MULTIVIRAL COMPOSITIONS AND USES THEREOF

Abstract: The invention provides improved compositions and methods for using them in gene therapy.

Description: MULTIVIRAL COMPOSITIONS AND USES THEREOF Background of the Invention A wide variety of systems have been suggested for the delivery of genes to mammals, especially human patients, to treat or prevent a number of diseases, including cystic fibrosis, genetic immune deficiencies, hemophilia A, hemophilia B, and many others. A number of systems have been evaluated and found wanting from a variety of standpoints including effectiveness of gene delivery and observed levels of gene expression. These systems include non- viral approaches as well as various configurations of viral systems including recombinant vaccinia, herpes, adeno and adeno associated viruses. Improved approaches for the delivery of transgenes and the more effective practice of gene therapy, preferably regulated gene therapy, would be of potentially immense clinical value. To that end, significant research has been committed to the development of a regulated gene therapy system involving transducing cells of the recipient mammal with (a) a target gene DNA construct and (b) a genetic switch represented by DNA constructs encoding a pair of fusion proteins which are capable, in the presence of a divalent ligand such as rapamycin, of dimerizing to form a complex which activates the transcription of the target gene. See e.g., Rivera et al, 1996, Nature Medicine 2, 1028-1032 and WO 96/41865 (Clackson et al). Those references disclose in detail, among other things, the design of a target gene construct comprising a target gene operably linked to an expression control sequence including a minimal promoter and 12 binding sites for the composite DNA-binding domain known as ZFHD1. An optimized bicistronic DNA construct encoding the requiste pair of fusion proteins is also disclosed there. The first fusion protein comprises a copy of the ZFHD1 DNA-binding domain linked to three FKBP12 domains in series. The second fusion protein comprises an FRB domain derived from human FRAP linked to the p65 transcription activation domain derived from NF-kB. As described in WO , the FRB domain may be engineered to contain one or more tions relative to the natrually ofccurring peptide sequence, including at position T2098, among others, which may be replaced, e.g. with Leu. While the operability of the foregoing switch mechanism has been clearly demonstrated (see e.g. Rivera et al, supra), improvements for deploying the switch may be of great additional value. Summary of the Invention More specifically, this invention provides an improved composition for the in vivo genetic engineering of cells within a mammal. The composition is of the type comprising a viral delivery vehicle containing (a) a target gene construct comprising a nucleic acid sequence encoding an erythropoietin or growth factor protein operably linked to an expression control sequence comprising 12 ZFHD1 sites; and (b) the bicistronic nucleic acid construct of the vector pAd-CMV- TF-1, which encodes a ZFHDl-3xFKBP12 DNA-binding fusion protein and an FRB T2098L-p65 transcription activation fusion protein. Our improvement involves using two different recombinant adenoviruses, adeno-associated viruses or hybrid adeno/ AAV viruses, in place of one recombinant virus. In our improved composition, the genome of the first such recombinant virus contains the bicistronic nucleic acid construct and the genome of the second recombinant virus contains the target gene construct. In our currently preferred embodiment, the genome of the second recombinant virus further comprises a nucleic acid sequence encoding another copy of the FRB T2098L-p65 transcription activation fusion protein. We have found that including a second copy of that construct leads to better target gene expression levels, although the reason for that is not completely clear at present. It is also preferred that in embodiments using recombinant adenoviruses, that the adenoviruses lack functional adenoviral El and E3 genes. Becuase the genetic payloads are apportioned among two recombinant viruses, the ratios of the respective viruses may be varied, e.g., such that one virus is present in excess of the other, to achieve optimal results. The recombinant viruses may be formulated with one or more pharmaceutically acceptable carriers, stabilizers, buffers or other excipients to provide a pharmaceutical preparation for delivery to mammals, including human patients. Such preparations may be packaged together with a package insert containing information concerning the administration of the viruses to recipient cells or patients. This invention thus provides a method for rendering a mammal capable of rapamycin-dependent transcription of an erythropoietin or growth hormone gene which comprises infecting the mammal with an improved composition as described herein. The improved composition may be administered to the mammal by intramuscular or intravenous administration. Transcription of an erythropoietin or growth factor target gene in genetically engineered cells within the mammal may then be induced by administering rapamycin to the mammal in an amount sufficient to induce expression of the desired target gene. Expression may be measured indirectly by measuring an increase in concentration of the target gene product in the mammal's serum or other tissues or by measuring an increase in hematocrit or animal growth following administration of rapamycin. It should be noted that the T2098 FRB domain provides the potential for use of rapamycin analogs in place of rapamycin, and that the use of such analogs may provide further practical advantages. Brief Description of the Figures Figure 1 Panel A shows the amount of human growth hormone (hGH) produced by HT1080 cells cotransfected with the adenovirus vector pAd-CMV- TF-1 or the AAV vector pAAV-CMV-TF-1 and one of the following vectors containing a gene encoding hGH: pAd-Z 12 I-hGH-1 (-1); pAd-Z 12 I-hGH-2 (- 2); pAd-Z 12 I-hGH-3 (-3); pAd-Z 12 I-hGH-3-ADl (-3AD1); pAAV-Z 12 I-hGH- 1 (-1); pAAV-Z 12 I-hGH-l-s2 (-l-s2); pAAV-Z 12 I-hGH-2-ADl (-2AD1); pAAV-Z 12 I-hGH-3 (-3); pAAV-Z 12 I-hGH-3-s3 (-3-s3); and pAAV-Z 12 I- hGH-3-ADl (-3AD1) and treated or not with 25 nM rapamycin. -3-TF1 represents cells transfected with the adenoviral vector pAd-CMV-TF-1 further including the reporter construct Z 12 I-hGH-3. See Table I in Example 1 for the description of these constructs. The numbers on the horizontal axis indicate the amount of hGH produced in each transfection in the absence of rapamycin. Figure 1 Panel B shows the amount of rhesus monkey growth hormone (rmGH) produced by HT1080 cells cotransfected with the adenovirus vector pAd-CMV-TF-1 or the AAV vector pAAV-CMV-TF-1 and one of the following vectors containing a gene encoding rmGH gene: pAd-Z 12 I-rmGH-3 (-3); pAd- Z 12 I-rmGH-3-ADl (-3AD1); pAAV-Z 12 I-rmGH-3 (-3); pAAV-Z 12 I-rmGH-3- s3 (-3-s3); and pAAV-Z 12 I-rmGH-3-ADl (-3AD1) and treated or not with 25 nM rapamycin. -3-TF1 represents cells transfected with the adenoviral vector pAd-CMV-TF-1 further including the reporter construct Z 12 I-rmGH-3. See Table I in Example 1 for the description of these constructs. The numbers on the horizontal axis indicate the amount of rmGH produced in each transfection in the absence of rapamycin. Figure 2 is a graphic representation of Ad.CMV-hGH-3 (Ad.CG-3), Ad.CMV-TFl (Ad.TFl), and Ad.Z 12 I-hGH-l (Ad.ZG-1). PAS stands for SV40 late poly A sequence. RBG stands for rabbit β-globin intron and polyA. These vectors are further described in Table 1 in the Examples. Figure 3 is a histogram representing the amount of human growth hormone (hGH) secreted from A549 cells infected with pAd-CMV-TFl (Ad.TFl), P Ad.Z 12 I-hGH-l (Ad.ZG-1), or Ad.TFl + Ad.ZG-1 in a 6:2 ratio and treated or not with 50 nM rapamycin. Figure 4 is a histogram representing the amount of human growth hormone (hGH) secreted from 84-31 cells infected with pAAV-CMV-TFl (TF1), pAAV.Z 12 I-hGH-l (ZG-1), or TF1 + ZG-1 in a 6:2 ratio and treated or not with 50 nM rapamycin. Figure 5 A is a graph showing the level of serum human growth hormone (hGH) in Balb/c nude mice injected i.v. with a high dose (1 x 10 π viral particles) or a low dose (2 x 10 10 ) of a 1: 1 mixture of Ad.TFl and Ad.ZG- 1, in experiment #1 and #2 (day 0), and injected i.p. with 5 mg/kg rapamycin at various days after viral administration. Also show in the serum level of hGH in a Balb/c mouse injected i.v. with 10 1 1 viral particles of the constitutive vector Ad.CG-3. Figure 5B is a graph showing the level of serum hGH in Balb/c nude mice injected i.v. with 1 x 10' ' viral particles of a 1: 1 mixture of Ad.TFl and Ad.ZG- 1 at day 0 and injected i.p. with various doses of rapamycin at different time points. Figure 6 is a graph showing the level of serum hGH in Balb/c nude mice injected i.m. with 1 x 10 11 viral particles of a 1: 1 mixture of Ad.TFl and Ad.ZG-1 at day 0 and injected i.p. with 5 mg/kg of rapamycin at different time points after viral infection or injected i.m. with 1 x 10 1 1 viral particles of Ad.CG-3. Figure 7 is a graph showing the hematocrit factor in Balb/c nude mice from one to 15 days after having been injected i.m. with 1 x 10 11 viral particles of pAAV.CMVmEpo (day 0). Figure 8 is a graph showing the hematocrit factor in Balb/c nude mice injected i.m. with a total 2 x 10 H viral particles of a 1: 1 mixture of pAAV-Z 12 I- mEpo-2-S2 (AAV.ZE2) or pAAV-Z 12 I-mEpo-3-S2 (AAV.ZE3) and pAAV- CMV-TF1 (day 0) and injected (+ rap) or not (- rap) i.p. with 1 mg/kg rapamycin every other day. Figure 9 is a graph showing the amount of serum hGH in mice at various times after i.v. injection of different amounts of Ad.CG-3 viral particles (day 0). Figure 10 is a graph showing the amount of serum hGH in mice after i.v. injection of various amounts of total viral particles of a mixture of Ad.TFl and Ad.ZGl in a 1: 1 ratio (day 0) and injection of 5mg/kg rapamycin at various times after viral infection. Figure 11 is a graph showing the amount of serum hGH in mice after i.v. injection of 1 x 10 1 1 total viral particles of a mixture of Ad.TFl and Ad.ZGl in a 1: 1 ratio (day 0) and injection of various amounts of rapamycin (0.05; 0.15; 0.5; 1.5; 5; and 15 mg/kg) 5 days after viral infection. Figure 12A shows serum erythropoietin (Epo) levels in two rhesus monkeys (RQ1564 and RQ1748) at various times after an i.m. injection of 10 13 viral particles/kg monkey of pAd-CMV-rmEpo-3. Figure 12B shows hematocrit factors in two rhesus monkeys (RQ1564 and RQ1748) at various times after an i.m. injection of 10 13 viral particles/kg monkey of pAd-CMV-rmEpo-3. Figure 12C shows serum erythropoietin (Epo) levels in a rhesus monkey (RQ1582) at various times after an i.m. injection of 10 13 viral particles/kg monkey of pAAV-CMV-rmEpo-3. Figure 12D shows hematocrit factors in a rhesus monkey (RQ1582) at various times after an i.m. injection of 10 13 viral particles/kg monkey of pAAV- CMV-rmEpo-3. Detailed Description of the Invention The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references including literature references, issued patents, published patent applications as cited throughout this application are hereby expressly incorporated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular CloningtA LaboratorytManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Exemplification Example 1: Bicistronic adenovirus encoding two chimeric proteins This Example describes the construction of an adenovirus containing a bicistronic sequence encoding a first chimeric protein having a ligand-binding domain (FRB T2098L) fused to a transcriptional activation domain (from p65) and a second chimeric protein having a ligand-binding domain (copies of FKBP) fused to a DNA-binding domain (ZFHD1). The two cistons are separated by an internal ribosome entry sequence (IRES). Expression of the chimeric proteins is under the control of a CMV promoter. A rabbit b-globin intron and poly A are located downstream of the bicistronic region. This adenovirus, referred to as pAd-CMV-TF-l,was constructed as follows. A plasmid encoding a chimeric protein comprising the DNA binding domain ZFHD1 fused to three copies of FKBP has been deposited at the ATCC under ATCC Accession No. 97399 and is described in Pomerantz et al. (1995) Science 267:93. To generate a chimeric protein containing an FRB domain fused to the activation domain of the human NF-kB p65 subunit (hereafter designated p65), two fragments were amplified by PCR from the plasmid pCG-p65. Primer 5' ATGCTCTAGAGATGAGTTTCCCACCATGGTG (p65/361 Xba) (SEQ ID NO: ) and primer 5' GCATGGATCCGCTCAACTAGTGGAGCTGATCTGACTCAG (SEQ ID NO: ), both flanked by 5' Xbal and 3' Spel BamHI sites, were used to amplify the nucleotide sequence encoding amino acids 361-550 of p65. The PCR product was digested with Xbal and BamHI and cloned into Xbal-BamHI digested pCGNN (Attar and Gilman (1992) Mol. Cell Biol. 12:2432) to yield pCGNN-p65. The constructs was verified by restriction analysis and DNA sequencing. The amino acid sequence of p65 transcription activation sequence (amino acids 361-550 is encoded by the following linear sequence: GATGAGTTTCCCACCATGGTGTTTCCTTCTGGGCAGATCAGCCAGGCCT CGGCCTTGGCCCCGGCCCCTCCCCAAGTCCTGCCCCAGGCTCCAGCCCCT GCCCCTGCTCCAGCCATGGTATCAGCTCTGGCCCAGGCCCCAGCCCCTGT CCCAGTCCTAGCCCCAGGCCCTCCTCAGGCTGTGGCCCCACCTGCCCCCA AGCCCACCCAGGCTGGGGAAGGAACGCTGTCAGAGGCCCTGCTGCAGCT GCAGTTTGATGATGAAGACCTGGGGGCCTTGCTTGGCAACAGCACAGA CCCAGCTGTGTTCACAGACCTGGCATCCGTCGACAACTCCGAGTTTCAG CAGCTGCTGAACCAGGGCATACCTGTGGCCCCCCACACAACTGAGCCCA TGCTGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGACAGCCCAGAG GCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCCCCAATG GCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTT CTCAGCCCTGCTGAGTCAGATCAGCTCCTAA. (SEQ ID NO: ) To obtain portions of the human FRAP gene, human thymus total RNA (Clontech #64028-1) was reverse transcribed using MMLV reverse transcriptase and random hexamer primer (Clontech 1st strand synthesis kit). This cDNA was used directly in a PCR reaction containing the primer 5' GCATGTCTAGAGAGATGTGGCATGAAGGCCTGGAAG (SEQ ID NO: ) and the primer 5' GCATCACTAGTCTTTGAG ATTCGTCGG A A C A C ATG (SEQ ID NO: ) and Pfu polymerase (Stratagene). The primers were designed to amplify the coding sequence for amino acids 2021-2113 inclusive of human FRAP: a 93 amino acid region containing an 89 amino acid region essentially corresponding to the minimal 'FRB' domain (amino acids 2025-2113) identified by Chen et al. (Proc. Natl. Acad. Sci. USA (1995) 92, 4947-4951) as necessary and sufficient for FKBP-rapamycin binding (hereafter named FRB) and an additional 4 N-terminal amino acids. The appropriately-sized band was purified, digested with Xbal and Spel, and ligated into Xbal-Spel digested pCGNN- GAL4. This construct was confirmed by restriction analysis (to verify the correct orientation) and DNA sequencing and designated pCGNN-GAL4-lFRB. An Xbal-BamHI fragment encoding 1 copy of FRB was recovered from the GAL4 fusion vector and ligated into Xbal-BamHI digested pCGNN to yield pCGNN-lFRB. The threonine at position 2098 of this FRAP portion was substituted with a Leucine (and is referred to as R H1 ). To generate an N-terminal fusion of an FRB domain with the p65 activation domain, plasmid pCGNN- R H1 was digested with Spel and BamHI. An Xbal-BamHI fragment encoding p65 was isolated from pCGNN-p65 and ligated into the Spel-BamHI digested vectors to yield the plasmid pCGNN- lFRB-p65. The internal ribosome entry sequence (IRES) from the encephalomyocarditis virus was amplified by PCR from pWZL-Bleo. The resulting fragment, which was cloned into pBS-SK+ (Stratagene), contains an Xbal site and a stop codon upstream of the IRES sequence and downstream of it, an Ncol site encompassing the ATG followed by Spel and BamHI sites. To facilitate cloning, the sequence around the initiating ATG of pCGNN-ZFHDl- 3FKBP was mutated to an Ncol site and the Xbal site was mutated to a Nhel site using the oligonucleotides 5'-GAATTCCTAGAAGCGACCATGGCTTCTAGC-3' (SEQ ID NO: ); and 5'-GAAGAGAAAGGTGGCTAGCGAACGCCCATAT-3' (SEQ ID NO: ) respectively. An NcoI-BamHI fragment containing ZFHD1-3FKBP was then cloned downstream of pBS-IRES to create pBS-IRES-ZFHDl-3FKBP. The Xbal-BamHI fragment from this plasmid was next cloned into Spel/BamHI-cut pCGNN- R m -p65 to create pCGNN- R H] -p65-IRES-ZFHDl-3FKBP. An Xbal- BamHI fragement from this plasmid was then cloned into pC 5 EN, a derivative of pCGNN that contains additional convenient restriction sites, to create pC 5 EN - R H] -p65-IRES-ZFHDl-3FKBP. To construct pAd-CMV-TF-1, a Bglll-Xhol fragment from pC 5 EN - R H1 -p65-IRES-ZFHDl-3FKBP, comprising the R H1 p65-IRES-ZFHDl- 3xFRBP12 coding region, and the rabbit b-globin intron and polyA, was cloned into into the linker of pAd.CMV-link (Kozarsky K.F. et al. (1993) Curr. Opin. Genetics Dev. 3:499). Thus, pAd-CMV-TF-1 comprises a CMV promoter which drives the expression of two chimeric proteins, one having a DNA binding domain and a second chimeric protein having a transcriptional activation domain. Example 2; Bicistronic AAV and p Ad/AAV vectors encoding two chimeric proteins An adenovirus-associated virus containing a bicistronic sequence encoding a first chimeric protein having a ligand-binding domain (FRB T2098L) fused to a transcriptional activation domain (from p65) and a second chimeric protein having a ligand-binding domain (copies of FKBP) fused to a DNA-binding domain (ZFHDl), is referred to as pAAV-CMV-TF-1, was constructed as follows. An Mlul-Xhol fragment from pC 5 EN - R H 1 -p65-IRES-ZFHDl-3FKBP, described in Example 1, comprising the CMV promoter, R H] p65-IRES- ZFHDl-3xFRBP12, and the rabbit b-globin intron and polyA, was inserted into pSub201 (Samulski et al. (1987) J. Virol. 61:3096). This insertion was done by first removing from the AAV vector an Xbal fragment located between the ITRs and containing the rap and cap genes of the virus and then inserting the fragment obtained from the pCGNN vector. The pAd/AAV vector is a pAd vector having two regions of an adenovirus: 0-1 map units and about 9-16 map units flanking two portions of AAV, one portion containing the 5' ITR and the other portion containing the 3' ITR. Thus, a portion of pCGNN-lFRB-p65(361-550)-IRES-ZFHDl-3FKBP, described above, comprising the CMV promoter, R H ,p65-IRES-ZFHDl- 3xFRBP12, and the rabbit b-globin intron and polyA was inserted between the AAV ITRs, as described above, to generate pAd/AAV-CMV-TF-1. Example 3: Construction of viral vectors containing a target gene Several viral vectors (adenovirus, adeno-associated virus, or hybrid adeno-adeno-associated virus, containing a target gene under the control of a minimal IL-2 promoter upstream of which are 12 copies of a ZFHDl binding site were constructed. These vectors are listed in Table I set forth below. First, pZHWTxl2-IL2-SEAP, containing a basal promoter from human interleukin-2 gene (Siebenlist et al. Mol. Cell. Biol. (1986) 6:3042-3049) and 12 tandem copies of a ZFHDl binding sites (Pomerantz et al., 1995) driving expression of a gene encoding secreted alkaline phosphatase (SEAP), was prepared by replacing the Nhel-Hindlll fragment of pSEAP Promoter (Clontech) with the following Nhel-Xbal fragment containing 12 ZFHD binding sites: GCTACTCTAATGATGGGCGCTCGAGTAATGATGGCTCGGTCCTACTAATGA TGGGCGCTCGAGTAATGATGGGCGTCTAGCTAATGATGGGCGCTCGAG TAATGATGGGCGGTCGACTAATGATGGGCGCTCGAGTAATGATGGGCG TCTAGCTAATGATGGGCGCTCGAGTAATGATGGGCGGTCGACTAATGA TGGGCGCTCGAGTAATGATGGGCGTCTAGA (SEQ ID NO: ) (the ZFHDl binding sites are underlined), and an Xbal-Hindlll fragment containing a minimal IL2 promoter (-72 to +45). pZHWTxl2-IL2-hGH, the activation of which leads to the production of human growth hormone (hGH), was constructed by replacing the Hindlll- BamHI (blunted) fragment of pZHWTxl2-IL2-SEAP (containing the SEAP coding sequence) with a Hindlll -EcoRI(blunted) fragment from pOGH (containing an hGH genomic clone; Selden et al., MCB 6:3171-3179, 1986). An MluI(blunted)-BamHI fragment from this plasmid containing the 12 tandem binding sites, the IL2 minimal promoter, the hGH gene (comprising all of the coding region), and the SV40 late poly A was inserted into the linker of the pAd link adenovirus cited above, to produce pAd-Z 12 I-hGH-l. The same portion of pZHWTxl2-IL2-hGH was also inserted into the AAV vector as described above, to produce pAAV-Z 12 I-hGH-l . As described in Table I, adenoviral, AAV and hybrid adenoviral/ AAV vectors containing other target genes were constructed. For example, one adenovirus was created which contained a cDNA encoding human GH (pAd- Z 12 I-hGH-2). The cDNA was obtained by PCR amplification of RNA from a cell line expressing the human genomic GH gene. Other vectors contained a cDNA encoding hGH, upstream of which an intron was inserted (chimeric intron from Promega pCI vector) (pAd-Z 12 I-hGH-3). Certain AAV vectors contained, in addition, a "stuffer DNA" to increase the total size of the vector. Yet other adenoviral and AAV vectors contained as a target gene the rhesus monkey growth hormone (e.g., pAd-Z 12 I-rmGH-3), murine erythropoietin (Epo) (e.g., pAd-Z 12 I-mEpo-2) (Shoemaker et al. (1986) Mol. Cell Biol. 6:849) or the rhesus monkey Epo (e.g., pAd-Z 12 I-rmEpo-2). Table I: Transcription Factor Constructs and Target Gene Constructs pAd-CMV-TF-1 Adenoviral vector containing CMV driving expression of RHlp65-IRES-ZlF3 transcription factor fusions; followed by rabbit B-globin intron and polyA pAAV-CMV-TF- AAV vector containing CMV driving expression of 1 RHlp65-IRES-ZlF3 transcription factor fusions; followed by rabbit B-globin intron and polyA (4799 bp including ITRs) pAd/AAV-CMV- Hybrid adenoviral AAV vector containing CMV driving TF-1 expression of RHlp65-IRES-ZlF3 transcription factor fusions; followed by rabbit B-globin intron and polyA pAd-CMV-hGH-1 CMV driving expression of genomic hGH; followed by SV40 late polyA pAd-CMV-hGH-2 CMV driving expression of hGH cDNA (no intron) followed by SV40 late polyA pAd-CMV-hGH-3 CMV driving expression of hGH cDNA with a 5' intron (SV40 intron from pAd.CMV-Link. l); followed by SV40 late polyA pAd-CMV-hGH-4 CMV driving expression of hGH cDNA with a 5' intron (chimeric intron from Promega pCI vector); followed by SV40 late polyA pAd-Z12I-hGH-l Adenoviral vector containing a genomic hGH downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAd-Z12I-hGH-2 Adenoviral vector containing a hGH cDNA downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA P Ad-Z12I-hGH-3 Adenoviral vector containing a hGH cDNA with a 5' intron (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA P Ad-Z12I-hGH- Adenoviral vector containing a hGH cDNA with a 5' 3-AD1 intron (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by CMV driving expression of the RHlp65 activation domain fusion (with 3'rabbit B-globin intron and polyA) pAd-Z12I-hGH- hGH cDNA with a 5' intron (chimeric intron from 3-TF1 Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by CMV driving expression of the RHlp65-IRES-ZlF3 transcription factor fusions (with 3'rabbit B-globin intron and polyA) pAAV-CMV- CMV driving expression of genomic hGH; followed by hGH-l-Sl SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4840 bp including ITRs) pAAV-CMV- CMV driving expression of hGH cDNA with a 5' intron hGH-3 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; ("l/2"size - 2472 bp including ITRs) pAAV-CMV- CMV driving expression of hGH cDNA with a 5' intron hGH-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4486 bp including ITRs) pAAV-Z12I- AAV vector containing a genomic hGH downstream of 12 hGH-1 ZFHDl binding sites and theminimal IL2 promoter; followed by SV40 late polyA pAAV-Z12I- AAV vector containing a genomic hGH downstream of 12 hGH-l-S2 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer pAAV-Z12I- AAV vector containing a hGH cDNA downstream of 12 hGH-2-ADl ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by a 2800 bp "stuffer" pAAV-Z12I- AAV vector containing a hGH cDNA with a 5' intron hGH-3 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAAV-Z12I- AAV vector containing a hGH cDNA with a 5' intron hGH-3-S3 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer pAAV-Z12I- AAV vector same as pAAV-Z12I-hGH-2-ADl, but hGH hGH-3-ADl cDNA has a 5' intron (chimeric intron from Promega pCI vector) (4761 bp including ITRs) pAAV-Z12I- AAV vector containing a hGH cDNA downstream of 12 hGH-2-S3 ZFHDl binding sites and the minimal EL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer pAd-CMV- CMV driving expression of rmGH cDNA with a 5' intron rmGH-3 (chimeric intron from Promega pCI vector); followed by SV40 late polyA pAd-CMV- CMV driving expression of rmGH cDNA with a 5' intron rmGH-4 (SV40 intron from pAd.CMV-Link. l); followed by SV40 late polyA (re-named to match my nomenclature) pAd-Z12I-rmGH- Adenovirus containing a rmGH cDNA with a 5' intron 3 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAd-Z12I-rmGH- Adenovirus containing a rmGH cDNA with a 5' intron 3-AD1 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by CMV driving expression of the RHlp65 activation domain fusion (with 3'rabbit B-globin intron and polyA) pAd-Z12I-rmGH- rmGH cDNA with a 5' intron (chimeric intron from Promega 3-TF1 pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by CMV driving expression of the RHlp65-IRES- Z1F3 transcription factor fusions (with 3'rabbit B-globin intron and polyA) pAAV-CMV- CMV driving expression of rmGH cDNA with a 5' intron rmGH-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4562 bp including ITRs) pAAV-Z12I- rmGH cDNA downstream of 12 ZFHDl binding sites and the rmGH-2-S3 minimal IL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence, the human growth hormone 3'UTR, and a portion of the 3'rabbit B-globin intron and polyA (4599 bp including ITRs) pAAV-Z12I- AAV vector containing rmGH cDNA with a 5' intron (chimeric rmGH-3 intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; ("l/2"size -2024 bp including ITRs) pAAV-Z12I- AAV vector containing rmGH cDNA with a 5' intron (chimeric rmGH-3-S3 intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer pAAV-Z12I- AAV vector containing rmGH cDNA with a 5' intron (chimeric rmGH-3-ADl intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed, head-to-tail, by CMV driving expression of the RHlp65 activation domain fusion (with 3'rabbit B-globin intron and polyA) pAd-CMV-mEpo-3 CMV driving expression of mEpo cDNA with a 5' intron (chimeric intron from Promega pCI vector); followed by SV40 late polyA pAd-Z12I-mEpo-2 Adenoviral vector containing mEpo cDNA downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAd-Z12I-mEpo-3 Adenoviral vector containing mEpo cDNA with a 5' intron (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAAV-CMV- CMV driving expression of mEpo cDNA with a 5' intron mEpo-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4289 bp including ITRs) pAAV-Z12I- mEpo cDNA downstream of 12 ZFHDl binding sites and mEpo-2 the minimal IL2 promoter; followed by SV40 late polyA; (<"l/2"size -1592 bp including ITRs) pAAV-Z12I- AAV vector containing mEpo cDNA downstream of 12 mEpo-2-S3 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer pAAV-Z12I- mEpo cDNA with a 5' intron (chimeric intron from mEpo-3 Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal JL2 promoter; followed by SV40 late polyA; (<"l/2"size -1751 bp including ITRs) pAAV-Z12I- AAV vector containing mEpo cDNA with a 5' intron mEpo-3-S3 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2.7 kb stuffer pAd-CMV-rmEpo- CMV driving expression of rmEpo cDNA with a 5' intron 3 (chimeric intron from Promega pCI vector); followed by SV40 late polyA pAd-Z12I-rmEpo- Adenoviral vector containing rmEpo cDNA downstream of 12 2 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAd-Z12I-rmEpo- Adenoviral vector containing rmEpo cDNA with a 5' intron 3 (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA pAAV-CMV- CMV driving expression of rmEpo cDNA with a 5' intron rmEpo-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4369 bp including ITRs) pAAV-Z12I- AAV vector containing rmEpo cDNA downstream of 12 rmEpo-2-S2 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR pAAV-Z12I- AAV vector containing rmEpo cDNA with a 5' intron (chimeric rmEpo-3-S2 intron from Promega p CI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer pAd/AAV-CMV- CMV driving expression of RHlp65-IRES-ZlF3 TF-1 transcription factor fusions; followed by rabbit B-globin intron and polyA (4799 bp including ITRs) pAd/AAV-CMV- CMV driving expression of rmEpo cDNA with a 5' intron rmEpo-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4383 bp including ITRs) pAd/AAV-Z12I- rmEpo cDNA downstream of 12 ZFHDl binding sites and rmEpo-2-S2 the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (3700 bp including ITRs) pAd/AAV-Z12I- Hybrid adenoviral/AAV vector containing rmEpo cDNA rmEpo-3-S2 with a 5' intron (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer pAd/AAV-CMV- CMV driving expression of rmGH cDNA with a 5' intron rmGH-3-S2 (chimeric intron from Promega pCI vector); followed by SV40 late polyA; followed by a 2 kb stuffer containing an internal portion of human placental alkaline phosphatase coding sequence and human growth hormone 3'UTR (4570 bp including ITRs) pAd/AAV-Z12I- Hybrid adenoviral/AAV vector containing rmGH cDNA rmGH-2-S2 downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer pAd/AAV-Z12I- Hybrid adenoviral/AAV vector containing rmGH cDNA rmGH-3-S2 with a 5' intron (chimeric intron from Promega pCI vector) downstream of 12 ZFHDl binding sites and the minimal IL2 promoter; followed by SV40 late polyA; followed by a 2 kb stuffer Example 4: Transfections indicate ligand inducible induction of the target gene This example demonstrates rapamycin-induced transcriptional activation of a target gene present on a viral vector in cells cotransfected with a viral vector described above encoding two chimeric proteins, one of which contains a transcriptional activation domain and the other which contains a DNA binding domain. HT1080 cells were transiently transfected as follows. 33 ng vector encoding the two chimeric proteins was mixed with 167 ng of vector containing the target gene and 0.75 μl Superfect reagent (Qiagen) and the volume brought to 50 μl with MEM medium. This mixture is incubated for 5 minutes at room temperature. Then 150 μl complete HT1080 medium was added. Two hundred μl of this mixture was added into a well of a 96 well dish containing 1.5 x 10 4 HT1080 cells and the cells were incubated for 3 hours. The medium was then replaced with fresh medium containing 25 nM rapamycin and the amount of GH secreted in the medium is measured 14-20 hours later using the Nichols hGH kit. Several vectors, adenoviral, AAV, and hybrid adenoviral/AAV vectors encoding two chimeric proteins were used and each contained R H1 p65-IRES- ZFHDl-3xFKBP12, described above. The results of the transfections are shown in Figure 1. The results indicate that in the absence of rapamycin, very low amounts of hGH are produced. As shown in the Figure, the addition of rapamycin to the cultures resulted in a significant increase in the production of hGH. The results also show that similar levels of hGH are obtained with all three types of vectors. Thus, the presence in cells of an adenoviral, AAV, or hybrid adenovirus/AAV vector encoding two chimeric proteins, one containing a ligand binding domain and a transcriptional activation domain and the other containing a ligand binding domain and a DNA-binding domain, results in transcriptional activation of a target gene under the control of a DNA sequence recognized by the DNA binding domain (present on a separate vector) in the presence of a dimerizer that brings the two chimeric proteins together. Example 5: Target gene expression following infection of a cell with a virus containing a bicistronic construct and a second virus containing a target gene This Example shows that infection of mice with a composition comprising two types of viral particles, a first viral particle containing a bicistronic viral vector encoding two chimeric proteins, and a second viral particle containing a target gene, results in efficient expression of the target gene upon administration of rapamycin to the mouse. The following two recombinant adenoviruses were used: Ad.TFl and Ad.ZG-1. These are graphically depicted in Figure 2. Briefly, Ad.TFl, which is also referred to as pAd-CMV-TFl is a bicistronic vector in which the expression of two chimeric proteins is under the control of the CMV promoter. The Ad.ZG-1, which is also referred to as pAd-Z 12 I-hGH-l contains a genomic DNA fragment containing the human growth hormone gene under the control of 12 ZFHDl binding sites. These vectors are further described in Table 1. These viral vectors were then encapsidated as described, e.g, in Kozarsky and Wilson (1993) 3:499. A human lung carcinoma cell line that does not support the replication of El-deleted virus, the A549 cell line, was infected with Ad.TFl, Ad.ZG-1 or a combination of Ad.TFl and Ad.ZG- 1 at a multiplicity of infection of 6:2, respectively and treated with 50 nM rapamycin for 24 hours. The amount of hGH produced was measured as described above. The results, which are presented in Figure 3 show that, whereas the addition of rapamycin to cells infected with only one type of virus did not result in an increase of hGH production, it resulted in an induction of a factor of about 518 in cells coinfected with both types of viruses. Thus, the addition of rapamycin induced transcription of the hGH gene. Various infection ratios of the two types of viruses were then compared. A multiplicity of infection of more than one was required for either virus to be effective in responding to rapamycin. Ratios of Ad.TFl and Ad.ZG-1 of 2:0.02; 2:0.06; 2:0.2; 2:0.6; 2:2; 2:6 and 2:20 were tested. The best induction was achieved with a ratio of Ad.ZG-1: Ad.TFl of 2:20 (264 fold induction). A ratio of Ad.ZG-1: Ad.TFl of 20:2 was also very efficient (with a 120 fold induction). Rapamycin also efficiently induces target gene expression in a two virus system using AAV vectors. 84-31 cells were infected with the viruses AAV.TF1, AAV.ZG-1 or a combination of these two viruses in a 1:1 ratio. 84-31 cells are derived from 293 cells and expresses E4. The trans functions provided in this cell line are necessary to augment AAV transduction in vitro. AAV.TF1 and AAV.ZG-1 are identical to pAd.TFl and pAd.ZG-1, described above, except that the viral backbone is AAV. These vectors are further described in Table 1. These vectors were then encapsidated. The cells were incubated with the viral particles and 50 nM rapamycin for 24 hours, at which time point, the amount of hGH in the culture medium was determined. As shown in Figure 4, coinfection of cells with the two types of viruses resulted in a 227 fold increase in the production of hGH following rapamycin treatment. Example 6: Rapamycin inducible gene expression following i.v. viral infection The adenoviral and AAV vectors were then tested in vivo in mice. Six to eight week old Balb/c nude mice were injected intravenously (i.v.) with either a total of 10 1 1 or 2 x 10 10 Ad.ZG-1 and Ad.TFl viral particles in a ratio of 1:1, or 10 11 Ad.CG-3 viral particles, in which the hGH gene is under the control of a constitutive promoter, i.e., the CMV promoter (see Figure 2). The viral particles were suspended in 100 μl PBS for the injection. Rapamycin was administered to the mice intraperitoneally (i.p.) at a dose of 5 mg/kg at various times (from 0 to 46 days) after viral administration. Serum hGH level was determined 24 hours after administration of rapamycin and at various time points thereafter. The results, which are shown in Figure 5A, indicate that administration of rapamycin to these mice resulted in a rapid increase in production of hGH, which progressively decreased to near background levels and increased again after readministration of rapamycin. hGH production could efficiently be increased by rapamycin administration at least as late as about 45 days after the viral infection. Figure 5B shows that repeated rapamycin administration resulted in induction of hGH production even when the challenge occurs at least 75 days after the viral infection. Administration of the constitutive hGH viral construct, i.e., Ad.GC-3, resulted in high levels of hGH production for at least 50 days after the viral infection and decreased only slowly with time (Figure 5A). The level of hGH production with the two systems, i.e., inducible versus constitutive, is similar. Thus, i.v. administration into mice of two adenoviruses, one containing a target gene under the control of an inducible promoter and the other containing genes encoding two chimeric proteins, and administration of rapamycin which is capable of cross-linking the two chimeric proteins to produce a transcription factor capable of binding to the inducible promoter, resulted in efficient expression of the target gene. Example 7; Rapamycin inducible gene expression following i.m. viral infection This example shows that intramuscular (i.m.) administration into mice of two types of viruses, one containing a target gene and the other encoding two chimeric proteins, followed by administration of rapamycin, results in efficient expression of the target gene. 10" Ad.ZG-1 and Ad.TFl viral particles in a ratio of 1: 1 or 10" Ad.CG-3 viral particles were administered i.m. to Balb/c mice. Various times (from 0 to 34 days) following the adenoviral infection with the two viruses, rapamycin was administered i.p. at a dose of 5 mg/kg and serum hGH levels were determined in all mice 24 hours later and at various time points thereafter. As shown in Figure 6, administration of rapamycin to the mice resulted in rapid increase of hGH production. Mice infected with the constitutive construct, i.e., AdCG-3, constitutively produced high levels of hGH until at least 50 days following viral infection. In another example, a direct comparison between i.m. and i.v. injection of adenoviruses containing a cDNA encoding the hGH gene under the control of CMV into Balb/c nude mice indicated that levels of hGH produced were higher in the case of an i.v. injection relative to an i.m. injection shortly and up until about 10 days after the injection, after which the level of hGH produced was similar in both cases. In a similar experiment, mice were injected i.m. with 2 x 10n viral particles containing an AAV vector having a cDNA encoding mouse erythropoietin (EPO) under the control of CMV (pAAV-CMV-mEpo-3-S2, set forth in Table 1). The murine Epo cDNA was cloned from Balb/c mouse kidney polyA+ RNA (Clontech). The gene was sequenced completely and found to differ from the Genbank sequence at only one position - amino acid 8 (which is in the leader sequence)- in the published sequence is a threonine (ace) while is an alanine (gcc) in this clone. The amount of Epo produced at 1, 8, and 15 days (please confirm) after the infection was determined indirectly by determining the hematocrit, i.e., the ratio of the volume of packed red blood cells to the volume of whole blood. An increasing hematocrit ratio is indicative of an increased production of Epo. The results, presented in Figure 7, show that increasing amounts of red blood cells were produced until at least 15 days after the viral infection. Thus, the transgene was expressed from the AAV vector in the injected mice. Similar results were obtained using hGH as the transgene. AAV viral particles injected i.m. were also used in the inducible system. Balb/c mice were injected i.m with a total of 2 x 10n viral particles of pAAV-Z 12 I-mEpo-2-S2 (AAV.ZE2) or pAAV-Z 12 I-mEpo-3-S2 (AAV.ZE3) and pAAV-CMV-TFl in a ratio of 1: 1. The target gene vectors are further described in Table 1. Rapamycin was administered i.p. to the mice at 1 mg/kg every other day, as indicated in Figure 8 and serum Epo levels were measured indirectly 24 hours later, by determining the hematocrit. The results, presented in Figure 8, show a dramatic increase in hematocrit upon rapamycin administration which was similar for the two different vectors, i.e., for AAV.ZE2 and AAV.ZE3. Similar results were obtained using hGH as the target gene in this inducible system. Example 8: Virus dose-response curves in vivo in a constitutive or inducible system This example investigates the relationship between the amount of virus used to infect a mouse and the level of hGH produced. Balb/c mice were injected i.v. with doses of 1.5 x 10 9 , 5 x 10 9 , 1.5 x 10i°, or 5 x 10 10 Ad.CG-3 viral particles and the amount of serum hGH was determined at various times after the viral infection. Measurement of the amount of hGH produced in these mice indicates that the production of hGH is proportional to the amount of virus administered to the mice (see Figure 9) and that hGH production continues at a relatively constant level until at least about 50 days after the viral infection. Similar results were obtained in an inducible expression system. In this case, Balb/c mice were injected i.v. with 3 x 10 9 , 1 x 10'0, 3 x 10 1( \ or 1 x ion Ad.ZG-1 and Ad.TFl viral particles in a ratio of 1: 1, rapamycin was administered to the mice at various times after the viral infection, as described above, and the amount of hGH was determined. The results, shown in Figure 10, indicate that there is a direct correlation between the amount of virus administered to the mice and the amount of hGH produced. Furthermore, the results indicate that expression following rapamycin administration is transient and can rapidly be reinduced upon readministration, even about 50 days after the viral infection of the mice. Example 9: Rapamycin dose-response curve in vivo in an inducible system Balb/c nude mice were infected i.v. with 1 x 10n Ad.ZG-1 and Ad.TFl viral particles in a ratio of 1: 1, various amounts of rapamycin (0.05; 0.15; 0.5; 1.5; 5; and 15 mg/kg) were administered to the mice days after the viral infection, and the amount of hGH was determined. The results, shown in Figure 11, indicate that there is a direct correlation between the amount of rapamycin administered to the mice and the amount of hGH produced when rapamycin concentration varies from 0.01 to 0.15 mg/kg. The level of hGH obtained upon administration of higher rapamyin doses does not significantly change from that obtained with doses of 0.05 and 0.15 mg/kg. Example 10: Somatic gene transfer and expression of the target gene in monkeys This Example demonstrates that administration of adenovirus or AAV virus containing a target gene under the control of a constitutive promoter, i.e., CMV, results in efficient expression of the target gene. Monkeys were injected in a skeletal muscle with 10 13 viral particles/kg monkey (each monkey had a weight of about 5 kg) of the adenovirus pAd- CMV-rmEpo-3 or the AAV virus pAAV-CMV-rmEpo-3-S2 (see Table 1). At various days after the viral infection, the serum level of Epo was determined using the human Epo i munoassay kit from R & D System (Cat.# DEP00) and hematocrits were also determined. Figure 12A shows that the target gene is efficiently expressed from the adenoviral vector until at least about 19 days after the viral infection. In agreement with the production of Epo, Figure 12B shows that the number of red blood cells increased until at least about 24 days after the viral infection. Similar results were obtained with the AAV vector. In fact, as shown in Figure 12C, the target gene is also expressed from the AAV vector, although the induction is much slower and weaker than that resulting from the adenoviral vector. The number of red blood cells in monkeys injected with the AAV vector also increased, as indicated by the hematocrit factor (Figure 12D).

Claims: Claims 1. An improved composition for the in vivo genetic engineering of cells within a mammal comprising a viral delivery vehicle containing (a) a target gene construct comprising a nucleic acid sequence encoding an erythropoietin or growth factor protein operably linked to an expression control sequence comprising 12 ZFHDl sites; and (b) the bicistronic nucleic acid construct of the vector pAd-CMV-TF-1, which encodes a ZFHDl-3xFKBP12 DNA-binding fusion protein and an FRB T2098L-p65 transcription activation fusion protein; the improvement comprising using two different recombinant adenoviruses or adeno-associated or hybrid adeno/AAV viruses, the genome of the first such recombinant virus containing the bicistronic nucleic acid construct and the genome of the second recombinant virus containing the target gene construct. 2. The improved composition of claim 1, whereing the genome of the second recombinant virus further comprises a nucleic acid sequence encoding another copy of the FRB T2098L-p65 transcription activation fusion protein. 3. The improved composition of claims 1 or 2, wherein the first and second recombinant viruses are an adenovirus lacking functional adenoviral El and E3 genes. 4. The improved composition of any of claims 1 - 3, wherein the respective recombinant viruses are provided as one or more pharmaceutical preparations comprising one or all of the recombinant viruses together with one or more pharmaceutically acceptable carriers, stabilizers, buffers or other excipients. 5. The improved composition of any of claims 1 - 4, wherein one of the recombinant viruses is provided in excess, relative to the other recombinant virus. 6. The improved composition of any of claims 1 - 5 which further comprises a package insert containing information concerning the administration of the viruses to recipient cells or patients. 7. A method for rendering a mammal capable of rapamycin-dependent transcription of an erythropoietin or growth hormone gene which comprises infecting the mammal with an improved composition of any of claims 1 - 6. 8. The method of claim 7, wherein the mammal is infected by intramuscular or intravenous administration of the improved composition. 9. A method for inducing transcription of an erythropoietin or growth factor target gene in genetically engineered cells in a mammal, which method comprises (a) providing a mammal which has been engineered in accordance with claim 7 or 8, and (b) administering rapamycin to the mammal in an amount sufficient to induce expression of the desired target gene.

Inventors: GILMAN MICHAEL, RIVERA VICTOR, WILSON JAMES, YE XUEHAI, GILMAN, MICHAEL, RIVERA, VICTOR, WILSON, JAMES, YE, XUEHAI
Applicants: ARIAD GENE THERAPEUTICS INC, UNIV PENNSYLVANIA, ARIAD GENE THERAPEUTICS, INC., UNIVERSITY OF PENNSYLVANIA
Application year: 1999
Main classification: C12N15
IPC codes: C12N15