiment, 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 25 for a vector used for gene transfer in vivo. In further preferred embodiments, 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. Moreover, adenoviral vectors for use in the invention derived from other group C or non-group C WO 99/36557 PCT/US99/00915 16 adenoviruses are also within the scope of the invention, including chimeric adenoviral vectors which contain nucleotide sequences from one or more serotypes. In order to construct an adenoviral vector for use in the invention, reference may be made to the substantial body of literature on how such vectors may 5 be designed, constructed and propagated using techniques from molecular biology and microbiology that are well-known to the skilled artisan. For example, 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. 10 Immunol. 158:39-66, 1992). For example, a plasmid containing a transgene and any operably linked CMV-derived promoter element of the invention inserted into 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 15 virus. In a preferred embodiment, the transgene and the operably linked CMV derived promoter element of the invention are inserted into the site of an El deletion. As a result, 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. 20 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, 25 e.g., 293 or A549 cells containing the deleted adenovirus El genomic sequences. HER3 cells (human embryonic retinoblasts transformed by Ad 12) may also be used. After amplification of plaques in suitable complementing cell lines, the viruses may be recovered by freeze-thawing and may subsequently be purified using cesium chloride centrifugation. Alternatively, virus purification may be performed using WO 99/36557 PCT/US99/00915 17 chromatographic techniques, 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 5 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 ACMV-Pgal-1, which comprises a CMV-derived 10 promoter element comprising nucleotides -295 to -14, operably linked to a P 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 15 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., John Wiley & Sons, Inc., New York, 1995). Plasmids may be delivered to target cells using such techniques as transfection, electroporation, microinjection, and other DNA transfer 20 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 25 a target cell in the form of naked DNA is also within the scope of the invention. Where 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 pCF 1-CAT (PCT publication WO 96/18372 Figure 18A), containing the chloramphenicol acetyltransferase (CAT) 30 gene operatively linked to the wild-type CMV promoter which may be truncated to WO 99/36557 PCT/US99/00915 18 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 P-galactosidase and luciferase. Proteins expressed from marker genes may be readily detected by standard techniques. 5 In a preferred embodiment, the plasmid pCFA-299/-19 CAT (Example 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 10 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. 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 15 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. 20 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 (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) (N-[1-( 2 ,3-dioletloxy)propyl].-N,N,N 25 trimethylammonium chloride); DOGS (dioctadecylamidoglycylspermine) (Behr et al., Proc. Natl. Acad. Sci. USA 86:6982-6986, 1989); DMRIE (1, 2 -dimyristyloxypropyl 3 -dimethyl-hydroxyethyl ammonium bromide) (Felgner et al., J. Biol. Chem. 269:2550-2561, 1994; and DC-chol (3B [N-N', N'-dimethylaminoethane) -carbamoyl] cholesterol) (U.S. Patent No. 5, 283,185). The use of other cationic amphiphiles WO 99/36557 PCT/US99/00915 19 recognized in the art or which may later be discovered is also within the scope of the invention. In preferred embodiments of the invention, the cationic amphiphiles useful to complex with and facilitate transfer of the vectors and plasmids of the 5 invention are those lipids disclosed in U.S. Patent No. 5,650,096 and in PCT Publication No. WO 96/18372, published June 20, 1996, both incorporated herein by reference. Preferred 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 10 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. Most preferably, 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 15 the following formula (I) (I) or N 4 -spermidine cholesteryl carbamate (GL-53) having the following formula (II) (II) 20 In the formulation of compositions comprising the adenoviral vectors and plasmids used in the invention, one or more cationic amphiphiles may be formulated with neutral co-lipids such as dileoylphosphatidylethanolamine (DOPE) to facilitate delivery of the vectors into a cell. Other co-lipids which may be used in WO 99/36557 PCT/US99/00915 20 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. 5 However, it is within the scope of the invention to vary this ratio, including also over a considerable range. In a particularly preferred embodiment of the invention, the cationic amphiphile N4-spermine cholesterol carbamate (GL-67) having the formula (I) (I) 10 and 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. In the formulation of complexes containing a cationic amphiphile with an adenoviral vector comprising the CMV-derived promoter element of the invention, a preferred range of 107 - 101" infectious units of virus may be combined with a range 15 of 104 - 106 cationic amphiphile molecules/viral particle. In the formulation of complexes containing a cationic amphiphile with a plasmid, a preferred range of from .4 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 20 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 25 control virus.

WO 99/36557 PCT/US99/00915 21 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 5 to target cells may be assayed by standard techniques. Where 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, S I analysis or reverse transcription-polymerase chain reaction (RT-PCR). The presence 10 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 15 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 20 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 25 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. In order to determine the persistence of transgene expression and 30 infection efficiency in vivo using constructs and compositions according to the present WO 99/36557 PCT/US99/00915 22 invention, animal models may be particularly relevant in order to assess transgene expression persistence against a background of potential host immune response. Such 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 5 status. Where the transgene encodes a protein whose absence or mutation is associated with a particular disease state, 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. For example, knockout mice (e.g. Fabry knockout mice (Ohshima et 10 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)) may be infected or transfected with 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 15 of a transgene of interest. Specifically, but not by way of limitation, an expression cassette of the present invention, comprising at least a CMV-derived promoter element and a-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. 20 Patent Application Serial No. 09/182,245, filed October 29, 1998 and PCT Application No. PCT/US98/22886, filed October 29, 1998, incorporated herein by reference). Similarly, 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, 25 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). It is also possible that particular adenoviral vectors may display 30 enhanced infection efficiency only in human model systems, e.g., using primary cell WO 99/36557 PCT/US99/00915 23 cultures, tissue explants, or permanent cell lines. In such circumstances where there is no animal model system available in which to model the infection efficiency of an adenoviral vector with respect to human cells, reference to art-recognized human cell culture models may be relevant and definitive. 5 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). 10 Where it is desirable to assess the host immune response to vector or plasmid administration, 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 15 expression, independent of an acquired host response. In a particular embodiment where the 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 20 mice, cotton rats, or Rhesus monkeys. Molecular markers which may used to determine expression include the measurement of CFTR mRNA, by, for example, Northern blot, S 1 analysis or RT PCR. The presence of the CFTR protein may be detected by Western blot, immunoprecipitation, immunocytochemistry, or other techniques known to those 25 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).

WO 99/36557 PCT/US99/00915 24 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. 5 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 10 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 15 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. Where primary cells are isolated from a tumor in an individual requiring transgene transfer, 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. 20 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. 25 The present invention is further directed to 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 30 to achieve a specific phenotypic result or to produce the biologically active protein.

WO 99/36557 PCT/US99/00915 25 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. The compositions can include physiologically acceptable carriers, 5 including any relevant solvents. As used herein, but not by way of limitation, "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. 10 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. 15 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 20 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 25 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 30 administration causes a specific phenotypic result, as measured by molecular assays or WO 99/36557 PCT/US99/00915 26 clinical markers. For example, 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, S 1 or RT-PCR analysis or the 5 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. Productive delivery of a transgene encoding 10 CFTR may also be demonstrated by detecting the presence of a functional chloride channel in cells of an individual with cystic fibrosis to whom the vector comprising the transgene has been administered (Zabner et al., 1996, J. Clin. Invest. 97:1504 1511 and Scaria, A. et al., 1998, Journal of Virology 72:7302-7309). Transgene expression and phenotypic alteration associated with transgene expression can be 15 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 20 approximately 108 infectious units (I.U.) to 10" I.U. for humans. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active 25 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 30 plasmid) is compounded for convenient and effective administration in effective WO 99/36557 PCT/US99/00915 27 amounts with the physiologically acceptable carrier in dosage unit form as discussed above. Maximum benefit and achievement of a specific phenotypic result from administration of the adenoviral vectors and plasmids of the invention may require 5 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. The practice of the invention employs, unless otherwise indicated, 10 conventional techniques of protein chemistry, molecular virology, microbiology, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc., New York, 1995, and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, 15 PA, 1985. The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention.

WO 99/36557 PCT/US99/00915 28 EXAMPLE 1: Effect of a CMV-derived promoter element on transgene expression in rat hepatocytes ACMVigal-1 is based on Ad2/3gal-5 (complete E4 deletion, Armentano et al. 1997, J. Virol. 71:2408-2416, 1997) and contains a promoter 5 element which is a truncated CMV promoter, containing nucleotide sequences -295 to -14 (see Figure 2; SEQ ID NO. 2). A pre-virus plasmid, pAdCMVigal (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 10 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, pAdACMVpgal-1, was cut with BstBI and recombined with PshAI digested Ad2/Pgal-5 DNA in VK2-20 cells to generate ACMVP3gal. Rat hepatocytes were isolated from Sprague-Dawley rats by perfusion 15 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/pgal-4, Ad2/Pgal-2 or ACMV[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 20 remove cells from the extracellular matrix. See Figure 7. Cells were pelleted, washed with PBS, pelleted again and resuspended in lysis buffer. The supernatant was analyzed for [-galactosidase activity by Galactolight assay and protein was determined by BCA. As shown in Figure 7, expression from Ad2/Pgal-2 is diminished and does not persist in comparison to Ad2/pgal-4. Expression from 25 ACMVigal-1 is not diminished compared to Ad2/Pgal-4 and also appears to persist. Because the ACMVPgal-1 vector is E4 deleted, the results indicate that P-gal expression from the ACMV promoter does not require E4.

WO 99/36557 PCT/US99/00915 29 EXAMPLE 2: Effect of a CMV-derived promoter element on transgene expression in human hepatocytes Human hepatocytes were obtained from Clonetics and were maintained 5 in Hepatocyte Maintenance media (Clonetics). Cultures were infected at an moi of 50 with Ad2/pgal-4, Ad2/pgal-2 or ACMV[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 3-galactosidase activity. 10 The results in Figure 8 indicate that expression from Ad2/3gal-4 could persist to day 11 and was not further enhanced by the co-infection of a virus that supplies E4 functions in trans. Expression from Ad2/pgal-2 was diminished on days 3 and 11 in comparison to that observed from Ad2/pgal-4. In addition, expression was enhanced on days 3 and 11 when cultures were co-infected with Ad2/CMVAAT and 15 reached levels detected in Ad2/pgal-4 infected cultures. Expression from ACMVigal-1 on days 3 and 11 was in the range of levels seen with Ad2/pgal-4 and was not further enhanced by co-infection with Ad2/CMVAAT. The results indicate that the ACMV promoter element does not require E4 for maintained elevated levels of expression and is no longer influenced by supplying E4 in trans. 20 EXAMPLE 3: Effect of a CMV-derived promoter element on transgene expression in Balb/c lungs. Balb/c nude mice were intranasally instilled with 3 x 109 i.u. of Ad2/pgal-4 or ACMVigal-1 or were co-infected with both vectors. Mice were sacrificed on days 3 and 14 post-instillation and the lungs were analyzed for P3 25 galactosidase activity by a Galactolight assay. Results of the single infections and co infections represent n=2 and n=3 per time point respectively. Expression from the ACMV promoter element persisted in the lungs of Balb/c mice which indicates that this promoter element can give rise to long-term expression in vivo in the absence of E4 ORFs. Although the ACMV promoter WO 99/36557 PCT/US99/00915 30 element can function independently of E4 in this experimental system, expression was enhanced on day 14 by approximately 4-fold by the co-infection of a vector that could supply E4 in trans (Figure 9). EXAMPLE 4: 5 Effect of a CMV-derived promoter element on plasmid-provided transgene expression in mice. Plasmid pCF1-299/-19-CAT was constructed by first digesting pCF 1 SEAP (pCF1 plasmid containing the gene for secreted alkaline phosphatase (SEAP) and an additional upstream polylinker called PCFA) (Yew et al., Hum Gene Ther. 10 8:575-584, 1997) with Pine 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 pCF 1-299-CAT. This 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 15 (SEQIDNO. 3). Cationic lipid: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 20 final concentration of 0.6 mM GL-67:1.2 mM DOPE: 3.6 mM pCFA-299/-19-CAT and allowed to sit 15 minutes at room temperature. Nude BALB/c mice were instilled intranasally with 100 gl of lipid:pDNA complex as described previously (Lee et al., Hum. Gene Ther. 7:1701-1717, 1996; U.S. Patent No. 5,650,096, WO 96/18372). Mice were instilled within 15 minutes of complex formation. At different days post 25 instillation, lungs were harvested and frozen at -80 0 C for later processing. CAT activity was assayed as described in the afore-mentioned references. The results show that the initial level of expression from pCF 1-299/ 19CAT is lower than pCF 1-CAT (1 ng of CAT from pCF 1-299/-19-CAT versus 26 ng of CAT from pCF 1-CAT at day 2 post-instillation). However, expression from pCF I - WO 99/36557 PCT/US99/00915 31 299/-19-CAT is more persistent than pCF 1-CAT, with approximately 40% of day 2 levels present at day 35 post-instillation versus less than 1% of day 2 expression from pCF1-CAT (Figure 10). EXAMPLE 5: 5 Effect of a human albumin gene-derived enhancer element operatively linked to CMV-derived promoter elements on adenoviral vector-provided transgene expression in 293 cells and Hep3B cells. Plasmids pBsl .7-2HI-AGAL, pBsl .7-3HI-AGAL, and pBs1.7-5HI AGAL were constructed as follows: A double stranded 65bp fragment comprising the 10 -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, e., 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' 15 overhangs that can be ligated into a Clal restriction site. Multiple copies were ligated into pBluescriptllsk+ (Strategene, La Jolla, CA) which was digested with Clal restriction enzyme. Pbluescriptllsk+ vectors containing 2, 3 or 5 copies were isolated and were digested with EcoRV and XbaI. The digested vectors were ligated to a SnaBI-XbaI digested fragment of the wild-type CMV promoter (-242 to -14) (SEQ ID 20 NO:4), a hybrid intron (from pADP, Clonetech), wild-type a-galactosidase cDNA and the SV40 polyadenylation signal. Plasmids pBs1.7-2HI-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 25 DMEM supplemented with 1 mM L-glutamine and 10% fetal bovine serum. Hep3B cells (hepatocellular cell line; ATCC) were maintained in 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 pyruvate and 10% fetal bovine serum.

WO 99/36557 PCT/US99/00915 32 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. After 48 hours post-transfection the cells were harvested by 5 centrifugation and the media supernatant was collected. The media supernatant was assayed using the fluorescent substrate 4 -methylumbelliferyl-a-D-galactopyranoside

(

4 -mu-a-gal) as described (Desnick, et al., 1973, J. Lab. Clin. Med 81:157 and Johnson, D.L. et al., 1975, Clin. Chim. Acta. 63:81). This method was modified as described by Mayes et al. (Clin. Chim. Acta. 112:247-251 (1981)) to include inhibitors 10 to a-galactosidase B. The results in Figure 11 indicate that in 293 cells no difference in a galactosidase activity is achieved with the -1.7kb enhancer element. Expression levels of constructs with a truncated CMV promoter linked to the -1.7kb enhancer element 15 are comparable to that obtained with full length CMV promoter (Figure 11 A). However, in Hep3B cells, the constructs with the truncated CMV promoter (-242 to 14) and 2, 3, or 5 copies of the -1.7kb enhancer element all gave significantly higher levels of expression than that obtained from the wild-type CMV promoter lacking the enhancer regions and the expression from the construct containing 5 copies of the 20 enhancer yielded the greatest expression levels. (Figure 11 B).

Claims (54)

1. A CMV-derived promoter element comprising a truncated portion of a wild-type cytomegalovirus promoter which confers persistent expression of a transgene operably linked thereto in a target cell. 5

2. The CMV-derived promoter element of Claim 1 wherein the cytomegalovirus promoter is the cytomegalovirus immediate early promoter.

3. The CMV-derived promoter element of Claim 1 having a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. 10

4. The CMV-derived promoter element of Claim 1 having the nucleotide sequence of SEQ ID NO:2.

5. The CMV-derived promoter element of Claim 1 having the nucleotide sequence of SEQ ID NO:3 15

6. The CMV-derived promoter element of Claim 1 having the nucleotide sequence of SEQ ID NO:4.

7. An expression cassette comprising the CMV-derived promoter element of Claim 1 operably linked to a transgene, wherein the CMV-derived promoter 20 element confers persistent expression of the transgene in a target cell.

8. The expression cassette of Claim 7 wherein the CMV-derived promoter element has a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. WO 99/36557 PCT/US99/00915 34

9. The expression cassette of Claim 7 wherein the CMV-derived promoter element has the nucleotide sequence of SEQ ID NO:2.

10. The expression cassette of Claim 7 wherein the CMV-derived promoter element has the nucleotide sequence of SEQ ID NO:3. 5

11. The expression cassette of Claim 7 wherein the CMV-derived promoter element has the nucleotide sequence of SEQ ID NO:4.

12. An adenoviral vector comprising the expression cassette of Claim 7.

13. The adenoviral vector of claim 12 comprising an adenovirus genome from which the E4 region of the adenovirus genome has been deleted. 10

14. The adenoviral vector of Claim 12 wherein the CMV-derived promoter element of the expression cassette has a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

15. The adenoviral vector of Claim 12 wherein the CMV-derived promoter element of the expression cassette has the nucleotide sequence of SEQ ID NO:2. 15

16. The adenoviral vector of Claim 12 wherein the CMV-derived promoter element of the expression cassette has the nucleotide sequence of SEQ ID NO:3.

17. The adenoviral vector of Claim 12 wherein the CMV-derived promoter element of the expression cassette has the nucleotide sequence of SEQ ID NO:4.

18. The adenoviral vector of Claim 12 wherein the transgene of the 20 expression cassette encodes a protein selected from the group consisting of human WO 99/36557 PCT/US99/00915 35 cystic fibrosis transmembrane conductance regulator protein and human a galactosidase.

19. The adenoviral vector of Claim 12 wherein the transgene of the expression cassette encodes human cystic fibrosis transmembrane conductance 5 regulator protein.

20. The adenoviral vector of Claim 12 wherein the transgene of the expression cassette encodes human a-galactosidase.

21. An plasmid comprising the expression cassette of Claim 7.

22. The plasmid of Claim 21 wherein the CMV-derived promoter element 10 of the expression cassette has a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

23. The plasmid of Claim 21 wherein the CMV-derived promoter element of the expression cassette has the nucleotide sequence of SEQ ID NO:2.

24. The plasmid of Claim 21 wherein the CMV-derived promoter element 15 of the expression cassette has the nucleotide sequence of SEQ ID NO:3.

25. The plasmid of Claim 21 wherein the CMV-derived promoter element of the expression cassette has the nucleotide sequence of SEQ ID NO:4.

26. The plasmid of Claim 21 wherein the transgene of the expression cassette encodes a protein selected from a group consisting of human cystic fibrosis 20 transmembrane conductance regulator protein and human a-galactosidase. WO 99/36557 PCT/US99/00915 36

27. The plasmid of Claim 21 wherein the transgene of the expression cassette encodes human cystic fibrosis transmembrane conductance regulator protein.

28. The plasmid of Claim 21 wherein the transgene of the expression cassette encodes human a-galactosidase. 5

29. A method of providing a transgene to a target cell or tissue and obtaining persistent expression therein comprising contacting the target cell or tissue with an adenoviral vector or plasmid comprising an expression cassette which comprises a CMV-derived promoter element operably linked to a transgene, wherein the CMV-derived promoter element confers persistent expression of the transgene in 10 the target cell or tissue, under conditions whereby the adenoviral vector or plasmid is taken up by the target cell or tissue and the transgene is expressed therein.

30. The expression cassette of claim 7 further comprising an enhancer element located to the 5' side of the CMV-derived promoter element, wherein the enhancer element confers increased expression of the transgene in a recipient cell. 15

31. The expression cassette of Claim 30 wherein the enhancer element is derived from the human albumin gene.

32. The expression cassette of Claim 30 wherein the enhancer element has the nucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. 20

33. The expression cassette of Claim 30 wherein the enhancer element has the nucleotide sequence of SEQ ID NO:5.

34. The expression cassette of Claim 30 wherein the enhancer element has the nucleotide sequence of SEQ ID NO:6. WO 99/36557 PCT/US99/00915 37

35. The expression cassette of Claim 30 wherein the enhancer element has the nucleotide sequence of SEQ ID NO:7.

36. The expression cassette of Claim 30 wherein the CMV-derived promoter element has the nucleotide sequence of SEQ ID NO:4 and the enhancer 5 element has the nucleotide sequence of SEQ ID NO:7.

37. An adenoviral vector comprising the expression cassette of Claim 36.

38. The adenoviral vector of claim 37 comprising an adenovirus genome from which the E4 region of the adenovirus genome has been deleted.

39. The adenoviral vector of Claim 37 wherein the transgene of the 10 expression cassette encodes a protein selected from the group consisting of human cystic fibrosis transmembrane conductance regulator protein human a-galactosidase.

40. The adenoviral vector of Claim 37 wherein the transgene of the expression cassette encodes human cystic fibrosis transmembrane conductance regulator protein. 15

41. The adenoviral vector of Claim 37 wherein the transgene of the expression cassette encodes human a-galactosidase.

42. An plasmid comprising the expression cassette of Claim 36.

43. The plasmid of Claim 42 wherein the transgene of the expression cassette encodes a protein selected from a group consisting of human cystic fibrosis 20 transmembrane conductance regulator protein human ca-galactosidase. WO 99/36557 PCT/US99/00915 38

44. The plasmid of Claim 42 wherein the transgene of the expression cassette encodes human cystic fibrosis transmembrane conductance regulator protein.

45. The plasmid of Claim 42 wherein the transgene of the expression cassette encodes human a-galactosidase. 5

46. A method of providing a transgene to a target cell or tissue and obtaining persistent expression therein comprising contacting the target cell or tissue with an adenoviral vector or plasmid comprising an expression cassette which comprises a CMV-derived promoter element operably linked to a transgene, wherein the CMV-derived promoter element confers persistent expression of the transgene in 10 the target cell or tissue, and an enhancer element located to the 5' side of the CMV derived promoter element, wherein the enhancer element confers increased expression of the transgene, under conditions whereby the adenoviral vector or plasmid is taken up by the target cell or tissue and the transgene is expressed therein.

47. A complex comprising the adenoviral vector of claim 12 and a cationic 15 amphiphile.

48. The complex of claim 47 wherein the cationic amphiphile is N4 spermine cholestryl carbamate (GL-67) having the following formula.

49. A complex comprising the adenoviral vector of claim 37 and a cationic amphiphile. 20

50. The complex of claim 49 wherein the cationic amphiphile is N4 spermine cholestryl carbamate (GL-67).

51. A complex comprising the plasmid of claim 21 and a cationic amphiphile. WO 99/36557 PCT/US99/00915 39

52. The complex of claim 51 wherein the cationic amphiphile is N4 Sspermine cholestryl carbamate (GL-67).

53. A complex comprising the plasmid of claim 42 and a cationic amphiphile. 5

54. The complex of claim 53 wherein the cationic amphiphile is N 4 spermine cholestryl carbamate (GL-67).