WO2003020879A2

WO2003020879A2 - Cells and methods for propagating adenoviral vectors

Info

Publication number: WO2003020879A2
Application number: PCT/US2002/024051
Authority: WO
Inventors: Douglas E. Brough; Jason G. D. Gall; Joseph T. Bruder; Imre Kovesdi
Original assignee: Genvec, Inc.
Priority date: 2001-07-23
Filing date: 2002-07-22
Publication date: 2003-03-13
Also published as: WO2003020879A3; AU2002327380A1

Abstract

The invention pertains to cells for the propagation of adenoviral vectors. The invention further provides methods of propagating a replication-deficient adenoviral vector which comprises providing an inventive cell, introducing a replication-deficient adenoviral vector into the inventive cell, and maintaining the cell to propagate the replication-deficient adenoviral vector.

Description

CELLS AND METHODS FOR PROPAGATING ADENOVIRAL VECTORS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] This patent application claims the benefit of U.S. Patent Application Nos. 09/911,011, 09/910,828, 09/911,020, and U.S. Provisional Patent Application No. 60/307,212, all of which were filed July 23, 2001.

FIELD OF THE INVENTION [0002] This invention pertains to cells and methods for the propagation of adenoviral vectors.

BACKGROUND OF THE INVENTION [0003] Recombinant eukaryotic viral vectors have become a preferred means of gene transfer for many researchers and clinicians. The human adenovirus is one of the most widely used recombinant viral vectors in current gene therapy protocols. As the use of adenoviral vectors becomes more prevalent, the need for systems that efficiently produce adenoviral vectors suitable for administration is increasingly important. [0004] A concern associated with recombinant adenoviral vectors is uncontrolled propagation of the vector upon administration. To address this concern, replication- deficient adenoviral vectors have been developed containing disruptions or deletions of all or part of at least one region of the adenoviral genome that is essential for viral replication including, for example, the El, E2, and E4 regions, the L1-L5 late regions, and the like. The need in the art to construct replication-deficient adenoviral vectors and the desire to incorporate large coding sequences has led to the generation of adenoviral amplicons, which lack all adenoviral sequences with the exception of the left and right inverted terminal repeats (ITRs) and the packaging signal.

[0005] In order for adenoviral vectors to be widely used in the industry, large quantities of adenoviral vectors must be available. Conceivably, industrial-scale production of gene therapy-quality gene transfer vectors will be required in order to successfully compete in the genetic therapeutics marketplace. In that replication-deficient adenoviral vectors are engineered not to replicate in host cells, complementing cells are commonly used to propagate adenoviral vectors. Problems associated with currently employed complementing cell lines include inefficient vector yields, toxicity, and the risk of recombination between the viral and cellular genomes to produce replication-competent adenovirus (RCA). For example, while the A549 cell line supports sufficient replication of wild-type adenovirus, adenoviral production is significantly reduced or nonexistent when A549 cells are engineered to constitutively express El gene products for complementation (see, e.g., Imler et al., Gene Ther., 1, 75-84 (1996), and Gao et al., Human Gene Ther., 11, 213-219 (2000)). Moreover, production of wild-type adenovirus on the widely used HEK 293 cell line (Graham et al., J. Gen. Virol, 36, 59-72 (1977)) is approximately 50-75% of the yield of wild-type adenovirus on A549 cells. In addition, there is risk associated with the use of HEK 293 cells to produce gene therapy-quality adenoviral vectors in that homologous recombination in regions of overlap between the cellular and viral genomes can result in an RCA-event. When complementing cells are insufficient in providing in trans all the gene functions required for replication of an adenoviral vector and, in particular, an adenoviral amplicons, helper viruses are used. In using an additional virus in a propagation system, however, there is a risk of helper virus contamination of stocks of gene therapy vectors. [0006] Accordingly, there remains a need for alternative cells and methods for propagating replication-deficient adenoviral vectors. The invention provides such cells and methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION [0007] The invention provides a cell comprising a heterologous nucleic acid sequence, which upon expression produces at least one non-adenoviral gene product. The heterologous nucleic acid sequence is contained within the cellular genome, and/or the cell is a transformed human cell. The non-adenoviral gene product complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when the replication-deficient adenoviral vector is present in the cell. [0008] The invention further provides a cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell. The cell (i) is a pleural effusion, large cell lung carcinoma, (ii) is epithelial, and (iii) comprises a wild-type p53 gene. Alternatively, the cell (i) is a lung carcinoma, (ii) comprises a homozygous deletion of the p53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation. The inventive cell preferably is an NCI-H460 cell or a Calu-1 cell.

[0009] The invention provides a cell for the propagation of a replication-deficient adenoviral vector having a cellular genome comprising a nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of an adenoviral genome. The nucleic acid sequence is operatively linked to a chimeric expression control sequence comprising at least a functional portion of a CMV immediate early promoter/enhancer region and at least a functional portion of an adenoviral promoter, wherein the chimeric expression control sequence is upregulated by one or more viral proteins not produced by the nucleic acid sequence.

[0010] The invention further provides a method of propagating a replication-deficient adenoviral vector which comprises providing the inventive cell, introducing a replication- deficient adenoviral vector into the inventive cell, and maintaining the cell to propagate the replication-deficient adenoviral vector.

[0011] The invention provides a method of propagating a replication-deficient adenoviral vector which comprises providing a cell with a cellular genome and a helper virus. The helper virus complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome, wherein the helper virus comprises a first packaging signal, and the cell and the helper virus do not produce a peptide that interacts with the first packaging signal to allow for packaging of the helper virus when the helper virus is present in the cell. A replication-deficient adenoviral vector also is provided that comprises an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome. The replication- deficient adenoviral vector comprises a second packaging signal that differs from the first packaging signal of the helper virus, and the cell or the helper virus produces a peptide that interacts with the second packaging signal to allow for packaging of the replication- deficient adenoviral vector. The method further comprises introducing the helper virus and the replication-deficient adenoviral vector into the cell, and maintaining the cell to propagate the replication-deficient adenoviral vector.

[0012] Alternatively, the method of propagating a replication-deficient adenoviral vector comprises providing a first cell with a cellular genome comprising a heterologous nucleic acid sequence encoding a first peptide required for packaging of an adenoviral vector, wherein the first peptide interacts with a first packaging signal. A helper virus is provided that comprises the first packaging signal. The helper virus encodes a second peptide required for packaging of an adenoviral vector, wherein the second peptide interacts with a second packaging signal, which is different from the first packaging signal. A replication-deficient adenoviral vector also is provided. The replication-deficient adenoviral vector comprises a second packaging signal. The method further comprises introducing the helper virus and the replication-deficient adenoviral vector into the first cell, and maintaining the first cell to propagate both the helper virus and the replication deficient adenoviral vector. The method then comprises providing a second cell not comprising the heterologous nucleic acid sequence encoding the first peptide and not producing the first peptide. The helper virus and the replication-deficient adenoviral vector are introduced into the second cell, and the second cell is maintained to selectively propagate the replication- deficient adenoviral vector.

[0013] The invention further provides a cell comprising a heterologous nucleic acid sequence which, upon expression, produces a first adenoviral IVa2 peptide possessing either encapsidation activity or transcription-activation activity but not both encapsidation activity and transcription-activation activity so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity possessed by the first adenoviral IVa2 peptide when the replication-deficient adenoviral vector is present in the cell. A method of propagating a replication-deficient adenoviral vector comprises (a) providing the aforesaid inventive cell, (b) introducing a replication- deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity possessed by the first adenoviral IVa2 peptide, and (c) maintaining the cell to propagate the replication- deficient adenoviral vector.

[0014] The invention also provides a method of propagating a replication-deficient adenoviral vector, wherein the method comprises (a) providing a cell with a cellular genome, (b) providing a helper virus comprising an adenoviral genome deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome, (c) providing a replication-deficient adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome, (d) introducing the helper virus and the replication-deficient adenoviral vector into the cell, and (e) maintaining the cell to propagate the replication-deficient adenoviral vector.

[0015] The invention additionally provides a cell, as well as a method of propagating an adenoviral vector with the cell, wherein the cell has a cellular genome comprising a nucleic acid sequence which upon expression produces a mutant DAI kinase that complements in trans for a deficiency in at least one essential gene function of at least one virus-associated RNA (VA-RNA) of an adenoviral genome so as to propagate an adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the at least one virus-associated RNA when present in the cell.

DETAILED DESCRIPTION OF THE INVENTION [0016] The invention provides cells that complement in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate (i.e., replicate the entire life cycle of, or replicate to any stage of the life cycle of) a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell. [0017] The inventive cell can be any suitable cell, preferably a cell that comprises a genome that incorporates and desirably retains, or a cell that is a transformed human cell that comprises, a nucleic acid that encodes at least one gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome. The cell desirably can propagate adenoviral vectors and/or adeno- associated viral (AAV) vectors when infected with such vectors or with nucleic acid sequences encoding the adenoviral or AAV genome. Most preferably, the cell can propagate a suitable replication-deficient adenoviral vector upon infection with an appropriate replication-deficient adenoviral vector or transfection with an appropriate replication-deficient viral genome.

[0018] Particularly desirable cell types are those that support high levels of adenovirus propagation. The cell preferably produces at least about 10,000 viral particles per cell and/or at least about 3,000 focus forming units (FFU) per cell. More preferably, the cell produces at least about 100,000 viral particles per cell and/or at least about 5,000 FFU per cell. Most preferably, the cell produces at least about 200,000 viral particles per cell and/or at least about 7,000 FFU per cell. In some embodiments, the cell desirably produces at least about 100% more wild-type adenovirus, preferably at least about 200% more wild-type adenovirus, and most preferably at least about 300% more wild-type adenovirus, than a 293 cell. The cell also desirably produces at least about 90% more wild-type adenovirus, more preferably at least about 100% more wild-type adenovirus, and most preferably at least about 130% more wild-type adenovirus, than an A549 cell.

[0019] Preferably, the cell is, or is derived from, an anchorage dependent cell, but which has the capacity to grow in suspension cultures. More preferably, the cell is a primary cell. By "primary cell" is meant that the cell does not replicate indefinitely in culture. Examples of suitable primary cells include, but are not limited to, human embryonic kidney (HEK) cells, human retinal cells, and human embryonic retinal (HER) cells. Most preferably, the cells are human embryonic lung (HEL) cells. Alternatively, the cell can replicate indefinitely in culture but maintain characteristics of primary cells. An example of such a cell is an ARPE- 19 cell.

[0020] Alternatively, the cell can be a transformed cell, especially a transformed human cell. The cell is "transformed" in that the cell has the ability to replicate indefinitely in culture. Examples of suitable transformed cells include renal carcinoma cells, CHO cells, KB cells, HEK-293 cells, SW-13 cells, MCF7 cells, and Vero cells. Preferably, the cell is a lung carcinoma cell, such as, for example, a non-small cell lung carcinoma cell. The non- small lung cell carcinoma cell can be a squamous/epidermoid carcinoma cell, an adenocarcinoma cell, or a large cell carcinoma cell. The adenocarcinoma cell can be an alveolar cell carcinoma cell or bronchiolo-alveolar adenocarcinoma cell. Other suitable non-small cell lung carcinoma cells include the cell lines NCI-H2126 (American Type Culture Collection (ATCC) No. CCL-256), NCI-H23 (ATCC No. CRL-5800), NCI-H322 (ATCC No. CRL-5806), NCI-H358 (ATCC No. CRL-5807), NCI-H810 (ATCC No. CRL- 5816), NCI-HI 155 (ATCC No. CRL-5818), NCI-H647 (ATCC No. CRL-5834), NCI-H650 (ATCC No. CRL-5835), NCI-H1385 (ATCC No. CRL-5867), NCI-H1770 (ATCC No. CRL-5893), NCI-H1915 (ATCC No. CRL-5904), NCI-H520 (HTB-182), and NCI-H596 (ATCC No. HTB-178). Also suitable are squamous/epidermoid carcinoma lines that include HLF-a (ATCC No. CCL-199), NCI-H292 (ATCC No. CRL-1848), NCI-H226 (ATCC No. CRL-5826), Hs 284.Pe (ATCC No. CRL-7228), SK-MES-1 (ATCC No. HTB- 58), and SW-900 (ATCC No. HTB-59), large cell carcinoma lines (e.g., NCI-H661 (ATCC No. HTB-183)), and alveolar cell carcinoma lines (e.g., SW-1573 (ATCC No. CRL-2170)). The most preferred cell is selected from the group consisting of an A549 cell (ATCC CCL- 185), an NCI-H1299 cell (ATCC CRL-5803), a Calu-1 cell (ATCC HTB-54), and an NCI- H460 cell (ATCC HTB-177). The transformed cell need not be a lung carcinoma cell. In this respect, the cell is preferably a HeLa cell (ATCC CCL-2) or an ARPE-19/HPV-16 cell (ATCC CRL-2502). In addition, the transformed cell can be any cell transformed by a viral gene isolated from a non-adenovirus family member, such as, for example, genes encoded by Papillomaviridae, Poxviridae, Polyomaviridae, Hepadnaviridae, Picorniviridae, Flaviviridae, or any other suitable virus family as defined by van Regenmortel et al., eds., Virus Taxonomy, Seventh Report on the International Committee on Taxonomy of Viruses, 2000.

[0021] Preferably, the cell comprises at least one nucleic acid sequence as described herein, i.e., the cell can comprise one nucleic acid sequence as described herein or more than one nucleic acid sequence as described herein (i.e., two or more of nucleic acid sequences). Such cell lines can be generated in accordance with standard molecular biological techniques as described in International Patent Application WO 95/34671 and U.S. Patent 5,994,106. The nucleic acid sequence preferably is stably integrated into the nuclear genome of the cell. The nucleic acid sequence preferably is retained in the cellular genome (and the nucleic acid sequence, upon expression, preferably produces a gene product complementing in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome) for at least about 10, more preferably at least about 20, passages in culture (e.g., at least about 30, 40, 100, or more passages). Genomic integration of the nucleic acid sequence encoding the complementing factor is a preferred method for generating stable cell lines for adenoviral vector production. The introduction and stable integration of the nucleic acid into the genome of the cell requires standard molecular biology techniques that are well within the skill of the art, such as those described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific American Books (1992), and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995).

[0022] The nucleic acid sequence can be of any suitable source and/or synthetically prepared. Preferably, the nucleic acid sequence is an adenoviral nucleic acid sequence or a heterologous nucleic acid sequence. An "adenoviral nucleic acid sequence" can be any nucleic acid sequence that is obtained from, derived from, or based upon an adenoviral nucleic acid sequence. A "heterologous nucleic acid sequence" can be any nucleic acid sequence that is not obtained from, derived from, or based upon a naturally occurring nucleic acid sequence of the precursor or host cell (i.e., the cell that is modified with the incorporation of the heterologous nucleic acid sequence to form the basis of the inventive cell). By "naturally occurring" is meant that the nucleic acid sequence can be found in nature and has not been synthetically modified. In some embodiments, the heterologous nucleic acid sequence also is not obtained from, derived from, or based upon an adenoviral nucleic acid sequence. For example, the heterologous nucleic acid sequence can be a viral, bacterial, plant, or animal nucleic acid sequence.

[0023] A sequence is "obtained" from a source when it is isolated from that source. A sequence is "derived" from a source when it is isolated from a source but modified in any suitable manner (e.g., by deletion, substitution (mutation), insertion, or other modification to the sequence) so as not to disrupt the normal function of the source gene. A sequence is "based upon" a source when the sequence is a sequence more than about 70% homologous (preferably more than about 80% homologous, more preferably more than about 90% homologous, and most preferably more than about 95% homologous) to the source but obtained through synthetic procedures (e.g., polynucleotide synthesis, directed evolution, etc.). Determining the degree of homology, including the possibility for gaps, can be accomplished using any suitable method (e.g., BLASTnr, provided by GenBank). Notwithstanding the foregoing, the nucleic acid sequence that makes up a heterologous nucleic acid sequence can be naturally found in the host cell, but located at a nonnative position within the cellular genome and/or operably linked to a nonnative promoter. [0024] In those embodiments where the nucleic acid sequence is an adenoviral nucleic acid sequence, the nucleic acid sequence can be obtained or derived from the same or different serotype of adenovirus as the adenoviral vector to be propagated in the cell. For instance, the nucleic acid sequence and the adenoviral vector can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype. Preferably, however, the nucleic acid sequence and adenoviral vector are of serotype 2 or 5. Moreover, the nucleic acid sequence, whether heterologous or adenoviral, can include one or more mutations (e.g., point mutations, deletions, insertions, etc.) from a corresponding naturally occurring adenoviral nucleic acid sequence or heterologous nucleic acid sequence. Thus, where mutations are introduced in the nucleic acid sequence to effect one or more amino acid substitutions in an encoded adenoviral or non-adenoviral gene product, such mutations desirably effect such amino acid substitutions whereby codons encoding positively-charged residues (H, K, and R) are substituted with codons encoding positively-charged residues, codons encoding negatively-charged residues (D and E) are substituted with codons encoding negatively-charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T, and Y) are substituted with codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V, and W) are substituted with codons encoding neutral non-polar residues. Such mutations can also be introduced to effect one or more amino acid substitutions in the N- or C- terminus of the encoded adenoviral or non-adenoviral gene product.

[0025] The adenoviral nucleic acid sequence or heterologous nucleic acid sequence can be any suitable nucleic acid sequence as described herein that, upon expression, produces one or more gene products that complement for one or more deficiencies in any adenoviral essential gene functions (i.e., functions necessary for adenovirus propagation). By "complements for a deficiency in an essential gene function of an adenoviral genome" is meant that the gene product encoded by the adenoviral nucleic acid sequence or heterologous nucleic acid sequence exhibits an adenoviral gene function that is essential (i.e., necessary) for an adenoviral vector to propagate in a cell. For example, the non- adenoviral gene product can induce transcription of promoters regulated by the El A protein, such as the E2A promoter.

[0026] The adenoviral gene product or non-adenoviral gene product can be an RNA sequence or a protein (e.g., a peptide or a polypeptide). Preferably, the adenoviral gene product or non-adenoviral gene product is a protein. By "adenoviral gene product" is meant that the gene product exhibits more than about 70% (preferably more than about 80%, more preferably more than about 90%, and most preferably more than about 99%) homology to a gene product encoded by an adenovirus. By "non-adenoviral gene product" is meant that the gene product exhibits less than about 50% (preferably less than about 30%, more preferably less than about 10%, and most preferably less than about 1%) homology to a gene product encoded by an adenovirus (preferably an adenovirus of serotype 2 or 5). The degree of homology can be determined using any suitable method known in the art (e.g., BLAST programs).

[0027] The nucleic acid, whether adenoviral or heterologous, preferably encodes a full- length gene product, especially a protein (e.g., an adenoviral protein or a non-adenoviral protein). Alternatively, the nucleic acid encodes a functional portion of a gene product (e.g., an adenoviral or non-adenoviral gene product), especially a protein. A "functional portion" is any portion of a gene product that complements for a deficiency in an adenoviral essential gene function at a measurable level. A functional portion of a gene product can be identified using any suitable method known in the art, such as the transfection experiments described herein.

[0028] The nucleic acid sequence, upon expression, produces at least one gene product that provides an adenoviral essential gene function, i.e., that complements in trans for one or more deficiencies in any adenoviral essential gene function (i.e., a function that is necessary for adenovirus propagation). The nucleic acid sequence, upon expression, can produce a gene product that complements for two or more deficiencies in adenoviral essential gene functions (from the same or different regions of the adenoviral genome). The nucleic acid sequence, upon expression, can produce two or more gene products, each of which complements for a deficiency (i.e., at least one deficiency, including but not limited to, two or more deficiencies) in adenoviral essential gene functions (from the same or different regions of the adenoviral genome).

[0029] Essential adenoviral gene functions are those gene functions that are required for propagation (i.e., replication) of a replication-deficient adenoviral vector. Essential gene functions are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA I and/or VA-RNA II). Thus, the gene product (e.g., an adenoviral gene product or a non-adenoviral gene product) complements for a deficiency in at least one adenoviral essential gene function encoded by the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including all adenoviral functions (e.g., to enable propagation of adenoviral amplicons comprising only inverted terminal repeats (ITRs) and the packaging signal or only ITRs and an adenoviral promoter).

[0030] The gene product desirably complements for a deficiency in at least one essential gene function of one or more regions of the adenoviral genome selected from the early regions, e.g., the El, E2, and E4 regions. Preferably, the gene product complements in trans for a deficiency in at least one essential gene function of the El region of the adenoviral genome. More preferably, the gene product complements in trans for a deficiency in at least one essential gene function of an adenoviral El A coding sequence and/or an adenoviral EIB coding sequence (which together comprise the El region). In that respect, one gene product can complement in trans for a deficiency in at least one essential gene function of the El A coding sequence and another (i.e., different) gene product can complement in trans for a deficiency in at least one essential gene function of the EIB coding sequence. In addition or alternatively to the gene product(s) complementing in trans for the aforementioned deficiencies in adenoviral essential gene functions, the same or different gene product(s) can complement for a deficiency in at least one essential gene function of the E2 (particularly the adenoviral DNA polymerase and terminal protein) and/or E4 regions of the adenoviral genome. Desirably, a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4- ORF6 protein. Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome.

[0031] Although not preferred, a helper virus can be provided to the inventive cell with a cellular genome comprising the adenoviral nucleic acid sequence or the heterologous nucleic acid sequence in the event that the inventive cell does not complement for all deficiencies in essential gene functions of the adenoviral genome of the adenoviral vector to be propagated. The helper virus contains coding sequences that, upon expression, produce gene products which provide in trans those gene functions that are necessary for adenoviral propagation (e.g., the IVa2 gene function). In other words, the helper virus can comprise any nucleic acid sequence that is not required in cis (e.g., the ITRs and packaging signal) for propagation.

[0032] The inventive cell, can further comprise an "enhancing" nucleic acid sequence which upon expression produces at least one gene product that enhances propagation of a replication-deficient adenoviral vector without necessarily complementing for a deficiency in an adenoviral essential gene function, so as to propagate more replication-deficient adenoviral vectors when present in the cell than when the "enhancing" nucleic acid sequence is absent from the cell. Although genomic integration of this "enhancing" nucleic acid sequence is preferred, the "enhancing" nucleic acid sequence also can be maintained in the cell extrachromosomally (e.g., on a plasmid). [0033] The "enhancing" nucleic acid sequence can be an adenoviral nucleic acid sequence that encodes at least one adenoviral gene product. In particular, the adenoviral gene product can be a protein encoded by, for example, the El , E2, or E4 regions. The adenoviral gene product also can be a protein encoded by the late regions of the adenoviral genome, such as those encoded by the L1-L5 regions. Alternatively, the "enhancing" adenoviral nucleic acid sequence can encode the adenoviral IVa2 protein, the pIX protein, or virus-associated RNA (e.g., VA-RNA I or II). Alternatively, the "enhancing" nucleic acid sequence also can be heterologous nucleic acid sequence. The "enhancing" nucleic acid sequence can encode, for example, an animal protein that inhibits and/or prevents apoptosis (e.g., Bcl-2). Moreover, the "enhancing" nucleic acid sequence can encode, for example, an RNA molecule or protein that improves the efficiency or rate of replication- deficient adenoviral vector propagation.

[0034] The expression of any of the nucleic acid sequences in the inventive cell is controlled by a suitable expression control sequence operably linked to the nucleic acid sequence. An "expression control sequence" is any nucleic acid sequence that promotes, enhances, or controls expression (typically and preferably transcription) of another nucleic acid sequence. Suitable expression control sequences include constitutive promoters, inducible promoters, repressible promoters, and enhancers. The nucleic acid sequence can be regulated by its endogenous promoter or by a nonnative promoter sequence. Examples of suitable nonnative promoters include the CMV immediate early promoter, the phosphoglycerate kinase (PGK) promoter, the long terminal repeat promoter of the Rous sarcoma virus (LTR-RS V), the sheep metallothionien promoter, and the human ubiquitin C promoter. Alternatively, expression of the nucleic acid sequence can be controlled by a chimeric promoter sequence. The promoter sequence is "chimeric" when it comprises at least two nucleic acid sequence portions obtained from, derived from, or based upon at least two different sources (e.g., two different regions of an organism's genome, two different organisms, or an organism combined with a synthetic sequence). In addition, the expression control sequence can be activated upon infection with a viral vector, such as a replication- deficient adenoviral vector, or contact with viral peptides. When the nucleic acid sequence that produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome is naturally found in the host cell but operably linked to a nonnative promoter, the nonnative promoter can be introduced into the inventive cell by homologous recombination (see, e.g., U.S. Patent 5,641,670) or by random promoter insertion (see, e.g., Harrington et al., Nature Biotechnology, 19, 440-445 (2001)). Suitable expression control sequences can be determined using eukaryotic expression systems such as are generally described in Sambrook et al., supra, and by using reporter gene systems (see, e.g., Taira et al., Gene, 263, 285-292 (2001)).

[0035] In one embodiment of the invention, the cell has a cellular genome comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell. [0036] Any suitable heterologous nucleic acid sequence that encodes a non-adenoviral gene product which complements for a deficiency in an adenoviral essential gene function can be used in the context of the invention. The heterologous nucleic acid sequence desirably is an animal nucleic acid sequence (e.g., a human or murine nucleic acid sequence, especially such a nucleic acid sequence that encodes a cellular protein) or a viral nucleic acid sequence (e.g., a viral nucleic acid sequence obtained from, derived from, or based upon CMV, EBV, HPV, or herpes simplex virus (HSV)). [0037] The identification of heterologous nucleic acid sequences that encode non- adenoviral gene products which complement for a deficiency in an adenoviral essential gene function is well within the skill of the art. In particular, the ordinarily skilled artisan can cotransfect cells that do not normally express any adenoviral gene products with an expression construct comprising the heterologous nucleic acid sequence and a construct comprising a reporter gene (e.g., chloramphenicol acetyltransferase (CAT)) whose expression is dependent (directly or indirectly) on the presence of an essential adenoviral gene product, e.g., whose expression is regulated by an adenoviral El A-responsive promoter such as the EIB or E2A promoter (Spergel et al., J. Virol, 66, 1021-1030 (1992)). Expression of the reporter gene, which can be determined by measuring reporter gene activity, indicates that the non-adenoviral gene product produced by the heterologous nucleic acid sequence complements for a deficiency in an adenoviral essential gene function, e.g., the transcription transactivating function of the El A region. Moreover, the ordinarily skilled artisan can determine whether the non-adenoviral gene product transforms cells through transfection experiments as described by Kimura et al., supra. Other experiments involving only standard molecular biology techniques, such as those described in Sambrook et al., supra, can be performed to determine whether the non-adenoviral gene product complements for a deficiency in other adenoviral essential gene functions. [0038] Examples of suitable heterologous nucleic acid sequences include viral and cellular nucleic acid sequences encoding a non-adenoviral gene product that complements for a deficiency in an adenoviral essential gene function in the El A region of a replication- deficient adenoviral genome. Preferred heterologous nucleic acid sequences encoding a non-adenoviral gene product that complements for a deficiency in an essential gene function of the El A region include the immediate early (IE) region genes I and II of human CMV, the E7 gene of HPV 16, and EBV nucleic acid sequences, as well as nucleic acids of the human HepG2 cell line, the mouse F9 teratocarcinoma stem cell line, and the mouse PCC4 teratocarcinoma stem cell line (see, e.g., Spergel et al., Imperiale et al., and La Thangue et al., supra). Other examples of heterologous nucleic acid sequences include viral and cellular nucleic acid sequences encoding a non-adenoviral gene product that complements for a deficiency in an essential gene function in the EIB region of a replication-deficient adenoviral genome. Preferred heterologous nucleic acid sequences encoding a non- adenoviral gene product that complements for a deficiency in an essential gene function of the EIB region, particularly the EIB- 19 kD protein and/or the E1B-55 kD protein, include nucleic acid sequences encoding the Bcl-2 protein (see, e.g., Rao et al., supra). Moreover, certain cells contain nucleic acid sequences that endogenously complement for adenoviral EIB essential gene function deficiencies, and the heterologous nucleic acid sequences can be those cellular nucleic acid sequences that provide such complementation, including HEK cells (see, e.g., Bernards et al., Virology, 150, 126-139 (1986)), A549 cells (ATCC No. CCL-185), IMR90 fibroblast cells (ATCC No. CCL-186) (see, e.g., Hay et al., Human Gene Ther., 10, 579-590 (1999)), H460 cells (ATCC No. HTB-177) (see, e.g., Lee et al., Int. J. Cancer, 88, 454-463 (2000)), and HCT116 cells (ATCC No. HCL-247) (see, e.g., Ries et al., Nature Medicine, 6, 1128-1133 (2000)). Moreover, a heterologous nucleic acid sequence encoding a non-adenoviral gene product that complements for a deficiency in an adenoviral essential gene function in the E1B-55 kD protein can be used to complement the overlapping functions of the E4-ORF6 protein (see, e.g., Goodrum et al., J. Virology, 72, 9479-9490 (1998)). Further examples of heterologous nucleic acid sequences include viral and cellular nucleic acid sequences encoding a non-adenoviral gene product that complements for a deficiency in an essential gene function of the E4 region of a replication- deficient adenoviral genome. Preferred heterologous nucleic acid sequences encoding a non-adenoviral gene product that complements for a deficiency in an essential gene function of the E4 region include nucleic acid sequences encoded by CMV. In particular, the non- adenoviral gene product can complement for a deficiency in an essential gene function of the E4 region that is not shared by an essential gene function of the EIB region. [0039] The heterologous nucleic acid sequence, however, is not limited to these exemplary sequences. Indeed, genetic sequences can vary between different animal and viral species and strains, and this natural scope of allelic variation is included within the scope of the invention. Once a candidate heterologous nucleic acid sequence (e.g., a CMV IE1 and/or IE2 gene region) is identified, other heterologous nucleic acid sequences encoding a non-adenoviral gene product with similar activity can be obtained by searching the myriad of available genetic sequence databases that enable DNA sequence searching based on homology. One such database is the GenBank sequence database provided by the National Center for Biotechnology Information (NCBI). Preferably, the heterologous sequence comprises a nucleic acid sequence which exhibits at least about 75%, desirably at least about 85%, and more preferably at least about 95%, nucleic acid sequence identity to (e.g., at least 97% identity to, or 100% identity with) any of the heterologous nucleic acids described herein. Determining the degree of homology, including the possibility for gaps, can be accomplished using any suitable method (e.g., BLASTnr, provided by GenBank). [0040] In addition to searching sequence databases, a candidate heterologous nucleic acid sequence encoding a non-adenoviral gene product can be used as a probe to identify homologous sequences from a genetic library via hybridization. An appropriate homologous sequence encodes a non-adenoviral gene product that functions similarly, if not identically, to the non-adenoviral gene product encoded by the candidate heterologous nucleic acid sequence. A suitable heterologous nucleic acid sequence is that which hybridizes to a reference nucleic acid sequence (e.g., a CMV IE1 and/or IE2 gene region) under at least moderate, preferably high, stringency conditions. Exemplary moderate stringency conditions include overnight incubation at 37° C in a solution comprising 20% formamide, 5x SSC (150 mM NaCl and 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing in lx SSC at about 37-50° C, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., supra. High stringency conditions are conditions that, for example (1) use low ionic strength and high temperature for washing, such as with a composition comprising 0.015 M sodium chloride and 0.0015 M sodium citrate, and 0.1% sodium dodecyl sulfate (SDS) at 50° C, (2) employ a denaturing agent during hybridization, such as a composition comprising formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA), 0.1% Ficoll, 0.1% polyvinylpyrrolidone (PVP), and 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C, or (3) employ a composition comprising 50% formamide, 5x SSC (0.75 M NaCl and 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, and sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C, with washes at (i) 42° C in 0.2x SSC, (ii) at 55° C in 50% formamide, and (iii) at 55° C in O.lx SSC (preferably in combination with EDTA). Additional details and explanation of stringency of hybridization reactions are provided in, e.g., Ausubel et al., supra.

[0041] Although primary cells, such as those described herein, are acceptable for use as complementing cell lines, the invention further provides a transformed human cell comprising a heterologous nucleic acid sequence, which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell. [0042] The cell is "transformed" in that the cell has the ability to replicate indefinitely in culture. The human transformed cells are advantageous over primary cells for generating complementing cell lines in some respects. In particular, transformation of primary cells with the El transcription unit may result in an El expression pattern that is optimal for transformation, but not complementation. Moreover, expression of the non-adenoviral gene product may be sufficient for complementation, but not transformation. In contrast, the use of transformed cells eliminates any uncertainty related to the transforming ability of a given gene product, and allows the skilled artisan to directly determine complementation by the non-adenoviral gene product.

[0043] The transformed human cell can be any suitable such cell that comprises a genome capable of incorporating and preferably retaining the heterologous nucleic acid encoding at least one non-adenoviral gene product that complements in trans for a deficiency in at least one adenoviral essential gene function. Preferably, the cell can produce adenoviral vectors and/or adeno-associated viral (AAV) vectors when infected with such vectors or with nucleic acid sequences encoding the adenoviral genome. Most preferably, the cell can produce a replication-deficient adenoviral vector upon infection with the virus or transfection with the viral genome. Particularly desirable cell types are those that support high levels of adenovirus propagation, with desired viral particles per cell and/or focus forming units per cell values as described herein with respect to the inventive cell with a cellular genome comprising the heterologous nucleic acid sequence. [0044] Preferably, the cells are, or are derived from, anchorage dependent cells, but which have the capacity to grow in suspension cultures. Examples of suitable human transformed cells include HEK-293 cells, SW-13 cells, MCF7 cells, and lung carcinoma cells such as those described herein with respect to the inventive cell with a cellular genome comprising the heterologous nucleic acid sequence. Most preferably, the cell is selected from the group consisting of an A549 cell, an NCI-H1299 cell, a Calu-1 cell, and an NCI- H460 cell. Alternatively, the cell need not be a lung carcinoma. In this respect the cell is preferably a HeLa cell or an ARPE-16/HPV-16 cell. In addition, the human transformed cell can be any human cell transformed by a viral gene isolated from a non-adenovirus family member, such as, for example, genes encoded by Papillomaviridae, Poxviridae, Polyomaviridae, Hepadnaviridae, Picorniviridae, Flaviviridae, or any other suitable virus family as defined by van Regenmortel et al., supra. The cell, however, is not limited to these specific examples. Indeed, the cell can be derived from, obtained from, or based upon any suitable human transformed cell.

[0045] The human transformed cell can comprise one heterologous nucleic acid sequence as described herein or more than one heterologous nucleic acid sequence as described herein (i.e., two or more of the heterologous nucleic acid sequence). The heterologous nucleic acid sequence can be integrated into the cellular genome or can be otherwise present in the cell. Desirably, the heterologous nucleic acid sequence is integrated into the cellular genome as described herein with respect to the inventive cell with a cellular genome comprising the heterologous nucleic acid sequence. When the heterologous nucleic acid sequence is not integrated into the cellular genome, the heterologous nucleic acid sequence can reside, for example, on a plasmid, liposome, or any other type of molecule that can harbor a heterologous nucleic acid sequence extrachromosomally. The transformed human cell can comprise one or more heterologous nucleic acid sequences in the cellular genome and one or more heterologous nucleic acid sequences that are not incorporated into the cellular genome. The descriptions of the heterologous nucleic acid sequence, non-adenoviral gene product, and complementation of deficiencies in adenoviral essential gene functions as described herein with respect to the inventive cell with a cellular genome comprising the heterologous nucleic acid sequence also apply to those same features of the human transformed cell.

[0046] The invention also provides a cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome of a replication-deficient adenoviral vector. The features of this cell as regards the adenoviral nucleic acid sequence, gene product, complementating of deficiencies in essential gene functions, and the like are as described above. The cell, however, has other particular characteristics in addition to the aforesaid features. [0047] The cell (i) is a large cell lung carcinoma derived from a pleural effusion (i.e., a pleural effusion, large cell lung carcinoma), (ii) is epithelial, and (iii) comprises a wild-type p53 gene. By "derived" from a pleural effusion is meant that the cell is isolated from a large cell lung carcinoma that originated from an effusion of the lung pleura. By "epithelial" is meant that the cell participates in lining the inner and outer surfaces of the organism from which it is isolated. The cell has a wild-type p53 gene in that the nucleic acid sequence encoding thcp53 gene does not comprise any alterations that change the normal function of t ep53 gene product in the inventive cell. Advantageously, the cell comprises a homozygous K-ras codon 12 mutation. The cell comprises a homozygous K- ras codon 12 mutation in that both alleles of the K-ras gene locus are mutated in the inventive cell. Moreover, the cell does not express the pl6INK4a protein. The cell also desirably exhibits adherent growth in culture, and comprises two X chromosomes and two Y chromosomes.

[0048] The cell alternatively (i) is a lung carcinoma, (ii) comprises a homozygous deletion of the p53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation. The cell comprises a homozygous deletion of ύιep53 gene in that both alleles of the p53 gene locus comprise deletions which, for example, prevent expression of the p53 gene product or render tiιep53 gene product non-functional. The cell is heterozygous for a K-ras codon 12 mutation in that the cell comprises a K-ras gene locus comprising a wild-type allele and a codon 12 mutation in the other allele. Advantageously, the cell does not express the pl6INK4a protein. The cell also desirably exhibits adherent growth in culture. Desirably, the antigen expression profile of the cell comprises (i) blood type A, (ii) Rh positive, and (iii) HLA antigens A10, A11, B15, and Bw35. By "antigen expression profile" is meant the collection of antigens that are expressed on the surface of the inventive cell. [0049] The invention also provides a cell, for the propagation of a replication-deficient adenoviral vector, wherein the cell has a cellular genome comprising a nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of an adenoviral genome. The nucleic acid sequence is operatively linked to a chimeric expression control sequence comprising at least a functional portion of a CMV immediate early promoter/enhancer region, at least a functional portion of an adenoviral promoter, or both. The chimeric expression control sequence is upregulated by one or more adenoviral proteins not produced by the nucleic acid sequence. The cell desirably is suitable for the propagation (i.e., the replication of the entire life cycle, or the replication to any stage of the life cycle) of an adenoviral vector , more preferably a replication-deficient adenoviral vector.

[0050] Preferably, the chimeric expression control sequence comprises two different nucleic acid sequence portions that exhibit less than about 40%, more preferably less than about 25%, and even more preferably less than about 10% nucleic acid sequence identity to one another (which can be determined by methods described elsewhere herein). Typically, the chimeric expression control sequence will comprise expression control sequences obtained or derived from at least two different eukaryotic viruses, preferably wherein only one of the expression control sequences is obtained from, derived from, or based upon the expression control sequence of an adenovirus. Preferably, the non-adenovirus portion of the expression control sequence is obtained or derived from a eukaryotic virus capable of infecting mammals, preferably humans, and desirably comprises a DNA genome, more desirably a double stranded DNA genome.

[0051] The chimeric expression control sequence can comprise any suitable type of individual expression control sequences, including promoters, enhancers, other regulatory sequences, or portions thereof (e.g., a Kozak consensus sequence, TATA box, or other DNA binding protein recognized sequence). The chimeric expression control sequence can comprise additional sequences which can exhibit expression control sequence activity alone or in conjunction with the other portions of the chimeric expression control sequence. Those additional sequences can be derived or obtained from additional sources (e.g., a third heterologous sequence obtained from a different source than the first and second sequences that are part of the chimeric expression control sequence). Preferably, the chimeric expression control sequence comprises a functional portion of a first sequence, which operates as an enhancer in the chimeric expression control sequence, from a first source, and a functional portion of a second sequence which operates as a promoter, from a second source.

[0052] In accordance with the invention, a "functional portion" is any portion of an expression control sequence that measurably promotes, enhances, or controls expression (typically transcription) of an operatively linked nucleic acid. Such regulation of expression can be measured via RNA or protein detection by any suitable technique, and several such techniques are known in the art. Examples of such techniques include Northern analysis (see, e.g., Sambrook et al., supra, and McMaster and Carmichael, PNAS, 74, 4835-4838 (1977)), RT-PCR (see, e.g., U.S. Patent 5,601,820, and Zaheer et al., Neurochem Res., 20, 1457-1463 (1995)), in situ hybridization methods (see, e.g., U.S. Patents 5,750,340 and 5,506,098), antibody-mediated techniques (see, e.g., U.S. Patents 4,367,110, 4,452,901, and 6,054,467), and promoter assays utilizing reporter gene systems such as the luciferase gene (see, e.g., Taira et al., Gene, 263, 285-292 (2001)). Eukaryotic expression systems in general are further detailed in Sambrook et al., supra.

[0053] An enhancer is any cw-acting polynucleotide sequence that promotes, induces, or otherwise controls (e.g., inhibits) expression (preferably transcription) of one or more operatively linked nucleic acid sequences. An enhancer that inhibits transcription also is termed a "silencer." The enhancer can function in either a direct or reverse orientation with respect to the nucleic acid sequence (e.g., from a position "downstream" of the operatively linked nucleic acid sequence) and over a relatively large distance (e.g., several kilobases (kb)) from an operatively linked nucleic acid sequence. Accordingly, the enhancer can be any nucleic acid sequence that can function to induce, promote, or control expression in either orientation and/or at various distances from the operatively linked nucleic acid sequence. In contrast, a promoter will operate in a sequence specific manner, typically in the same orientation with and upstream from the nucleic acid sequence, and in a more localized manner. For example, most eukaryotic promoters recognized by RNA polymerase II have a TATA box that is centered around position 25-30 upstream of the transcription start site and has the consensus sequence TATAAAA. Several promoters have a CAAT box around position 90 with the consensus sequence GGCCAATCT. Typically, the promoter will exhibit greater control over the operatively linked nucleic acid sequence. Thus, the promoter can be any nucleic acid sequence which exhibits localized control over the operatively linked nucleic acid.

[0054] In a preferred embodiment of the invention, the chimeric expression control sequence comprises a non-adenoviral functional portion. Preferred functional portions of non-adenoviral expression control sequence portions in this respect are obtained or derived from a cytomegalovirus (CMV), preferably a human CMV, and more particularly from the human CMV immediate early (IE) promoter/enhancer region. Advantageously, the CMV IE portion exhibits an enhancer activity in the chimeric expression control sequence. Enhancer activity can be determined by any suitable method, such as an enhancer trap as described in Boshart et al., Cell, 41, 521-530 (1985). Preferably, the CMV IE enhancer portion exhibits upregulation of operatively linked adenoviral genes in the presence of an adenoviral protein not expressed by the cellular genome and/or adenoviral vectors which lack adenoviral vector proteins expressed by the cellular genome. The CMV IE enhancer portion can be of any suitable size and comprise any suitable sequence derived from, based upon, or obtained from the wild-type CMV IE promoter/enhancer sequence (as described in Thomsen et al., PNAS, 81, 659-663 (1984), Jahn et al., J. Virol, 49, 363-370 (1984), Jahn et al., In: Herpseviruses, F. Rapp, ed., Alan Liss, Inc., New York, pp. 455-463 (1984), and Boshart et al., Cell, 41, 521-530 (1985)). Preferably, the CMV IE enhancer comprises a nucleic acid sequence which exhibits at least about 75%, desirably at least about 85%, and more preferably at least about 95% nucleic acid sequence identity to (e.g., at least 97% identity to, or 100% identical with) SEQ ID NO: 1. However, the invention is not limited to this exemplary sequence. Indeed, genetic sequences can vary between different strains, and this natural scope of allelic variation is included within the scope of the invention. Determining the degree of homology, including the possibility for gaps, can be accomplished using any suitable method (e.g., BLASTnr, provided by GenBank). [0055] Additionally and alternatively, the CMV IE enhancer desirably includes any sequence that hybridizes to SEQ ID NO:l under at least moderate, preferably high, stringency conditions, as set forth above with respect to identification of a heterologous nucleic acid sequence and in, e.g., Ausubel et al., supra.

[0056] Preferably, a portion of the chimeric expression control sequence is obtained or derived from an adenovirus, and optionally (though not necessarily) from an adenovirus of the same serotype as the adenovirus from which the nucleic acid encoding the adenoviral protein is obtained from, derived from, or based upon, when the nucleic acid sequence is obtained from, derived from, or based upon an adenovirus. Preferred adenovirus genomes are obtained, derived, or based upon Group C adenoviruses and more preferably are obtained or derived from a serotype 2 or serotype 5 adenovirus. By incorporation of an adenoviral expression control sequence portion into the chimeric expression control sequence, stable cell lines are more readily obtained (as described further herein). The presence of the adenoviral expression control sequence portion is further believed to provide better control over expression of the operatively linked adenoviral-protein-encoding gene.

[0057] The chimeric expression control sequence can exhibit any suitable type of expression confrol sequence activity. Preferably, the chimeric expression control sequence exhibits a promoter activity. Preferably, the chimeric expression control sequence comprises an adenoviral promoter TATA box-associated sequence. A TATA-box associated sequence can be any sequence comprising the consensus sequence TATA (SEQ ID NO:2), preferably positioned about 20-30 nucleotides upstream of a protein-encoding gene's transcription start site, which directs RNA polymerase binding and transcription of the operatively linked nucleic acid sequence. Desirably, the adenoviral TATA-box associated sequence promotes the production of stable cell lines compared to cell lines comprising only a heterologous promoter/enhancer region (e.g., a cell line which is capable of survival and adenoviral production after at least about 3, more preferably at least about 5, even more preferably at least about 10, advantageously at least about 20, and optimally at least about 100 passages). The adenoviral promoter TATA box-associated sequence can be obtained from any suitable adenoviral promoter. The TATA box-associated sequence can be of any suitable length. Typically, the adenoviral TATA box-associated sequence will be 135 nucleotides in length.

[0058] El A TATA box-associated sequences are particularly preferred in cells of the invention in which the adenoviral promoter portion is linked to a portion of the El A region of the adenoviral genome. Desirably, the El A TATA box-associated sequence exhibits at least about 75% nucleic acid sequence identity, more preferably at least about 80% sequence identity, even more preferably at least about 90% nucleic acid sequence identity, and optimally at least about 95% sequence identity to (e.g., at least 97% identity to, or 100% identical with) SEQ ID NO:3. However, the invention is not limited to this exemplary sequence. Indeed, genetic sequences can vary between different strains, and this natural scope of allelic variation is included within the scope of the invention. Additionally and alternatively, the El A TATA box-associated sequence can include any sequence that hybridizes to SEQ ID NO: 3 under at least moderate, preferably high, stringency conditions. Determining the degree of homology and performing nucleic acid hybridizations can be accomplished using the methods discussed herein or any other suitable method. [0059] The chimeric expression control sequence can be generated using standard molecular biology techniques, such as those described in Sambrook et al., supra. The chimeric expression control sequence can be inserted in the cellular genome using any suitable technique, such as, for example, those described in Sambrook et al., supra. Suitable transfection methods include but are not limited to calcium phosphate or DEAE- dextran-mediated transfection, polybrene transfection, protoplast fusion, electroporation, liposome-mediated transfection, or direct microinjection of DNA.

[0060] The function of the chimeric expression control sequence is induced, promoted, or enhanced (i.e., "upregulated") by the presence of one or more adenoviral proteins not produced by the nucleic acid sequence of the inventive cell, typically and preferably resulting in inducing or promoting expression of the operatively linked adenoviral protein- encoding gene, as detected by standard methods such as those described herein. [0061] The adenoviral protein(s) not produced by the nucleic acid sequence and that upregulates the chimeric expression control sequence can be any suitable protein obtained from, derived from, or based upon, a protein produced by an adenovirus. The precise adenoviral protein or combination of proteins will vary depending upon the components of the chimeric expression control sequence. Identification of suitable proteins can be determined by simple experimentation to determine whether administration or expression of the protein in the cell results in upregulation of the chimeric expression control sequence. Examples of suitable adenoviral proteins are discussed by Thomas Shenk, supra, and M. S. Horwitz, supra.

[0062] The one or more adenoviral proteins can be introduced to the cell independent from a viral particle or, typically and preferably, by their presence in or expression from a viral particle. The viral particle can be any suitable viral particle, including a non-intact viral particle (e.g., an incomplete viral particle) or a virus-like particle (VLP). Preferably, the viral particle is an intact adenoviral vector particle, and more preferably an intact replication-deficient adenoviral vector particle. The adenoviral vector particle can be a modified adenoviral vector particle, such as an adenoviral vector particle that exhibits a targeting function, such as the adenoviral vectors described in U.S. Patents 5,559,099, 5,731,190, 5,712,136, 5,770,442, 5,846,782, 5,962,311, 5,965,541, and 6,057,155 and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, and WO 00/15823. Other suitable adenoviral vectors are those comprising protein modifications that decrease the potential for immunological recognition by the host and resultant coat-protein directed neutralizing antibody production, as described in, e.g., International Patent Applications WO 98/40509 and WO 00/34496.

[0063] A suitable adenoviral protein can be introduced to the cell independent from a viral particle via expression of a nucleic acid encoding the protein delivered to the cell using vectors and transfection procedures in accordance with standard molecular biological techniques. Any suitable vector can be used for such a purpose. Suitable expression vectors are exemplified in Sambrook et al., supra, and can include a naked DNA or RNA vector (including, for example, a linear expression element or a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119) or a precipitated nucleic acid vector construct (e.g., a CaPO₄ precipitated construct). The vector also can be a shuttle vector able to replicate and/or be expressed (desirably both) in both eukaryotic and prokaryotic hosts (e.g., a vector comprising an origin of replication recognized in both eukaryotes and prokaryotes). The vectors can be associated with salts, carriers (e.g., PEG), formulations which aid in transfection (e.g., sodium phosphate salts, Dextran carriers, iron oxide carriers, or gold bead carriers), and/or other pharmaceutically acceptable carriers, some of which are described herein. Alternatively or additionally, the vector can be associated with one or more transfection-facilitating molecules such as a liposome (preferably a cationic liposome), a transfection facilitating peptide or protein-complex (e.g., a poly(ethylenimine), polylysine, or viral protein-nucleic acid complex), a virosome, a modified cell or cell-like structure (e.g., a fusion cell), or a viral vector. Any suitable transfection method may be used to introduce the vector into the cell, such as, for example, those described in Sambrook et al., supra, and those described elsewhere herein.

[0064] In an alternative embodiment, nucleic acid sequences encoding the one or more adenoviral proteins can reside in the cell episomally or as stable integrants in the cellular genome. In this context, the production of the one or more adenoviral proteins can be controlled by an inducible promoter system operatively linked to the nucleic acid sequences encoding the one or more adenoviral proteins.

[0065] The one or more adenoviral proteins not produced by the nucleic acid sequence of the inventive cell (e.g., the adenoviral vector particle comprising or expressing such a protein) can upregulate the chimeric expression control sequence in any suitable manner and to any suitable degree. For example, the presence of the one or more adenoviral proteins not produced by the nucleic acid sequence can result in an at least about a 10%, 20%, or 30% increase in the expression of the chimeric expression sequence-linked adenoviral coding sequence. Preferably, the presence of the adenoviral protein results in at least about a 40%, and more preferably at least about a 50%, increase (e.g., at least about a 60%, 70%, 100%, 200%, or even a 1, 000-fold increase) in expression of a nucleic acid sequence operatively linked to the chimeric expression confrol sequence. The adenoviral protein can "induce" expression of the operatively linked nucleic acid sequence from non- detectable levels or merely enhance expression levels over "constitutive" expression levels. The adenoviral protein can upregulate the chimeric expression confrol sequence directly by, for example, physical interaction of the adenoviral protein with the chimeric expression control sequence. Alternatively, the adenoviral protein can upregulate the chimeric expression control sequence indirectly. For example, the adenoviral protein can interact with a molecule that represses expression from the chimeric expression control sequence. Such an interaction releases transcriptional repression by the molecule, resulting in upregulation of expression from the chimeric expression control sequence. Preferably, the chimeric expression control sequence is operatively linked to the adenoviral El A gene. More preferably, the adenoviral protein induces El A expression to levels sufficient to induce expression of the EIB protein. The degree and manner of upregulation associated with the chimeric expression confrol sequence can be determined by any suitable technique, such as the techniques described elsewhere herein or otherwise known in the art for measuring expression confrol sequence activity and/or gene expression. [0066] The invention further provides a method of propagating a replication-deficient adenoviral vector. The method comprises providing a cell of the invention, introducing the replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of the one or more regions, and maintaining the cell (e.g., under conditions suitable for adenoviral propagation) to propagate the adenoviral vector. [0067] The adenoviral vector is deficient in at least one gene function (of the adenoviral genome) required for viral propagation (i.e., an adenoviral essential gene function), thereby resulting in a "replication-deficient" adenoviral vector. The adenoviral vector is deficient in the one or more adenoviral essential gene functions complemented for by the inventive cell to allow for propagation of the replication-deficient adenoviral vector when present in the cell.

[0068] Preferably, the adenoviral vector is deficient in at least one essential gene function of the El region, e.g., the Ela region and/or the Elb region, of the adenoviral genome that is required for viral replication. The recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. More preferably, the vector is deficient in at least one essential gene function of the El region and at least part of the nonessential E3 region (e.g., an Xba I deletion of the E3 region). The adenoviral vector can be "multiply deficient," meaning that the adenoviral vector is deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome, such as the El, E2, and/or E4 regions of the adenoviral genome. For example, the aforementioned El -deficient or E1-, E3 -deficient adenoviral vectors can be further deficient in at least one essential gene function of the E4 region and/or at least one essential gene function of the E2 region (e.g., the E2A region and or E2B region). Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses. Examples of suitable adenoviral vectors include adenoviral vectors that lack (a) all or part of the El region and all or part of the E2 region, (b) all or part of the El region, all or part of the E2 region, and all or part of the E3 region, (c) all or part of the El region, all or part of the E2 region, all or part of the E3 region, and all or part of the E4 region, (d) at least part of the Ela region, at least part of the Elb region, at least part of the E2a region, and at least part of the E3 region, (e) at least part of the El region, at least part of the E3 region, and at least part of the E4 region, and (f) all essential adenoviral gene products (e.g., adenoviral amplicons comprising ITRs and the packaging signal only). The adenoviral vector can contain a wild-type pIX gene. Alternatively, although not preferably, the adenoviral vector also can contain a pIX gene that has been modified by mutation, deletion, or any suitable DNA modification procedure.

[0069] The replication-deficient adenoviral vector can be generated by using any species, strain, subtype, or mixture of species, sfrains, or subtypes, of an adenovirus or a chimeric adenovirus as the source of vector DNA. The adenoviral vector can be any adenoviral vector capable of growth in a cell, which is in some significant part (although not necessarily substantially) derived from or based upon the genome of an adenovirus. For instance, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype. The adenoviral vector preferably comprises an adenoviral genome of a wild-type adenovirus of group C, especially of serotype (i.e., Ad5). Adenoviral vectors are well known in the art and are described in, for example, U.S. Patents 5,559,099, 5,712,136,

5.731.190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106,

6.020.191, and 6,113,913, International Patent Applications WO 95/34671, WO 97/21826, and WO 00/00628, and Thomas Shenk, "Adenoviridae and their Replication," and M. S. Horwitz, "Adenoviruses," Chapters 67 and 68, respectively, in Virology, B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996).

[0070] The construction of adenoviral vectors is well understood in the art and involves the use of standard molecular biological techniques, such as those described in, for example, Sambrook et al., supra, Watson et al., supra, Ausubel et al., supra, and other references mentioned herein. Moreover, adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 5,965,358 and International Patent Applications WO 98/56937, WO 99/15686, and WO 99/54441.

[0071] When the cell has a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome, and is used to propagate a replication-deficient adenoviral vector, it is desirable to avoid a recombination event between the cellular genome (of the cell) and the adenoviral genome (of the adenoviral vector) that would result in the generation of a replication-competent adenovirus (RCA). As such, there is preferably insufficient overlap between the genome of the cell and the replication-deficient adenoviral vector genome to mediate a recombination event sufficient to result in a replication-competent adenovirus. If overlap exists, the overlapping sequences desirably are predominantly located in the nucleic acid flanking the coding region of the complementation factor (the "trans-complementing region") in the cellular genome and the nucleotide sequences adjacent to the missing region(s) of the adenoviral genome. Ideally, there is no overlap between the cellular genome and the adenoviral vector genome. However, it is acceptable that partial overlap exists between the cellular genome and the adenoviral vector genome on one side of the trans-complementing region. In such an event, the region of homology preferably is contiguous with the trans-complementing region. For example, when the cell comprises a trans-complementing region comprising a nucleotide sequence of the adenoviral El region, the cell desirably lacks homologous sequences on the 5' side (left side) of the trans- complementing region corresponding to the adenoviral inverted terminal repeats (ITRs) and packaging signal sequences, but contains homologous sequences on the 3' side (right side) of the trans-complementing region. The region of homology is at least about 2000 base pairs, preferably at least about 1000 base pairs (e.g., at least about 1500 base pairs), more preferably at least about 700 base pairs, and most preferably at least about 300 base pairs. [0072] When the cell has a cellular genome comprising at least one nucleic acid sequence, which is operatively linked to a chimeric expression control sequence comprising at least a functional portion of a CMV immediate early promoter/enhancer region, at least a functional portion of an adenoviral promoter, or both, the cell preferably is characterized by lacking the 5' ITR, the packaging sequence, and the El A enhancer of the adenoviral genome. The preferred cell is further characterized by desirably comprising the nucleic acid sequences encoding El A, EIB, protein IX, and IVa2/partial E2B. In particular, the prefeπed cell comprises at least one adenoviral nucleic acid sequence which lacks nucleotides 1-361, yet comprises adenoviral nucleotides 3325-5708 located 3' to the complementing region. Not to adhere to any particular theory, it is believed that a single recombination event in such a homologous region will not give rise to a replication competent adenoviral vector due to the absence of the 5' ITR and packaging sequence. In a similar manner, a prefeπed cell that contains both the El and E4 regions sufficient to propagate E1-, E4-deleted adenoviral vectors can comprise a region of homology between the cellular genome and the adenoviral genome located 5' or 3' to the nucleic acid sequence encoding the E4 region.

[0073] The generation of RCA desirably is diminished such that (a) the cell produces less than about one replication-competent adenoviral vector for at least about 20 passages after infection with the adenoviral vector, (b) the cell produces less than about one replication-competent adenoviral vector in a period of about 36 hours post-infection, (c) the cell produces less than about one replication-competent adenoviral vector per 1 x 10¹⁰ total viral particles (preferably 1 x 10 total viral particles, more preferably 1 x 10 total viral particles, and most preferably 1 x 10¹³ total viral particles), or (d) any combination of (a)- (c). Optimally, the amount of overlap between the cellular genome and the adenoviral genome (i.e., the genome of the adenoviral vector being propagated in the cell) is insufficient to mediate a homologous recombination event that results in a replication- competent adenoviral vector such that replication-competent adenoviruses are eliminated from the vector stocks resulting from propagation of the replication-deficient adenoviral vector in the cell. Virus growth yield and virus plaque formation have been previously described (see, e.g., Burlseson et al., Virology: a Laboratory Manual, Academic Press Inc. (1992)), and measuring RCA as a function of plaque forming units is described in U.S. Patent 5,994,106.

[0074] In addition to manipulation of the essential gene functions of the adenoviral genome, manipulation of the packaging signal of the adenoviral genome and/or the manipulation of peptides required for efficient packaging of the adenoviral genome into capsids provides a unique opportunity for development of propagation systems for helper virus-free stocks of replication-deficient adenoviral vectors. Another embodiment of the invention is predicated, at least in part, on the surprising discovery that the adenoviral IVa2 coding sequence can be manipulated to produce a IVa2 protein unable to participate in encapsidation of an adenoviral vector, but which retains the ability to activate the major late promoter (MLP), so as to produce a replication-deficient adenoviral vector and that a complementary cell can utilize a portion of the nucleic acid sequence encoding the IVa2 protein to complement for the deficiency in the adenoviral vector that renders that vector replication-deficient.

[0075] The invention provides a method of propagating a replication-deficient adenoviral vector using, in part, a helper virus. The method provides a means of preferentially encapsidating the replication-deficient adenoviral vector to form a stock of adenoviral particles free from contamination with the helper virus. The invention further provides a complementary cell for propagating a replication-deficient adenoviral vector that has an adenoviral IVa2 peptide lacking either encapsidation activity or transcription- activation activity. A propagation method involving the use of such a complementary cell are also provided.

[0076] In particular, the invention provides a method of propagating a replication- deficient adenoviral vector. The method comprises providing a cell comprising a cellular genome. A helper virus also is provided that complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome. The helper virus comprises a first packaging signal, and the cell and the helper virus do not produce a peptide that interacts with the first packaging signal to allow for packaging of the helper virus when the helper virus is present in the cell. A replication-deficient adenoviral vector also is provided that comprises an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome. The replication-deficient adenoviral vector comprises a second packaging signal that differs from the first packaging signal of the helper virus, and the cell or the helper virus produces a peptide that interacts with the second packaging signal to allow for packaging of the replication-deficient adenoviral vector. The method further comprises introducing the helper virus and the replication-deficient adenoviral vector into the cell, and maintaining the cell to propagate the replication-deficient adenoviral vector. [0077] Descriptions of the cell, the heterologous nucleic acid sequence, the adenoviral vector, and components thereof set forth above in connection with other embodiments of the invention also are applicable to those same aspects of the aforesaid inventive method. For example, the cell comprises a cellular genome that preferably comprises a nucleic acid sequence (e.g., a heterologous nucleic acid sequence) that, when expressed, produces a gene product (e.g., a protein or RNA) that complements in trans for a deficiency in at least one essential gene function of one or more early regions and/or late regions of an adenoviral genome as described elsewhere herein. Desirably, the heterologous nucleic acid sequence, when expressed, produces one or more gene product(s) that complement in trans for the all the essential gene functions lacking in an adenoviral vector amplicon, i.e., a "gutted" adenoviral vector lacking all native adenoviral sequences except at least one adenoviral ITR and the packaging signal. Ideally, the nucleic acid sequence(s) that, when expressed, produce gene products that complement in trans for a deficiency in at least one essential gene function of one or more genes of the early and/or late regions, are adenoviral nucleic acid sequences.

[0078] A helper virus and a replication-deficient adenoviral vector also are provided in the inventive method. The helper virus complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome. Indeed, in a prefeπed embodiment, the helper virus and cell together provide the necessary gene functions for propagation of the replication-deficient adenoviral vector. Accordingly, the helper virus can be any virus capable of producing factors that provide essential gene functions of an adenoviral genome. The helper virus can complement in trans for a deficiency in one or more essential gene functions by expressing endogenous or heterologous nucleic acid sequences that complement for one or more adenoviral gene functions required for viral replication. Examples of suitable helper viruses include, for instance, parvovirus (i.e., adeno-associated virus), retrovirus, herpes simplex virus (HSV), and adenovirus. Desirably, the helper virus comprises regions of the adenoviral genome. Most preferably, the helper virus is an adenovirus. The helper virus and replication-deficient adenoviral vector can be derived or based on any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenovirus as described elsewhere herein. The helper virus can be constructed and/or purified using the same methods set forth above in connection with the construction of the replication-deficient adenoviral vector.

[0079] The helper virus complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome. The helper virus preferably comprises adenoviral nucleic acid sequences encoding gene functions required for propagating a particular replication-deficient adenoviral vector. For instance, the helper virus can comprise an adenoviral El A coding sequence, an adenoviral EIB coding sequence, an adenoviral E2 coding sequence (e.g., an adenoviral E2A coding sequence), an adenoviral E4 coding sequence (e.g., E4-ORF6), the coding sequences of the late region of the adenoviral genome, an adenoviral IVa2 coding sequence, combinations of any of the foregoing, and the like, to efficiently complement replication-deficient adenoviral vectors. However, as described herein, non-adenoviral-based factors have been identified that effectively complement for essential adenoviral gene functions, and nucleic acid sequences encoding such factors also can be present in the helper virus.

[0080] The helper virus comprises a first packaging signal. The replication-deficient adenoviral vector comprises a second packaging signal that differs from the first packaging signal of the helper virus. For example, the second packaging signal is a wild-type adenovirus packaging signal, in which case the first packaging signal is not a wild-type adenovirus packaging signal. Alternatively, the second packaging signal is not a wild-type adenovirus packaging signal, but still differs from the first packaging signal. By "packaging signal" is meant any adenoviral DNA that has a role in packaging of the adenoviral genome into viral capsids, e.g., any DNA that binds a peptide that is part of the adenoviral packaging machinery. An example of a suitable packaging signal is that located at, for example, nucleotides 194-358 of adenovirus serotype 5 (Grabel and Hearing, J Virol, 64(5), 2047-2056 (1990)). The region comprises at least five functionally redundant domains, the A repeats, with Al, All, AV, and AVI being critical. Each repeat fits a consensus motif and can function independently (Zhang and Imperiale, J. Virol, 74(6), 2687-2693 (2000)). The packaging signal of the adenoviral genome has been identified for most adenoviral serotypes. Location of the packaging signal of non-serotype 5 adenoviruses can be determined by searching the GenBank computer database (provided by NCBI). [0081] Preferably, the first and/or second packaging signal are mutated in the target sequence that interacts with a peptide required for packaging of the adenoviral genome into viral capsids (e.g., a peptide associated with the adenoviral packaging machinery), such that at least one wild-type adenoviral peptide responsible viral packaging is incapable of interacting with the packaging sequence. For example, the first and/or second packaging signal (preferably the first packaging signal) can comprise a mutated IVa2 peptide binding site. Alternatively, the first and/or second packaging signal (preferably the first packaging signal) can comprise a mutated adenoviral preterminal protein (pTP) binding site. The first and/or second packaging sequence can be constructed using routine molecular biology techniques. For example, the wild-type packaging sequence can be present in the helper virus or the replication-deficient adenoviral vector. By "wild-type packaging signal" is meant a packaging signal as found in nature, as well as a wild-type packaging signal comprising conservative mutations such that the packaging signal retains the ability to interact with that packaging signal's DNA-binding peptide. A wild-type packaging signal can be mutated such that native binding of an adenoviral packaging peptide to the DNA sequence is ablated, which is confirmed using standard peptide-DNA binding assays. Such mutations include point mutations of the packaging signal, as well as additions, substitutions, or deletions within the packaging signal. A mutated packaging signal is also meant to include instances wherein the complete target sequence of the packaging signal is removed and, if desired, replaced with a target sequence for a different DNA-binding peptide (e.g., the target sequence for IVa2 binding is replaced with the target sequence for Gal4 binding). Alternatively, a packaging sequence specific for a particular DNA-binding peptide (or DNA-binding domain of a DNA-binding peptide) can be synthetically generated. Ideally, the packaging signal comprises a target sequence for a native or nonnative DNA-binding peptide. By inserting a nonnative target sequence in the packaging signal (e.g., a target sequence that is not naturally found in the packaging signal) of the helper virus or the replication-deficient adenoviral vector, a DNA-binding peptide associated with adenoviral packaging can effectively be targeted to the helper virus or replication-deficient adenoviral vector, respectively.

[0082] The replication-deficient adenoviral vector and, in some embodiments, the helper virus are deficient in at least one gene function required for viral replication (i.e., an "essential" gene function) as set forth above. Preferably, the adenoviral vector and/or helper virus is "multiply deficient," as defined herein. In instances where the helper virus is replication-deficient, the cell provides the missing gene functions. [0083] In view of the above, the invention further provides a method of propagating a replication-deficient adenoviral vector. The method comprises providing a cell with a cellular genome, a helper virus comprising an adenoviral genome deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome, and a replication-deficient adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome. The method further comprises introducing the helper virus and the replication-deficient adenoviral vector into the cell, and maintaining the cell to propagate the replication-deficient adenoviral vector.

[0084] The replication-deficient adenoviral vector and/or helper virus can comprise modifications other than disruption of replication-associated regions of the adenoviral genome. The adenoviral vector can be modified, for example, to alter coat proteins to modulate the binding specificity of the vector for host cells (i.e., a targeted adenoviral vector), or those as set forth above comprising protein modifications that decrease the potential for immunological recognition by the host.

[0085] In the inventive method, the cell and the helper virus do not produce a peptide that interacts with the first packaging signal to allow for packaging of the helper virus when the helper virus is present in the cell. However, the cell or the helper virus does produce a peptide that interacts with the second packaging signal to allow for packaging of the replication-deficient adenoviral vector. Peptides associated with packaging of the adenoviral genome into viral capsids include, but are not limited to, the adenoviral IVa2 peptide, the preterminal protein (pTP), and the adenoviral 52/55 kD peptide. The adenoviral IVa2 peptide and pTP directly interact with the viral genome to promote encapsidation into viral capsids. Thus, the peptide that interacts with the second packaging signal can be an adenoviral IVa2 peptide or an adenoviral pTP.

[0086] The adenoviral IVa2 peptide plays a role in both encapsidation of the adenoviral genome into viral particles and in activation of the MLP that drives expression of the late region genes encoding adenoviral structural proteins. The IVa2 peptide has been shown to associate with the adenoviral LI 52/55 kD peptide during viral infection (Gustin et al., J. Virol, 70(9), 6463-6467 (1996)). The 52/55 kD peptide is required for viral assembly and it is believed that the protein may aid in forming a stable interaction between viral DNA and the viral capsid (Gustin and Imperiale, J. Virol, 72(10), 7860-7870 (1998)). Sequence homology between the DE of the MLP and the packaging signal has led to the discovery that IVa2 is required for viral packaging, and it has been proposed that IVa2 aids in capsid assembly (Zhang and Imperiale, J. Virol, 74(6), 2687-2693 (2000)). The IVa2-52/55 kD complex is believed to be required for viral replication. The adenoviral nucleic acid sequence encoding the adenoviral IVa2 peptide is found at the left end of the adenoviral genome situated at nucleotides 4060-5838 of the human adenovirus type 5 genome (as set forth in GenBank Accession No. X02996.1) or nucleotides 4050-5695 of the human adenovirus type 2 genome (as set forth in GenBank Accession No. J01917.1). The adenoviral nucleic acid sequence encoding the 52/55 kD peptide is found in the LI region of the adenoviral genome, e.g., at nucleotides 6039-14113 of the human adenovirus serotype 2 genome (as set forth in GenBank Accession No. NC 001405.1) and nucleotides 11565- 12297 of the human adenovirus serotype 5 genome (as set forth by GenBank Accession No. NC 001406.1). The adenoviral nucleic acid sequence encoding the pTP is found in the E2 region of the adenoviral genome, as is known in the art. For example, the pTP coding sequence is found at nucleotides 8582-10544 of the adenovirus serotype 5 genome. [0087] The peptide that interacts with the second packaging signal can be produced by either the helper virus or the cell. The helper virus can comprise an endogenous or heterologous nucleic acid sequence (as defined previously herein) encoding a peptide required for the adenoviral packaging, e.g., the adenoviral IVa2 peptide and/or the adenoviral pTP. Alternatively, a heterologous nucleic acid sequence encoding a peptide required for adenoviral packaging that interacts with the second packaging signal can be introduced into the cell. One of ordinary skill in the art will appreciate that other nucleic acid sequences encoding gene products with similar biological activity as the heterologous nucleic acid sequence may also be employed, and can be identified using the myriad of available genetic sequence databases or via hybridization techniques, described previously with respect to the inventive cell.

[0088] Preferably, the heterologous nucleic acid sequence encoding a first peptide required for packaging an adenoviral vector is of adenoviral origin. Most preferably, the heterologous nucleic acid sequence is derived from the adenoviral IVa2 coding sequence. In instances where the second packaging signal is not a wild-type adenoviral packaging signal, a peptide required for viral packaging can be manipulated at the DNA level to generate a peptide that interacts with the second packaging signal, thereby promoting encapsidation of the replication-deficient adenoviral genome. For example, it is believed that the first 25%-30% of the adenoviral IVa2 coding sequence is responsible for encapsidation activity. One of ordinary skill in the art has the ability to mutate the region encoding encapsidation activity and screen candidate peptides for binding to the second packaging sequence using, for example, standard DNA-peptide binding assays as well as those assays described in Zhang and Imperiale, supra, Gustin et al., supra, and Gustin and Imperiale, supra. Alternatively, the DNA-binding region of a packaging peptide, such as a IVa2 peptide, can be replaced with a nonnative DNA binding motif (thereby forming a nonnative peptide), such that the packaging peptide is redirected to a previously unrecognized packaging signal.

[0089] Preferably, the peptide that interacts with the packaging signal is capable of interacting with additional peptides (adenoviral and cellular) associated with the adenoviral packaging machinery such that efficient packaging of the replication-deficient adenoviral genome or the helper virus genome occurs. For example, as described above, the IVa2 peptide interacts with the adenoviral 52/55 kD peptide in the packaging process. Preferably, the IVa2 peptide that binds to the first or second packaging sequence, if appropriate, retains the ability to bind to the necessary peptides for efficient viral packaging, e.g., the 52/55 kD peptide. In one embodiment, the binding sites for interaction of a DNA-binding peptide required for viral packaging (e.g., a IVa2 peptide) and an associating peptide, an additional peptide associated with viral packaging that does not bind adenoviral DNA but, instead, assembles to form a protein-based packaging complex required for incorporation of the genome into viral capsids (e.g., a 52/55 kD peptide), are mutated. By requiring a nonnative associating peptide (e.g., an associating peptide with a mutated binding site for the DNA- binding peptide) for encapsidation of the adenoviral genome, inadvertent encapsidation of helper virus due to "leaky" binding of the first packaging signal is abolished when the nonnative associating peptide is not present. Indeed, by manipulating the requirements for protein-protein interactions associated with adenoviral packaging in addition to manipulating the requirements for protein-DNA interactions, the risk of contamination of a stock of replication-deficient adenoviral vectors with helper virus is further minimized. [0090] Helper virus for use in the inventive method can be produced by (a) providing a cell that produces a peptide that interacts with the first packaging signal, (b) introducing the helper virus into the cell, and (c) maintaining the cell to propagate the helper virus. The helper virus can then be isolated for use in the inventive method. In a prefeπed embodiment, the first packaging signal is not a wild-type packaging signal. Accordingly, the peptide that interacts with the first packaging signal is desirably a mutated adenoviral IVa2 peptide or a mutated adenoviral pTP that does not bind a wild-type adenoviral packaging signal, but binds the first packaging signal such that packaging of the helper virus occurs.

[0091] Alternatively, the inventive method of propagating a replication-deficient adenoviral vector comprises providing a first cell with a cellular genome comprising a heterologous nucleic acid sequence encoding a first peptide required for packaging of an adenoviral vector, wherein the first peptide interacts with a first packaging signal. A helper virus is provided that comprises the first packaging signal. The helper virus encodes a second peptide required for packaging of an adenoviral vector, wherein the second peptide interacts with a second packaging signal, which is different from the first packaging signal. The helper virus does not encode the first peptide. A replication-deficient adenoviral vector also is provided. The replication-deficient adenoviral vector comprises a second packaging signal. The cell, helper virus, replication-deficient adenoviral vector, and components thereof are described above. The method further comprises introducing the helper virus and the replication-deficient adenoviral vector into the first cell, and maintaining the first cell to propagate both the helper virus and the replication deficient adenoviral vector. This first round of replication allows propagation of both the helper virus and the replication-deficient adenoviral vector, thereby creating a large pool of vectors. The inventive method further comprises providing a second cell not comprising the heterologous nucleic acid encoding the first peptide and which does not produce the first peptide. The second cell is infected with the helper virus and the replication-deficient adenoviral vector. The second cell is maintained to selectively propagate the replication-deficient adenoviral vector (i.e., as opposed to the helper virus), thereby effectively purifying the stock of replication-deficient adenoviral vectors from any helper virus contamination. In that the second cell and replication-deficient adenoviral vector do not provide the first peptide, the helper virus is not packaged in the second cell. Preferably, the first packaging signal is not a wild-type packaging signal, and the second packaging signal is a wild-type packaging signal. The first and or second peptide required for packaging of the adenoviral vector preferably is either the adenoviral IVa2 peptide or the adenoviral pTP.

[0092] In addition, the invention provides a cell for use in methods of propagating replication-deficient adenoviral vectors. In particular, the inventive cell comprises a heterologous nucleic acid sequence which, upon expression, produces a first adenoviral IVa2 peptide. The first adenoviral IVa2 peptide possesses either encapsidation activity or transcription-activation activity but not both encapsidation activity and transcription- activation activity. The production of the first adenoviral IVa2 peptide allows the cell to propagate a replication-deficient adenoviral vector comprising an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity possessed by the first adenoviral IVa2 peptide when the replication-deficient adenoviral vector is present in the cell. The replication-deficient adenoviral vector is propagated (i.e., replicated) when present in the cell.

[0093] As set forth herein, the adenoviral IVa2 peptide plays a role in both encapsidation of the adenoviral genome into viral particles and in activation of the MLP that drives expression of the late region genes encoding adenoviral structural proteins. With respect to the transcription-activation activity of the IVa2 peptide, IVa2 forms part of the DEF-A and DEF-B protein complexes, which bind to the downstream elements (DEs) of the MLP and, thus, activate transcription. DEF-B consists of a homodimer of two IVa2 peptides, while DEF-A consists of a heterodimer of a IVa2 peptide associated with another adenoviral peptide. [0094] Two distinct DNA binding motifs have been identified in the IVa2 peptide. The amino acid sequence of the DNA binding motifs is capable of amphipathic helix formation. In this regard, it appears that the amino acid residues spanning from about 50 to about 100 and about 357 to about 449 of the IVa2 amino acid sequence are required for DNA-binding activity (Lutz and Kedinger, J. Virol, 70(3), 1396-1405 (1996)). However, at least with respect to the MLP promoter, a single IVa2 peptide does not appear to have transcription- activation activity itself, but does activate transcription when dimerized or associated with another viral protein. Sequence homology between the DE of the MLP and the packaging signal has led to the discovery that IVa2 also is required for viral packaging, and it has been proposed that IVa2 aids in capsid assembly (Zhang and Imperiale, J. Virol. , 74(6), 2687- 2693 (2000)). The encapsidation activity of the adenoviral IVa2 peptide is described above. [0095] The inventive cell comprises a heterologous nucleic acid sequence encoding a first adenoviral IVa2 peptide. "Heterologous nucleic acid sequence" is defined above and indicates that that the nucleic acid sequence is not obtained from, derived from, or based upon a naturally occurring nucleic acid sequence of the inventive cell. When the heterologous nucleic acid sequence encodes a peptide required for packaging of an adenoviral vector, the heterologous nucleic acid sequence preferably is of adenoviral origin. Most preferably, the heterologous nucleic acid sequence is derived from the adenoviral IVa2 coding sequence.

[0096] The heterologous nucleic acid sequence encodes a first adenoviral IVa2 peptide that has either encapsidation activity or transcription-activation activity, but not both encapsidation activity and transcription-activation activity. Complete elimination of a particular activity is not required so long as an adenoviral vector comprising the IVa2 peptide cannot be efficiently propagated without complementation of the missing IVa2 peptide activity. The native adenoviral IVa2 coding sequence can be altered in any fashion to separate the encapsidation and transcription-activation functions of the resulting IVa2 peptide. For example, the nucleic acid sequence can be altered outside the coding regions for the DNA binding motifs described above to retain transcription-activation functions. It appears that the amino terminus of the IVa2 protein is dispensable for MLP function, but is required for packaging.

[0097] Separating the function of the IVa2 peptide in the method described herein lessens the risk of obtaining helper virus contamination in the stock of replication-deficient adenoviral vectors. For instance, the cell of the inventive method preferably comprises a cellular genome comprising a heterologous nucleic acid encoding a first adenoviral IVa2 peptide, wherein the first adenoviral IVa2 peptide comprises encapsidation activity but not transcription-activation activity, and binds the second packaging signal. Also preferably, the helper virus encodes a second adenoviral IVa2 peptide comprising transcription- activation activity, but not encapsidation activity. Alternatively, the cellular genome of the cell of the inventive method comprises a heterologous nucleic acid sequence encoding a first adenoviral IVa2 peptide comprising transcription-activation activity, but not encapsidation activity. In this instance, the helper virus encodes a second adenoviral IVa2 peptide comprising encapsidation activity, but not transcription-activation activity, and binds the second packaging signal. By allowing the helper virus to encode only one IVa2 function, it is less likely that replication-competent helper virus will contaminate the final stock of replication-deficient adenoviral vectors.

[0098] Appropriate heterologous nucleic acid sequences for insertion into the inventive cell can be identified and obtained using routine laboratory techniques, such as those known in the art and described herein. For example, a native adenoviral IVa2 coding sequence can be mutated at the level of individual base pairs, truncated at the N- or C-terminus, deleted of various regions of open reading frames, manipulated as regards splicing, inserted with stop codons, etc., to create a series of mutants that can subsequently be screened for the desired activity. Alternatively, a nucleic acid sequence can be generated synthetically and expressed, with the resulting products screened for desired activity. [0099] Moreover, the heterologous nucleic acid sequence can include one or more mutations (e.g., point mutations, deletions, insertions, etc.) from a conesponding naturally occurring heterologous nucleic acid sequence as set forth above. The heterologous nucleic acid sequence also can encode fragments of an adenoviral IVa2 peptide with encapsidation activity or transcription-activation activity, determined using the methods described herein. It will be appreciated that any of the methods described herein pertaining to identification of heterologous nucleic acid sequences can be adapted to identify and construct suitable packaging signals.

[0100] Heterologous nucleic acid sequences can be screened for use in the inventive cell using routine laboratory methods such as those described in Zhang and Imperiale, supra; Gustin et al, supra; and Lutz and Kedinger, supra. For example, the ability of an encoded adenoviral IVa2 peptide to form DEF-A and DEF-B complexes can be determined. Transcription-activation activity can be characterized by first producing the adenoviral IVa2 peptide and monitoring MLP activation in response to the adenoviral IVa2 peptide by (a) operably linking a reporter gene to the MLP and assaying for expression, or (b) detecting late protein (L1-L5) production using standard protein detection techniques, such as SDS- PAGE and Western blots. With respect to identifying encapsidation activity, the candidate heterologous nucleic acid sequence is expressed, and, for instance, the ability of the produced adenoviral IVa2 peptide to associate with the 52/55 kD protein can be assessed. The ability of the encoded adenoviral IVa2 peptide to promote assembly of adenoviral capsid proteins can be determined as set forth in Gustin and Imperiale, supra. In addition, binding of the mutated IVa2 peptide to either the adenoviral MLP nucleotide sequence or to the adenoviral packing signal can be determined using standard laboratory techniques. [0101] The description of the introduction and location of (e.g., integrated or episomal) the heterologous nucleic acid sequence in a host cell and components thereof set forth above in connection with the inventive cell also are applicable to the same aspects of the inventive method. Moreover, the heterologous nucleic acid sequence typically is operably linked to expression control sequences that are necessary for efficient transcription of the heterologous nucleic acid sequence as described herein.

[0102] The adenoviral vector is deficient in one IVa2 gene function, i.e., either encapsidation activity or transcription-activation activity, and is thereby unable to replicate in cells which do not complement in trans for the deficient gene function. Adenoviral vectors containing a deficiency in the encapsidation activity or transcription-activation activity of the adenoviral IVa2 peptide can be generated using any suitable method, such as those genetic manipulation techniques known in the art. For example, adenoviral vectors containing a series of mutations can be generated. The mutant viruses are screened initially using growth as a means of selection. The desired adenoviral vector is unable to replicate in the absence of complementation. Transcription-activation activity can be determined by monitoring late protein expression using standard protein detection techniques, such as SDS-PAGE and Western blots. A deficiency in the encapsidation function of the IVa2 peptide can be detected using marker rescue assays, as is commonly used in the art. [0103] The inventive method of propagating a replication-deficient adenoviral vector comprises providing the aforesaid inventive cell, introducing a replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide, and maintaining the cell to propagate the replication-deficient adenoviral vector. Descriptions of the cell, the heterologous nucleic acid sequence, the adenoviral vector, and components thereof set forth above in connection with the other embodiments of the invention also are applicable to those same aspects of the aforesaid inventive method to the extent not inconsistent therewith. [0104] The invention also provides a method of propagating a replication-deficient adenoviral vector, wherein the method comprises (a) providing a cell with a cellular genome, (b) providing a helper virus comprising an adenoviral genome deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome, (c) providing a replication-deficient adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome, (d) introducing the helper virus and the replication-deficient adenoviral vector into the cell, and (e) maintaining the cell to selectively propagate the replication-deficient adenoviral vector (i.e., as opposed to the helper virus). The adenoviral genome of the helper virus desirably is deficient in one or more essential gene functions of the early regions. The adenoviral genome of the helper virus preferably is deficient in one or more essential gene functions of two or more early regions selected from the group consisting of the El region, the E2 region, and the E4 region. The adenoviral genome of the helper virus, alternatively or in addition, can be deficient in one or more essential gene functions of the late regions. [0105] The invention further provides a cell having a cellular genome comprising a nucleic acid sequence which upon expression produces a mutant DAI kinase that complements in trans for a deficiency in at least one essential gene function of at least one virus-associated RNA (VA RNA) of an adenoviral genome so as to propagate an adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the at least one virus-associated RNA when present in the cell. [0106] Descriptions of the cell, the nucleic acid sequence, the adenoviral vector, and components thereof set forth above in connection with the other embodiments of the invention also are applicable to those same aspects of the aforesaid inventive cell to the extent not inconsistent therewith.

[0107] The inventive cell has a cellular genome comprising a nucleic acid sequence which upon expression produces a mutant DAI kinase. The cellular response to viral infection typically involves repression of translation initiation by the inhibitory activity of double-stranded RNA-activated kinase (DAI kinase), which, as its name implies, is activated by virus-encoded double-stranded RNA (see, e.g., Lloyd et al., J. Virol., 66, 6878- 94 (1992)). To evade the cellular response to viral infection, adenoviruses encode two double-stranded virus-associated RNAs, VA-RNA I and VA-RNA II. Upon adenoviral infection, VA-RNA I interacts with and inhibits DAI kinase, thereby allowing viral translation to proceed.

[0108] Preferably, the nucleic acid sequence encodes a mutant DAI kinase. A DAI kinase is mutant when it exhibits a function that is distinct from a naturally occurring DAI kinase. A mutant DAI kinase is typically and preferably produced by introducing one or more mutations (e.g., point mutations, deletions, insertions, etc.) into the nucleic acid sequence encoding a naturally occurring DAI kinase. Such mutations are introduced in the nucleic acid sequence to effect one or more amino acid substitutions in an encoded DAI kinase. Methods for introducing such mutations into a nucleic acid sequence are described above. [0109] Preferably, the DAI kinase mutant is generated by introducing one or more mutations into the nucleic acid sequence encoding a naturally occurring DAI kinase that confer a dominant negative phenotype. A mutation produces a dominant negative phenotype when it results in a gene product (i.e., a polypeptide or protein) that, when overexpressed, disrupts the activity of a simultaneously expressed wild-type gene product in a cell, thereby rendering the cell deficient in the function of the wild type gene product (see, e.g., Herskowitz, I., Nature, 329, 219-222 (1987)). Such dominant negative mutations are desirably produced by introducing mutations which disrupt the one or more functional domains of a monomeric or multimeric (e.g., dimeric) protein. For example, with respect to a monomeric protein such as an enzyme, a dominant negative mutation may introduce an amino acid substitution which inactivates the catalytic site of the enzyme, but does not affect native substrate binding. As such, the mutant enzyme acts as a competitive inhibitor of the wild-type enzyme. With respect to a multimeric protein, a dominant negative mutation can introduce an amino acid alteration that does not affect multimerization with the wild-type protein, but results in inactive multimers when formed. In the context of the invention, a dominant negative mutant DAI kinase can function to prevent activation of wild-type DAI kinase upon adenoviral infection of a host cell. Alternatively, the dominant negative DAI kinase mutant can function to prevent phosphorylation of the DAI target, elF- 2α. Any dominant negative DAI kinase mutation that disrupts cellular translation inhibition is within the scope of the invention.

[0110] Dominant negative mutations can be generated in accordance with any of the techniques described herein for inducing mutations in nucleic acid sequences. Once a candidate dominant negative DAI kinase mutant has been generated, the phenotype of that particular candidate can be assessed by, for example, introducing the DAI kinase mutant into a cell. The cell is then infected with a wild-type adenovirus, and translation of adenoviral mRNAs can be detected using routine assays that are within the skill of the art. The absence of adenoviral translation products (i.e., adenoviral proteins) indicates that the cellular translational machinery has been inhibited, and thus, the candidate DAI kinase mutant is not a dominant negative. Alternatively, detection of adenoviral translation products indicates that the cellular translational machinery was not inhibited upon viral infection, which strongly suggests that the DAI kinase mutant is a dominant negative. [0111] The expression of the nucleic acid sequence encoding the mutant DAI kinase in the inventive cell is controlled by a suitable expression confrol sequence operably linked to the nucleic acid sequence. An "expression control sequence" has been defined previously herein. Suitable expression control sequences include constitutive promoters, inducible promoters, repressible promoters, chimeric promoters, and enhancers, as described above. The nucleic acid sequence encoding the mutant DAI kinase can be regulated by its endogenous promoter or by a nonnative promoter sequence.

[0112] As described above, the inventive cell comprising a nucleic acid sequence which upon expression produces a mutant DAI kinase can be generated in accordance with standard molecular biological techniques as described in, for example, International Patent Application WO 95/34671 and U.S. Patent 5,994,106. The nucleic acid sequence can be stably integrated into the nuclear genome of the cell, and is preferably retained in the cellular genome for multiple passages (e.g., at least about 30, 40, 100, or more passages). The introduction and stable integration of the nucleic acid into the genome of the cell requires standard molecular biology techniques that are well within the skill of the art, such as those described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific American Books (1992), and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995). When the nucleic acid sequence is not integrated into the cellular genome, the nucleic acid sequence can reside, for example, on a plasmid, liposome, or any other type of molecule that can harbor a nucleic acid sequence extrachromosomally.

[0113] The mutant DAI kinase, when expressed in the inventive cell, complements in trans for a deficiency in at least one essential gene function of at least one virus-associated RNA (VA-RNA) of an adenoviral genome. The nucleic acid sequence, upon expression, produces at least one gene product that provides an adenoviral essential gene function, i.e., that complements in trans for a deficiency in at least one essential gene function (i.e., a function that is necessary for adenovirus propagation) of at least one virus-associated RNA (VA RNA) of an adenoviral genome. The nucleic acid sequence, upon expression, can produce a gene product that complements for two or more deficiencies in at least one adenoviral VA-RNA (e.g., VA-RNA I and/or VA-RNA II). Alternatively, the nucleic acid sequence, upon expression, can produce two or more gene products, each of which complements for a deficiency (i.e., at least one deficiency, including but not limited to, two or more deficiencies) in at least one adenoviral VA-RNA.

[0114] In addition to complementing in trans for a deficiency in at least one essential gene function of at least one VA-RNA of an adenoviral genome, the cellular genome of the inventive cell can further comprise a nucleic acid sequence which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more other regions of the adenoviral genome. [0115] Essential adenoviral gene functions are described above with respect to other embodiments of the invention. The gene functions of VA-RNA are essential in that deletion of VA-RNA I results in a two-log reduction in adenoviral propagation as compared to wild- type adenovirus. Moreover, an adenovirus containing a double deletion of VA-RNA I and VA-RNA II has yet to be isolated. The nucleic acid sequence that produces at least one gene product that complements in trans for a deficiency in at least one essential gene function of one or more other regions (i.e., other than VA-RNA) of the adenoviral genome desirably complements for a deficiency in at least one essential gene function of one or more regions of the adenoviral genome selected from the early regions, e.g., the El, E2, and E4 regions. Preferably, the gene product complements in trans for a deficiency in at least one essential gene function of the El region of the adenoviral genome. More preferably, the gene product complements in trans for a deficiency in at least one essential gene function of an adenoviral El A coding sequence and/or an adenoviral EIB coding sequence (which together comprise the El region), as described elsewhere herein in connection with other embodiments of the inventive cell. In addition or alternatively to the gene product(s) complementing in trans for the aforementioned deficiencies in adenoviral essential gene functions, the same or different gene product(s) can complement for a deficiency in at least one essential gene function of the E2 and/or E4 regions of the adenoviral genome. Desirably, a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein. Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome. [0116] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1 [0117] This example describes the construction of a primary cell having a cellular genome comprising the CMV immediate early gene regions 1 and 2 heterologous nucleic acid sequences, which, upon expression, produce at least one non-adenoviral protein (i.e., the IE1 and IE2 proteins).

[0118] Plasmid pCMVXbaEpuro contains the Xbal-E fragment of HCMV DNA (0.68 - 0.77 map units), which includes the CMV immediate early (IE) regions 1 and 2 inserted into the Nrul - Apal digested pSMTpuro-ORF6 plasmid. The pSMTpuro-ORF6 plasmid contains the adenovirus 5 E4-ORF6 gene under the control of the sheep metallothionein promoter (see, e.g., International Patent Application WO 95/34671). [0119] Primary human embryonic lung (HEL) cells are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, 100 μg of penicillin per milliliter (all components from Life Technologies, Gaithersburg, MD). The HEL cells are then transfected with pCMVXbaEpuro by the calcium phosphate method (Sambrook et al., supra). Following transfection, 2.5 μg/ml of puromycin is added to the culture medium for selection of the CMV IE1 and IE2 expressing cells, which are clonally isolated and propagated (see Sambrook et al., supra). Approximately 24 hours post-transfection, expression of CMV IE1 and IE2 genes is assayed via Northern and Western blotting. Integration of the CMV sequences is confirmed by Southern blotting.

EXAMPLE 2 [0120] This example describes a method for demonstrating the ability of an inventive cell having a cellular genome comprising a heterologous nucleic acid sequence encoding the CMV IE1 and IE2 proteins to support propagation and production of a replication-deficient adenoviral vector.

[0121] The HEL cells of Example 1 are cultured using routine tissue culture techniques. Monolayers at passages 5 and 10 are screened for El A complementation by a virus production assay (see, e.g., Burlseson et al., Virology: A Laboratory Manual, Academic Press Inc. (1992)). In that respect, the cells are infected with a replication-deficient adenoviral vector wherein the El A region has been deleted from the adenoviral genome thereof. Specifically, the cells are infected with an El A-deficient adenoviral vector, which contains the EIB region encoding the protein AdElB, at a multiplicity of infection (MOI) of 10. Cell lysates are prepared at 3 days post-infection (d.p.i.), and the amount of active virus in the lysates is determined by a focal forming unit (FFU) assay (Cleghon et al., Virology, 197, 564-575 (1993)). The detection of significant yields of AdElB for the cells at passages 5 and 10 evidences the ability of the cells to complement in trans for deficiencies in adenoviral essential gene functions of the El A region of the adenoviral genome.

EXAMPLE 3 [0122] This example describes the construction of a transformed human cell having a cellular genome comprising the CMV immediate early gene regions 1 and 2 heterologous nucleic acid sequences, which, upon expression, produce at least one non-adenoviral protein (i.e., the IE1 and IE2 proteins).

[0123] HeLa cells (ATCC CCL-2) are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, 100 μg of penicillin per milliliter (all components from Life Technologies, Gaithersburg, MD). The HeLa cells are then transfected with the pCMVXbaEpuro plasmid of Example 1 by the calcium phosphate method (Sambrook et al., supra). Following transfection, 2.5 μg/ml of puromycin is added to the culture medium for selection of the CMV IE1 and IE2 expressing cells, which are clonally isolated and propagated (see Sambrook et al., supra). Approximately 24 hours post-transfection, expression of CMV IE1 and IE2 genes is assayed via Northern and Western blotting. Integration of the CMV sequences is confirmed by Southern blotting. EXAMPLE 4 [0124] This example describes a method for demonstrating the ability of an inventive cell having a cellular genome comprising a heterologous nucleic acid sequence encoding the CMV IE1 and IE2 proteins to support propagation and production of a replication-deficient adenoviral vector.

[0125] The HeLa cells of Example 3 are cultured using routine tissue culture techniques. Monolayers at passages 5 and 10 are screened for El A complementation by a virus production assay (see, e.g., Burlseson et al., supra). In that respect, the cells are infected with a replication-deficient adenoviral vector wherein the El A region has been deleted from the adenoviral genome thereof. Specifically, the cells are infected with an El A-deficient adenoviral vector, which contains the EIB region encoding the protein AdElB, at a multiplicity of infection (MOI) of 10. Cell lysates are prepared at 3 days post- infection (d.p.i.), and the amount of active virus in the lysates is determined by a focal forming unit (FFU) assay (Cleghon et al., supra). The detection of significant yields of AdElB for the cells at passages 5 and 10 evidences the ability of the cells to complement in trans for deficiencies in adenoviral essential gene functions of the El A region of the adenoviral genome.

EXAMPLE 5 [0126] This example demonstrates the ability of an NCI-H460 cell and a Calu-1 cell to support high levels of wild-type adenovirus growth.

[0127] Calu-1 cells, NCI-H460 (H460) cells, A549 cells (ATCC CCL-185, Manassas, VA), and 293 cells (Graham et al., supra) were separately cultured using routine tissue culture techniques. Sub-confluent monolayers of each cell culture were infected with wild- type adenovirus 5 (Ad5) at a multiplicity of infection (MOI) of 5. Cells were harvested 48 hours post infection (h.p.i.), and the infected cell lysates were titered for infectious adenovirus by a focus forming unit (FFU) assay (Cleghon et al., supra). The viral yields of each cell type are set forth in Table 1.

Table 1 : Wild-type Adenovirus Yield in Cells

Cell Line Wild-type Adenovirus Yield (FFU/cell)

293 8,511

A549 14,638

NCI-H460 9,173

Calu-1 > 35,000 [0128] This example demonstrates the ability of an NCI-H460 cell and a Calu-1 cell to support high levels, e.g., higher than 293 cells or A549 cells, respectively, of wild-type adenovirus production.

EXAMPLE 6 [0129] This example describes the construction of an H460 cell and a Calu-1 cell having a cellular genome that comprises an adenoviral El A coding sequence, an adenoviral EIB coding sequence, and ORF6 (and no other ORF) of the E4 region of the adenoviral genome. [0130] The adenoviral El region, conesponding to Ad2 nucleotides 490-3505, is amplified via polymerase chain reaction (PCR) (Innis et al., eds., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. (1990)) using primers JG104 (5'- GGACTAGTAAGAGGCCACTCTTGAGTG-3' (SEQ ID NO: 4)) and JG105 (5'- AAAAGTACTGGCCGGCCTCAATCTGTATC-3' (SEQ ID NO: 5)). After confirming that the amplified sequences are the expected Ad2 El sequence by DNA sequencing, the El sequence is cloned between the HIV-1 5' long terminal repeat (LTR) and the murine μM polyadenylation sequence at the Hpal/Spel cloning sites in the plasmid pHIV.13s.μM.zeo to generate the plasmid pHIV.2El.μM.zeo. H460 cells and Calu-1 cells are transfected with pHIV.2El.μM.zeo via the calcium phosphate method and grown under zeocin selection. Zeocin resistant colonies are isolated and propagated under zeocin selection. Northern and Western blotting and mRNA detection assays are performed to detect El A and EIB expression. Genomic integration of the El coding sequences is confirmed via Southern blotting.

[0131] The primers A5s(33190)P and A5a(34084)P are used to amplify the ORF-6 region of the Ad5 E4 region by PCR and generate Pad sites at the ends for cloning. The amplified fragment is blunt-ended with Klenow large fragment of DNA polymerase I and cloned into pCR-Script SK(+) (Sfratagene, La Jolla, CA). The resulting plasmid, pCR/ORF-6, is sequenced. The ORF-6 insert is transfened into the pSMT/puro expression vector, which is generated by ligation of a blunt-ended EcoRI - Hindlll fragment containing the SMT promoter into the blunt-ended Mlul-Hindlll site in pRCpuro, to generate pSMT/ORF-6, which also contains the puromycin resistance gene as a selectable marker. [0132] Calu-1 cells and H460 cells containing the pHIV.2El .μM.zeo plasmid are cultured using standard techniques and transfected with pSMT/ORF-6 via the calcium phosphate method (see, e.g., Sambrook et al., supra). Colonies of transformed cells are subcloned and propagated under puromycin selection for at least 20 passages in culture, to ensure stable retention of the pSMT/ORF-6 construct. Expression of El A, EIB, and E4- ORF6 gene products is assayed via Northern and Western blotting. Genomic integration of the El genes and E4-ORF6 is confirmed via Southern blotting. EXAMPLE 7 [0133] This example describes a method for demonstrating the ability of an NCI-H460 cell and a Calu-1 cell, each comprising the El region and ORF-6 of the E4 region of an adenoviral genome, to complement in trans for a deficiency in at least one essential gene function of one or more regions (e.g., El and E4) of an adenoviral genome of a replication- deficient adenoviral vector.

[0134] The Calu-1 cells and H460 cells of Example 6, which comprise the El region and ORF6 of the E4 region of an adenoviral genome, are separately cultured using routine tissue culture techniques. Monolayers at passages 5 and 10 are screened for adenoviral El and E4 region gene function complementation by a virus production assay (see, e.g., Burlseson et al., supra). In that respect, cells are separately infected with wild-type adenovirus 5 and a replication-deficient adenoviral vector (AdRSVβ-gal.11) wherein the El and E4 regions have been deleted from the adenoviral genome thereof (Brough et al., J. Virol, 70, 6497-6501 (1996)). Specifically, the cells are infected with AdRSVβ-gal.l 1 at a multiplicity of infection (MOI) of 10. Cell lysates are prepared at 3 days post-infection (d.p.i.), and the amount of active virus in the lysates is determined by a focal forming unit (FFU) assay (Cleghon et al., supra). The detection of significant yields of AdRSVβ-gal.l 1 for each cell line at passages 5 and 10 evidences the ability of the cell line to complement in trans for deficiencies in adenoviral essential gene functions of the El and E4 regions of the adenoviral genome.

EXAMPLE 8 [0135] This example demonstrates the construction of a nucleic acid comprising an adenoviral El coding sequence.

[0136] An Ad2 El expression cassette was assembled in pKS (Sfratagene) to generate pKSCMVEl . initially, Ad2 nucleotides 362-917, including the El A TATA box-associated sequence comprising nucleotides 362-497, was amplified by PCR, and the resulting Eco RI to Cla I fragment was cloned into pKS. Subsequently, the Ad2 sequences from 918-5708 were incorporated into pKS as a PCR-generated Cla I fragment. Finally, the CMV IE enhancer comprising nucleotides 174254-173843 of the human CMV genome was incorporated into pKS as a PCR-generated EcoR I fragment to generate pKSCMVEl. pZeoEl (Invifrogen) was constructed by replacing the SV40 driven expression cassette with the El expression cassette from pKSCMVEl. Thus, pZeoEl comprises the human CMV IE enhancer sequence, the Ad2 El A TATA box-associated sequence comprising nucleotides 362-497, and the Ad2 El A and EIB coding sequences. [0137] The adenoviral vectors comprising coding sequences for β-glucoronidase, no transgene, β-galactosidase, TNF-α, secretory alkaline phosphatase, or vascular endothelial growth factor (VEGF) 121 (AdG, Adnull, AdZ, AdTNF, AdS, and AdVEGF121, respectively) under the control of a CMV IE promoter are known in the art. Each adenoviral vector comprises a deletion of nucleotides 356-3328 of the El region of the adenoviral genome.

[0138] The El expression cassette plasmid was tested for functionality by infection- transfection experiments in A549 cells. The cells were infected with AdG and thereafter transfected with pZeoEl . At 20 hours post-infection (h.p.i.) the amount of transgene expression (glucoronidase (gus) activity) was quantified, and vector DNA replication was detected by Southern blot analysis. The presence of El gene products expressed in trans from pZeoEl increased transgene expression approximately 100-fold. The increase in gus activity was accompanied with adenovirus vector DNA replication. Thus, the expression cassette El gene products provided functional complementation.

[0139] The results of this example confirm the construction and proper functioning of a nucleic acid comprising a chimeric expression control sequence, (illustrated by a CMV IE enhancer, an El TATA-box associated sequence (Ad2 nucleotides 362-497), and the Ad2 El A and EIB coding regions (nucleotides 498-5708)), which upon expression produces a gene product that complements in trans an adenoviral vector comprising a deficiency in at least one essential gene function (illustrated by a deficiency in the El region) of the adenoviral genome.

EXAMPLE 9 [0140] This example demonstrates the construction of a complementing cell using the A549 cell line as the parent cell line.

[0141] A549 cells are continuous tumor cells derived from a human lung carcinoma with properties of type II alveolar epithelial cells (Lieber et al., Int. J. Cancer, 17, 62-70, (1976)). A549 cells support productive wild-type adenovirus replication, and are adaptable to growth in serum free suspension culture (ATCC, CCL-185.1). A549 cells were transfected with linearized pZeoEl, placed under Zeocin selection, and resistant colonies were isolated. The cells were subsequently cultured using routine tissue culture techniques. Monolayers at passages 5 and 10 were screened for El complementation by a virus production assay (see, e.g., Burlseson et al., supra). In that respect, cells were infected with Adnull at a multiplicity of infection (MOI) of 10, cell lysates were prepared at 3 days post- infection (d.p.i.), and the amount of active virus in the lysates was determined by a focal forming unit (FFU) assay (Cleghon et al., supra). The detected yields of Adnull for each cell line at passages 5 and 10, which evidence the ability of the cell line to complement for an El -deficiency in an adenoviral genome, are set forth in Table 2.

Table 2: Yield of Adnull from El cell lines at 3 d.p.i. (FFU/cell)*

Passage Passage Passage

Cell line 5 10 Cell line 5 10 Cell line 5 10

P 118 T 52 69 T R10 1681 65

1 136 53 53 454 250 Rll 111 12

2 256 45 55 36 T R13 134 0

3 153 0 57 39 T R14 133 26

5 42 T 61 108 25 R15 63 0

9 179 21 63 141 25 R24 125 125

11 20 T 70 133 0 R26 175 150

16 25 T 71 75 T R29 650 900

18 80 T 75 46 T R36 212 120

21 218 57 76 8 T R38 25 12

24 42 T 77 166 42 R50 62 26

25 830 700 80 311 0 R52 83 12

28 280 46 81 79 T R57 37 35

31 218 10 85 358 12 R59 40 23

32 410 360 88 51 0 R60 55 28

33 275 54 Rl 47 21 R65 37 25

46 708 550 R9 55 45 R66 125 142

*P=Zeocin-resistant cell line; the prefix AE' was added to the isolate number for the final name of each cell line; T=termination of the cell line due to problems with maintenance of the monolayer

[0142] As is apparent from the results set forth in Table 2, complementing cells of the invention were produced and confirmed to complement in trans an adenoviral genome comprising a deficiency in at least one essential gene function. In particular, AE25 and AE29 (lines 25 and R29 in Table 2, respectively) were the highest producing cell lines at passages 5 and 10 and formed high quality monolayers. EXAMPLE 10 [0143] This example demonstrates the ability of complementing cells of the invention to support viral replication and viral production.

[0144] AE25 and AE29 cells of Example 9 (lines 25 and R29 in Table 2, respectively) were infected with Adnull. The growth kinetics of Adnull in AE25 and AE29 cells were compared to the growth of the virus in 293 cells and A549 cells over a five-day time course. Virus growth yield, virus plaque formation, Southern blot, Northern blot, and PCR analyses have been previously described (see, e.g., Burlseson et al., supra, Sambrook et al., supra, and Innis et al., supra).

[0145] Adnull replication in AE25 cells had a one-day lag period compared to replication in 293 or AE29 cells. The overall yield of active particles in AE25 cells ranged from 25-50% of the yield from 293 cells. Adnull growth in AE29 cells did not show a lag period, and the number of active particles produced per cell was within 2-fold of 293 cells at 2 and 3 days post infection. Therefore, both AE25 and AE29 functionally complement for growth of El deleted virus to within at least about 50% of 293 cells. The results ofthese experiments are set forth in Table 3.

Table 3: Adnull growth kinetics in different host cells (number of active particles produced per cell (pfu/cell))

Days post-infection cell line 1 2 3 5

293 1000 4000 3000 2000

AE25 0 550 700 1000

AE29 160 2500 1500 850

A549 0 0 0 0

[0146] The ability of AE25 and AE29 cells to support productive infection was measured by plaque formation assays. The plaque forming unit (pfu) titers of AdZ, AdTNF, and AdS were determined. The efficiency of plaque formation on AE25 and AE29 cells was about 5-60% of the best available plaque-forming 293 monolayer cell line. The results ofthese experiments are set forth in Table 4. Table 4: Plaque formation efficiency of El -complementing cell lines

Vector titer (pfu/ml)

Cell line AdZ titer AdTNF titer AdS titer

293 1.0 x lO¹⁰ 2.0 x lO¹⁰ 2.6 x 10⁹

AE10 1.5 x lO⁸ 1.0 x lO⁸ 1.0 x lO⁸

AE25 2.0 x lO⁹ 2.5 x 10⁹ 1.0 x lO⁹

AE29 2.5 x 10⁹ 2.0 x lO⁹ 3.0 x lO⁹

A549 0 0 0

[0147] The results of this example demonstrate the ability of A549-derived complementing cells (specifically, AE25 and AE29) to support the production of an El - deleted Ad vector to within at least about 50% of the efficiency of 293 cells.

EXAMPLE 11 [0148] This example demonstrates upregulation of the chimeric expression control sequence by adenoviral infection.

[0149] The number of copies of the El expression cassette integrated into the cellular genome of the AE25 and AE29 cells of Examples 9 and 10 was determined by Southern blot analysis. There was approximately one copy of the El expression cassette per cell integrated in both AE25 and AE29 cell lines. AE25 and AE29 cells were infected with an El- and protein IX-deleted mutant adenoviral vector, H5dl313 (Jones and Shenk, Cell, 17, 683-89 (1979)). The expression of E1A and EIB adenoviral proteins in the AE25 and AE29 cells was induced 2-fold to 5-fold by infection with H5dl313 as compared to uninfected cells. Presumably, the upregulation of the El A and EIB adenoviral proteins by infection occuπed via activation of the chimeric CMV/adeno virus expression control sequence in the integrated El cassette.

[0150] This example confirms the upregulation of the chimeric expression control sequence by one or more adenoviral proteins not produced by the nucleic acid sequence that is incorporated into the cellular genome and provides, upon expression, the complementing function. Moreover, this example confirms that the adenoviral protein upregulating the chimeric control expression sequence can be provided by the replication-deficient adenoviral vector to be propagated in the cell line. [0151] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0152] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0153] Prefened embodiments of this invention are described herein, including the best mode known to the inventors for caπying out the invention. Variations of those prefened embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A cell having a cellular genome comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when the replication-deficient adenoviral vector is present in the cell.

2. The cell of claim 1 , wherein the cell is a primary cell.

3. The cell of claim 1, wherein the cell is a human embryonic lung (HEL) cell or an ARPE-19 cell.

4. The cell of claim 1 , wherein the cell is a transformed cell.

5. A transformed human cell comprising at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when the replication-deficient adenoviral vector is present in the cell.

6. The cell of claim 4 or claim 5, wherein the cell is a lung carcinoma cell.

7. The cell of claim 6, wherein the cell is selected from the group consisting of an A549 cell, an NCI-H1299 cell, a Calu-1 cell, and an NCI-H460 cell.

8. The cell of claim 4 or claim 5, wherein the cell is a HeLa cell or an ARPE- 19/HPV-16 cell.

9. The cell of any of claims 1-8, wherein the one or more regions of the adenoviral genome are selected from the group consisting of the El, E2, and E4 regions.

10. The cell of claim 9, wherein at least one non-adenoviral gene product complements in trans for a deficiency in at least one essential gene function of the El A region of an adenoviral genome.

11. The cell of claim 9 or claim 10, wherein at least one non-adenoviral gene product complements in trans for a deficiency in the essential gene function of the E4- ORF6 of an adenoviral genome.

12. The cell of any of claims 1-11, wherein at least one non-adenoviral gene product is a viral protein or a cellular protein.

13. The cell of any of claims 1-12, wherein the cell further comprises a heterologous nucleic acid sequence which upon expression produces a non-adenoviral gene product that enhances propagation of the replication-deficient adenoviral vector, so as to produce more replication-deficient adenoviral vectors when the heterologous nucleic acid sequence is present in the cell than when it is absent from the cell.

14. A cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when the replication-deficient adenoviral vector is present in the cell, wherein either (a) the cell (i) is a pleural effusion, large cell lung carcinoma, (ii) is epithelial, and (iii) comprises a wild-type p53 gene, (b) the cell (i) is a lung carcinoma, (ii) comprises a homozygous deletion of the p53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation, (c) the cell is an NCI-H460 cell, or (d) the cell is a Calu-1 cell.

15. The cell of claim 14, wherein the cell (i) is a pleural effusion, large cell lung carcinoma, (ii) is epithelial, and (iii) comprises a wild-type p53 gene.

16. The cell of claim 15, wherein the cell comprises a homozygous K-ras codon 12 mutation.

17. The cell of claim 15 or claim 16, wherein the cell does not express the pl6INK4a protein.

18. The cell of any of claims 15-17, wherein the cell exhibits adherent growth in culture.

19. The cell of any of claims 15-18, wherein the cell comprises two X chromosomes and two Y chromosomes.

20. The cell of claim 14, wherein the cell (i) is a lung carcinoma, (ii) comprises a homozygous deletion of i ep53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation.

21. The cell of claim 20, wherein the cell does not express the pl6INK4a protein.

22. The cell of claim 20 or claim 21, wherein the cell exhibits adherent growth in culture.

23. The cell of any of claims 20-22, wherein the cell has an antigen expression profile comprising (i) blood type A, (ii) Rh positive, and (iii) HLA antigens AlO, A11, B15, and Bw35.

24. The cell of any of claims 20-23, wherein the cell produces at least about 300% more wild-type adenovirus than a 293 cell, and at least about 130% more wild-type adenovirus than an A549 cell.

25. The cell of claim 14, wherein the cell is an NCI-H460 cell.

26. The cell of claim 14, wherein the cell is a Calu-1 cell.

27. The cell of any of claims 14-26, wherein the one or more regions of the adenoviral genome are selected from the group consisting of the El, E2, and E4 regions.

28. The cell of any of claims 14-27, wherein the adenoviral nucleic acid sequence comprises an adenoviral El A coding sequence and an adenoviral EIB coding sequence.

29. The cell any of claims 14-28, wherein the one or more regions of the adenoviral genome are the El region and at least one additional region.

30. The cell of claim 29, wherein the at least one additional region comprises the E4 region.

31. The cell of claim 30, wherein the cellular genome comprises at least ORF6 of the E4 region of the adenoviral genome.

32. The cell of claim 31 , wherein the cellular genome comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome.

33. A cell for the propagation of a replication-deficient adenoviral vector having a cellular genome comprising a nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions an adenoviral genome, and which is operatively linked to a chimeric expression control sequence comprising at least a functional portion of a CMV immediate early promoter/enhancer region, at least a functional portion of an adenoviral promoter, or both, wherein the chimeric expression control sequence is upregulated by one or more adenoviral proteins not produced by the nucleic acid sequence.

34. The cell of claim 33, wherein the chimeric expression control sequence comprises at least a functional portion of a CMV immediate early promoter/enhancer region and at least a functional portion of an adenoviral promoter.

35. The cell of claim 33, wherein expression of the nucleic acid sequence complements in trans an adenoviral genome comprising deficiencies in at least one essential gene function of the El region of the adenoviral genome.

36. The cell of claim 35, wherein the nucleic acid sequence comprises an adenoviral El A coding sequence and an adenoviral EIB coding sequence.

37. The cell of claim 36, wherein the cellular genome further comprises a nucleic acid sequence comprising an adenoviral E2 region, E4 region, or both.

38. The cell of claim 37, wherein the cellular genome comprises a nucleic acid sequence encoding an E4-ORF6 gene product.

39. The cell of any of claims 33-38, wherein the chimeric expression control sequence comprises a CMV immediate early enhancer and/or an El A TATA box-associated sequence.

40. The cell of claim 39, wherein the chimeric expression control sequence comprises a CMV immediate early enhancer, and the CMV immediate early enhancer comprises a sequence which exhibits at least about 80% identity to SEQ ID NO: 1.

41. The cell of claim 39 or claim 40, wherein the chimeric expression confrol sequence comprises an El A TATA box-associated sequence, and the El A TATA box- associated sequence comprises a sequence which exhibits at least about 80% identity to SEQ ID NO:3.

42. The cell of any of claims 33-41, comprising a replication-deficient adenoviral vector having an adenoviral genome deficient in an essential gene function of an early region of the adenoviral genome.

43. The cell of claim 42, wherein the amount of overlap between the cellular genome and the adenoviral genome of the adenoviral vector is such that

(a) the cell produces less than about one replication-competent adenoviral vector for at least about 20 passages after infection with the adenoviral vector,

(b) the cell produces less than about one replication-competent adenoviral vector in a period of about 36 hours post infection,

(c) the cell produces less than about one replication-competent adenoviral vector per 1 x 10¹⁰ total viral particles, or

(d) any combination of (a)-(c).

44. The cell of claim 42, wherein the amount of overlap between the cellular genome and the adenoviral genome is insufficient to mediate a recombination event that results in a replication-competent adenoviral vector.

45. The cell of claim 42, wherein there is a region of homology between the cellular genome and the adenoviral genome located 5' or 3' to the nucleic acid sequence.

46. The cell of any of claims 33-45, wherein the cell is an A549 cell.

47. The cell of any of claims 33-45, wherein the cell is a human embryonic kidney cell, a human embryonic retinal cell, a renal leiomyoblastoma, a renal adenocarcinoma cell, a retinal cell, or a small cell lung carcinoma cell.

48. The cell of any of claims 33-45, wherein the cell is a non-small cell lung carcinoma cell.

49. A method of propagating a replication-deficient adenoviral vector, which method comprises:

(a) providing the cell of any of claims 1 -48,

(b) introducing a replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of the one or more regions, and (c) maintaining the cell to propagate the replication-deficient adenoviral vector.

50. A method of propagating a replication-deficient adenoviral vector, wherein the method comprises:

(a) providing a cell with a cellular genome,

(b) providing a helper virus that complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome, wherein the helper virus comprises a first packaging signal, and the cell and the helper virus do not produce a peptide that interacts with the first packaging signal to allow for packaging of the helper virus when the helper virus is present in the cell,

(c) providing a replication-deficient adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome, wherein the replication-deficient adenoviral vector comprises a second packaging signal that differs from the first packaging signal of the helper virus, and wherein the cell or the helper virus produces a peptide that interacts with the second packaging signal to allow for packaging of the replication-deficient adenoviral vector,

(d) introducing the helper virus and the replication-deficient adenoviral vector into the cell, and

(e) maintaining the cell to propagate the replication-deficient adenoviral vector.

51. The method of claim 50, wherein the first and/or second packaging signal comprises a mutated IVa2 peptide binding site.

52. The method of claim 51 , wherein the first packaging signal comprises a mutated IVa2 peptide binding site.

53. The method of any of claims 50-52, wherein the peptide that interacts with the second packaging signal is an adenoviral IVa2 peptide.

54. The method of any of claims 50-52, wherein the peptide that interacts with the second packaging signal is a nonnative peptide.

55. The method of claim 54, wherein the nonnative peptide further associates with an additional peptide involved in packaging of the replication-deficient adenoviral vector.

56. The method of any of claims 50-55, wherein the peptide that interacts with the first packaging signal is a mutated adenoviral IVa2 peptide that does not bind a wild- type adenoviral packaging signal.

57. The method of claim 50, wherein the first and/or second packaging signal comprises a mutated preterminal protein (pTP) binding site.

58. The method of claim 57, wherein the first packaging signal comprises a mutated pTP binding site.

59. The method of any of claims 50-52 and 56-58, wherein the peptide that interacts with the second packaging signal is an adenoviral pTP.

60. The method of any of claims 50-55, and 57-59, wherein the peptide that interacts with the first packaging signal is a mutated adenoviral pTP peptide that does not bind a wild-type adenoviral packaging signal.

61. The method of any of claims 50-60, wherein the cellular genome complements in trans for a deficiency in one or more essential gene functions of an adenoviral genome.

62. The method of any of claims 50-61 , wherein the cellular genome complements in trans for a deficiency in at least one essential gene function of the El region, E2 region, and/or E4 region of the adenoviral genome.

63. The method of any of claims 50-62, wherein the cellular genome comprises a nucleic acid sequence comprising an adenoviral El A coding sequence and an adenoviral EIB coding sequence.

64. The method of claim 63, wherein the cellular genome further comprises a nucleic acid sequence comprising an adenoviral E2 coding sequence, an E4 coding sequence, or both.

65. The method of any of claims 50-64, wherein the cellular genome comprises a nucleic acid sequence encoding an E4-ORF6 gene product.

66. A method of propagating a replication-deficient adenoviral vector, wherein the method comprises:

(a) providing a first cell with a cellular genome comprising a heterologous nucleic acid sequence encoding a first peptide required for packaging of an adenoviral vector, wherein the first peptide interacts with a first packaging signal,

(b) providing a helper virus comprising the first packaging signal and encoding a second peptide required for packaging of an adenoviral vector, wherein the second peptide interacts with a second packaging signal, which is different from the first packaging signal, and wherein the helper virus does not produce the first peptide,

(c) providing a replication-deficient adenoviral vector comprising a second packaging signal,

(d) introducing the helper virus and the replication-deficient adenoviral vector into the first cell,

(e) maintaining the first cell to propagate both the helper virus and the replication- deficient adenoviral vector,

(f) providing a second cell not comprising the heterologous nucleic acid sequence encoding the first peptide and not producing the first peptide,

(g) introducing the helper virus and the replication-deficient adenoviral vector into the second cell, and

(h) maintaining the second cell to selectively propagate the replication-deficient adenoviral vector.

67. The method of claim 66, wherein the first packaging signal is not a wild-type packaging signal.

68. The method of claim 66 or claim 67, wherein the second packaging signal is a wild-type packaging signal.

69. The method of any of claims 66-68, wherein the peptide required for packaging of an adenoviral vector is an adenoviral IVa2 peptide or an adenoviral pTP.

70. The method of any of claims 66-69, wherein the first cell and/or the second cell comprises a cellular genome comprising a heterologous nucleic acid sequence that, when expressed, produces one or more gene products that complement in trans for a deficiency in one or more essential gene functions of an adenoviral genome.

71. The method of any of claims 66-70, wherein the first cell and/or the second cell comprises a cellular genome comprising a heterologous nucleic acid sequence that, when expressed, produces one or more gene products that complement in trans for a deficiency in at least one essential gene function of the El region, E2 region, and/or E4 region of the adenoviral genome.

72. The method of claim 70 or claim 71, wherein the heterologous nucleic acid sequence comprises an adenoviral El A coding sequence and an adenoviral EIB coding sequence.

73. The method of claim 72, wherein the heterologous nucleic acid sequence further comprises a nucleic acid sequence comprising an adenoviral E2 coding sequence, an E4 coding sequence, or both.

74. The method of any of claims 70-73, wherein the heterologous nucleic aid sequence comprises a nucleic acid sequence encoding an E4-ORF6 gene product.

75. The method of any of claims 50-74, wherein the helper virus comprises an adenoviral genome deficient in one or more essential gene functions required for viral replication.

76. The method of claim 75, wherein the adenoviral genome of the helper virus is deficient in one or more essential gene functions of the El region, E2 region, and/or E4 region.

77. The method of any of claims 50-76, wherein the adenoviral genome of the replication-deficient adenoviral vector is deficient in one or more essential gene functions of the early regions.

78. The method of claim 77, wherein the adenoviral genome of the replication- deficient adenoviral vector is deficient in one or more essential gene functions of the El region, E2 region, and/or E4 region.

79. The method of any of claims 50-78, wherein the replication-deficient adenoviral vector is an adenoviral amplicon.

80. The method of any of claims 50-79, wherein (i) the cellular genome comprises a heterologous nucleic acid sequence encoding a first adenoviral IVa2 peptide, wherein the first adenoviral IVa2 peptide comprises encapsidation activity, but not transcription-activation activity, and binds the second packaging signal, and (ii) the helper virus encodes a second adenoviral IVa2 peptide comprising transcription-activation activity, but not encapsidation activity.

81. The method of any of claims 50-79, wherein (i) the cellular genome comprises a heterologous nucleic acid sequence encoding a first adenoviral IVa2 peptide, wherein the first adenoviral IVa2 peptide comprises transcription-activation activity, but not encapsidation activity, and (ii) the helper virus encodes a second adenoviral IVa2 peptide comprising encapsidation activity, but not transcription-activation activity, and binds the second packaging signal.

82. The method of any of claims 50-81 , wherein the helper virus is produced by (a) providing a cell that produces a peptide that interacts with the first packaging signal, (b) introducing the helper virus into the cell, and (c) maintaining the cell to propagate the helper virus.

83. The method of claim 82, wherein the peptide that interacts with the first packaging signal is a nonnative peptide.

84. The method of claim 83, wherein the nonnative peptide further associates with an additional peptide involved in packaging of the helper virus.

85. The method of any of claims 82-84, wherein the peptide is a mutant adenoviral IVa2 peptide that does not interact with a wild-type adenoviral packing signal.

86. A cell comprising a heterologous nucleic acid sequence which, upon expression, produces a first adenoviral IVa2 peptide possessing either encapsidation activity or transcription-activation activity but not both encapsidation activity and transcription- activation activity so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity possessed by the first adenoviral IVa2 peptide when the replication-deficient adenoviral vector is present in the cell.

87. The cell of claim 86, wherein the cell comprises a cellular genome into which is integrated the heterologous nucleic acid sequence.

88. The cell of claim 86 or claim 87, wherein the cell comprises a heterologous nucleic acid sequence that, when expressed, produces one or more gene products that complement in trans for a deficiency in one or more essential gene functions of one or more regions of an adenoviral genome selected from the group consisting of the El, E2, and E4 regions.

89. The cell of any of claims 86-88, wherein the cell comprises a heterologous nucleic acid sequence that, when expressed, produces one or more gene products that complement in trans for all essential gene functions lacking in an adenoviral vector amplicon.

90. The cell of any of claims 87-89, wherein there is insufficient overlap between the cellular genome and the adenoviral genome to mediate a recombination event that results in a replication-competent adenovirus.

91. The cell of any of claims 86-90, wherein the adenoviral vector comprises a packaging signal comprising a mutated IVa2 binding sequence.

92. A method of propagating a replication-deficient adenoviral vector, which method comprises:

(a) providing the cell of any of claims 86-91 ,

(b) introducing a replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome comprising a nucleic acid sequence that, when expressed, produces a second adenoviral IVa2 peptide possessing either the encapsidation activity or the transcription-activation activity not possessed by the first adenoviral IVa2 peptide and lacking either the encapsidation activity or the transcription-activation activity possessed by the first adenoviral IVa2 peptide, and

(c) maintaining the cell to propagate the replication-deficient adenoviral vector.

93. A method of propagating a replication-deficient adenoviral vector, wherein the method comprises:

(a) providing a cell with a cellular genome,

(b) providing a helper virus comprising an adenoviral genome deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome,

(c) providing a replication-deficient adenoviral vector comprising an adenoviral genome deficient in one or more essential gene functions complemented for in trans by the helper virus alone or in combination with the cellular genome,

(e) maintaining the cell to selectively propagate the replication-deficient adenoviral vector.

94. The method of claim 93, wherein the adenoviral genome of the helper virus is deficient in one or more essential gene functions of the early regions.

95. The method of claim 93 or claim 94, wherein the adenoviral genome of the helper virus is deficient in one or more essential gene functions of two or more early regions selected from the group consisting of the El region, the E2 region, and the E4 region.

96. The method of any of claims 93-95, wherein the adenoviral genome of the helper virus is deficient in one or more essential gene functions of the late regions.

97. A cell having a cellular genome comprising a nucleic acid sequence which upon expression produces a mutant DAI kinase that complements in trans for a deficiency in at least one essential gene function of at least one virus-associated RNA (VA-RNA) of an adenoviral genome so as to propagate an adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the at least one virus- associated RNA when present in the cell.

98. The cell of claim 97, wherein the mutant DAI kinase is a dominant negative mutant.

99. The cell of claim 97, wherein the virus-associated RNA is VA-RNA I and/or VA-RNA II.

100. The cell of claim 97, wherein the cellular genome further comprises a nucleic acid sequence which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of the adenoviral genome.

101. The cell of claim 100, wherein the one or more regions of the adenoviral genome are selected from the group consisting of the El, E2, and E4 regions.

102. The cell of claim 101, wherein the nucleic acid sequence comprises an adenoviral El A coding sequence and an adenoviral EIB coding sequence.

103. The cell of claim 101, wherein the one or more regions of the adenoviral genome are the El region and at least one additional region.

104. The cell of claim 103, wherein the at least one additional region comprises the E4 region.

105. The cell of claim 104, wherein the cellular genome comprises at least ORF6 of the E4 region.

106. The cell of claim 105, wherein the cellular genome comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome.

107. A method of propagating a replication-deficient adenoviral vector, which method comprises:

(a) providing the cell of any of claims 97- 106,

(b) introducing a replication-deficient adenoviral vector into the cell, wherein the replication-deficient adenoviral vector comprises an adenoviral genome deficient in the at least one essential gene function of at least one virus-associated RNA of the adenoviral genome, and