WO2009006300A2 - Production cell lines for adenoviral manufacturing - Google Patents

Abstract

An adenoviral vector system for growth of E1A/E1B deficient adenovirus that has produces replication competent adenovirus (RCA) at a rate significantly below that seen with 293 cells, are provided. Methods for producing adenovirus with reduced levels of RCA compared to 293 cells are also provided, wherein the deficient adenovirus is grown in a cell line containing coding sequences for adenovirus E1A and E1B that retain significant but limited areas of homology to both the 5' and 3' ends of the E1 deletion in the defective vector. Also provided are specific cell lines useful in this adenoviral production system and vectors and pharmaceutical preparations produced with the system.

Description

PRODUCTION CELL LINES FOR ADENOVIRAL MANUFACTURING

TECHNICAL FIELD

The present invention relates to the production of adenoviral vectors and more specifically to reducing the probability of production of contaminating replication competent adenovirus in preparations of adenoviral vectors whiie maintaining vector yieid.

BACKGROUND OF THE INVENTION

Adenoviral vectors have been used in clinical trials for indications such as cystic fibrosis, cancer, infectious disease and cardiovascular disease (NR Hacket and RG Crystal "Adenoviral vectors for gene therapy" ch2, pp17- 41 in: Gene and Cell Therapy, NS Templeton Ed. Marcel Dekker, New York and Basel., 2004). Compared to most other vira! vectors, adenoviruses appear relatively straightforward to manufacture, process and store. In addition, despite the high profile death in 1999 of a clinical trial subject receiving an experimental adenoviral-vector-based therapy, it is now clear that, used appropriately, these vectors are quite safe. For example the drug company Merck is currently involved in two large 3000 patient clinical trials of an adenoviral vector based HIV vaccine, given to healthy individuals, (DH Barouch and GJ Nabet "Adenovirus vector-based vaccines for Human Immunodeficiency Virus Type 1" Hum Gene Ther 16:149-156 2005) (http://chi.ucsf.edu/vaccines/vaccines?page=vc-03-00). Indeed, much valuable experience has been accumulated in manufacturing and corresponding clinical use over the past few years, but some issues become more problematic as these products progress in ciinicai trials and towards the market. A key problem is the occurrence of RCA in vector preparations (H Lochmϋller, A Jani, J Huard et al. "Emergence of early region 1 -containing replication competent adenovirus in stocks of replication defective adenovirus recombinants (ΔE1+ΔE3) during multiple passages in 293 cells" Hum. Gene Ther. 5:1485-1491 ,1994 ; J Zhu, M Grace, J Casale et al. "Characterization of replication-competent adenovirus isolates from large scale production of a recombinant adenoviral vector" HumGeneTher10:113-121 1999; GJ Duigou and CSH Young "Replication competent adenovirus formation in 293 cells: the recombination-based rate is influenced by structure and location of the transgene cassette and not increased by overproduction of hsRad51 , Rad51 -interacting, or E2F family proteins" J.Virol. 79:5437-5444 2005; KM Hehir, D Armentano, LM Cardoza et al. "Molecular characterization of replication competent variants of adenovirus vectors and genetic modifications to prevent their occurrence" J. Virol. 70: 8459-8467 1996; Y Haj- Ahmad and FL Graham "Development of a helper-independent human adenovirus vector and its use in the transfer of the Herpes thymidine kinase gene" J.Virol. 57: 267- 274 1986). 293 cells (Y Haj-Ahmad and FL Graham "Development of a helper- independent human adenovirus vector and its use in the transfer of the Herpes thymidine kinase gene" J.Virol. 57: 267-274 1986) have been the reliable workhorse production system for first generation adenoviral vectors for many years and for many groups. Currently FDA has allowed vector preparation with less than 1 RCA/3x10e10 viral particles (vp) to be used in the clinic h ttp://www. fda. go v/ohrms/dockets/ac/01 /brief inα/3768b 1 01.pdf and generally about 10-50% of vector batches do not meet this criterion (FJ Failaux, AJ van der Eb, and RC Hoeben "Whose afraid of replication competent virus?" Gene Therapy 6: 709-712 1999). In the hands of the inventors, with a vector we have used in clinical trials (Aguilar LK, Aguilar-Cordova E. "Evolution of a gene therapy clinical trial. From bench to bedside and back." J Neurooncol. 2003 Dec;65(3):307-15.), the failure rate has been about 10% for preparations using 2x10e9 cells. For large scale production of vectors, it is not necessary to go to a scale of thousands of liters as is used for monoclonal antibodies. Scales 10 to 100 fold larger than this pilot scaie are sufficient to produce thousands of doses. The appearance of RCA is^a stochastic event that depends on the occurrence of recombination (usually homologous) between the vector and the endogenous adenoviral sequences in the helper line (Zhu et al. 1999, op. cit.). Therefore the larger the scale, the more likely it is that an RCA contamination event will occur, with subsequent batch failure. FDA requires manufacturers to use a controlled process to make therapeutic biologies. A rule of thumb is that if more than 10% of batches fail, then the process is not controlled. Use of the 293 cell line is a limiting factor in this context, and a replacement is needed.

The only replacement for which public data suggest a measured improvement over 293 cells performance, is the PER.C6 cell line (FJ Failaux, A Bout, I Van der Yelde, "New helper cells and matched early region 1 -deleted adenovirus vectors prevent generation of replication competent adenoviruses" Hum Gen Ther. 9: 1909-1917 1998). PER.C6 is used by Merck to manufacture adenoviral vectors for their HIV vaccine trials to be used in thousands of subjects. Most current clinical adenoviral vectors are derived from Adenovirus serotypes 5 or 2, have deletions in the E1 and E3 region of the genome, and so have about 8.5 kb of space before vector capacity is reached. They are made initially by transfecting a packaging cell line (usually the 293 cell line, as described above) that supplies the E1a and E1b regions and proteins in trans. The vector stock is expanded by transduction of the viral vector onto further cultures of the same packaging cells. !n these cells the vectors can replicate like wild- type adenovirus due to the complementing functions in the cell (the E3 proteins are not needed for replication in culture), and the viral burst size can be tens of thousands per cell (typically 50-100,000vp/cell). The 293 cell line carries the first 4344 nucleotides of

Adenovirus ( N Louis, C Evelegh and FL Graham "Cloning and sequencing of the cellular - viral junctions from the human adenovirus type 5 transformed 293 cell line" Virology 233: 423-429 1997) but may also have further poorly characterized adenovirus sequences (Hehir et al 1996 op.cit.; LAiello, R GuiiFoyle, K Huebner and R Weinmann "Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells" Virol. 94:460-469 (1979). L.Aiello, R GuiiFoyle, K Huebner and R Weinmann "Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells" Virol. 94:460-469 1979) or may even no longer be completely the same in all laboratories. The number of groups that have recently described alternative systems claimed to give satisfactory vector production and reduced RCA compared to the standard 293 cells demonstrates the ongoing nature of the RCA issue (Table 1). However, other than the PER.C6 ceils, none have been quantifiably characterized and clinically qualified.

Key Criteria for adenoviral vector production systems

As part of the accumulation of experience in adenoviral vector production, referred to above and discussed in at least two recent reviews (M Lusky "Good manufacturing practice production of adenoviral vectors for clinical trials." Hum Gene Then 16:2812-91 2005; NE Altaras, JG Aunins, RK Evans et al. "Production and formulation of adenovirus vectors" Adv Biochem Engin/Biotechnol 99: 193-260 2005) key criteria for an adenoviral vector production system include: a) probability of contamination with RCA; and b) productivity (usually measured as viral particles produced/cell and called the 'burst size'). The frequency of RCA occurrence is influenced by a number of factors, including the configuration of the vector (Duigou and Young 2005 op.cit.) For example, vectors with genomes longer than the native viral genome, by even a couple of percentage points, are known to be more likely to generate RCA; the size of the initial inoculums of starter viral vector onto producer cells can also be a factor. Adenoviruses naturally suppress homologous recombination to avoid complications during viral replication (MD Weitzman, CT Carson, Raschwartz et al. "Interactions of viruses with cellular DNA repair machinery" DNA Repair 3: 1165-1173 2004) and this makes the frequency of RCA occurrence lower than expected from other studies of homologous recombination, like those looking at gene knock-outs or knock-ins. Several attempts have been made to generate improved cell lines compared to 293 cells (Table 1). As discussed above, with the exception of the PER.C6 line, there has been insufficient data and follow-up to know whether any of these reports represent genuinely improved lines. The key obstacles in generating such lines have been: a) finding cells that will tolerate constitutive expression of the E1 a and E1b proteins; b) overall yield (per cell) of vector; and c) freedom from RCA. These are all non-trivial obstacles. Most of these reported lines have data on a) and b) and show only preliminary data on RCA, and no follow-up descriptions are widely available. Instead, these groups have relied mainly on eliminating sequence overlap between the helper genome and the E1 deletion in the vector. In particular the 5^J end of the helper/deletion has been a target for this strategy, as this seems fairly straightforward with conventionally deleted Adenoviral vectors (a typical deletion is from about 455 to 3228, see Figure 1 , Figure 2 and SEQ ID NO:1) to eliminate (in the vector) and retain (in the helper genome) the complete E1A coding region. However, at the 3' end, such a vector has sequences that include E1B coding sequences and so it has required more complex manipulations to eliminate this overlap, including the use of custom engineered vectors. Although it is known that sequences as small as 10-25 nucleotides overlap can result in products that have apparently undergone homologous recombination (E Otto, A Jones-Trower EF Vanin et al. "Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences" Hum Gene Ther. 5:567-75. 1994; D Ayares, L Chekuri, K-Y Song et a!. "Sequence homology requirements for intermolecular recombination in mammalian cells" Proc Natl. Acad Sci 83: 5199-5203 1986) in most human ceils it appears that this frequency drops rapidly with size of overlap below the kilobase range (C Deng and M Capecchi "Re-examination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus MoI cell boil 12:3365-3371 1992)and then plateaus somewhere below about 300 bp overlap. Toxicity resulting from the expression of the adenovirus E1A protein(s) has been a bottleneck that only a few cell types seem able to pass through (e.g. KB, HeIa, A549, Human Embryonic Kidney [HEK] cells, Human Embryonic Retina! [HER] ceils). The ■ requirement for vector yields has also been hard to meet, and only extensive screening has yielded useful cell lines (as productive as 293 cells). In this context, it was thought at one time that high yields per cell might predispose to RCA generation, based on mass action effects, but additional data suggest these phenomena may not be closely linked. The probability of RCA generation has not been as well-defined. It is possible, even likely, that the cell lines in Table 1 will produce RCA less frequently than 293 cells. However, certification by experimentation that one cell line is less likely to produce RCA than another requires fairly large and repeated experiments, and these have not been performed. In fact, for the cell lines in Table 1 , data that wouϊd give such assurance is only available for PER.C6 cells. Even there, the information available is limited. Although there have recently been a number of publications from Merck ( Altaras et al 2005 op.cit. ; R Youii, TJ Toner, Q Su et al. "Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses" Hum Gene Ther. 14:1017-1034, 2003; L Maranga, JG Aunins and W Zhou "Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector." Biotechnol Bioeng. 90:645-55, 2005). evaluating PER.C6, often some details are not provided. The PER.C6 system also poses several scientific and technical hurdles. Firstly most adenoviral vectors that have been clinically used are not PER.C6 compatible, in that there is extensive overlap at the 3' end of the helper vector. In turn thus has lead to generation of anomalous cytopathic non- replicating vectors in the PER.C6 cell line (P Murakami, E Pungor, J Files, et al. "A single short stretch of homology between adenoviral vector and packaging eel! line can give rise to cytopathic effect-inducing, helper dependent E 1 -positive particles" Hum. Gene Ther. 13:909-920, 2002). This is an adventitious agent that needs to be characterized and eliminated or minimized, before undertaking commercial manufacturing. Thus there is still a great need for a novel, well characterized adenoviral vector production cell line with Sow risk of RCA or deleterious products of homologous recombination, and, crucially, that gives good yields and can be used with a range of vectors. It is important to emphasize that a criterion for a commercially useful vector producer line is not that RCA can never be generated (and in any case such a negative can never be proven). A productive cell line with a modest, but reliable ten fold reduction of RCA compared to 293 is very useful at a commercial level.

Table 1 : Deletions and complementing functions in various Adenoviral packaging systems

Note: § This is the commonly used system for manufacturing adenoviral vectors.

Statistical treatments and experimental approaches that can be adapted to aliow the determination and comparison of RCA frequencies are available (Duigou and Young 2005 op.cit; C Wang and F Gheyas " Sampling strategies for detecting rare impurities: an application in gene therapy products" J Biopharm Stat 15:241-252 2005) A cell line tested in such a way can then be used, with confidence, as the basis for further product development and manufacturing for adenoviral vectors.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the unexpected finding that it is possible to make adenoviral vectors with a productivity equivalent to 293 cells but with a 10 fold or more reduced frequency of RCA generation compared to 293 cells, while retaining significant levels of homologous nucleic acid sequence overlap between the vector and the helper genome in a adenovirus packaging cell (PC). Novel configurations for adenovirus packaging cells are described that allow efficient production of adenoviral vectors, with yields comparable to standard 293 cells but with a much lower rate of contamination with RCA.

In one embodiment of the invention an adenoviral packaging system is supplied with an adenoviral vector deleted in the E1A and E1 B regions and a packaging cell which provides sufficient complementary E1A and E1 B functions that the vector is produced with an efficiency of at least 20% of that produced on 293 cells and with an RCA rate at least 10-fold less than the same vector on 293 cells. In a further embodiment of the invention the system has a productivity of least 50% of 293 cells but with rates of RCA generation at least 10-fold less than the rate of generation of RCA with the same vector in 293 cells. In a further embodiment the system has a productivity of at least 50% of 293 cells but with rates of RCA generation ≤ 1% that of 293 cells. In a further preferred embodiment, PCs are supplied with productivity of at least 50% of 293 ceils and a rate of RCA generation < 10% that in 293, where the PC carries a helper gene or genes that encode the adenoviral E1A and E1B genes, and maintain an overlap with the vector being produced of between 1 and 350 base pair at the left (5¹) end and an overlap of between 1 to 600bp at the right (3¹) end. In a further preferred embodiment, a PCL is supplied with productivity of at least 50% of 293 cells and a rate of RCA generation ≤ 10% that in 293, where the helper gene encodes the adenoviral E1A and E1 B genes, and maintain an overlap with the vector being produced of between 1 to 200 base pair at the left (5^J) end and an overlap of between 1 and 210bp, 300bp, 400bp or 500bp at the right (3') end. In a further preferred embodiment, a system for generating adenoviral vectors is supplied with productivity of at least 50% of 293 cells with the same vector, and a rate of RCA generation ≤ 10% that in 293, where the helper gene encodes the adenoviral E1 A and E1B genes, and maintain an overlap with the vector being produced of between 1 and 100 base pair at the left (5') end and an overlap of between 1 and 210bp, 300bp, 400bp or 500bp at the right {3') end. In a further preferred embodiment, a PCL is supplied with productivity of at least 50% of 293 cells and a rate of RCA generation ≤ 10% that in 293, where the helper gene encodes the adenoviral E1A and E1 B genes, and maintain an overlap with the vector being produced of between 1 and 97 base pair at the left (5') end and an overlap of between 1 and 210bp at the right (3') end. In a further preferred embodiment the PCL in the adenoviral production system with the any of the above properties is derived from the A549 cell line. In a further preferred embodiment the PCL used in the system is the c24 cell line.

In a further embodiment of the invention methods are provided for producing adenoviral vectors using such PCLs as described above and corresponding adenoviral vectors with the described overlap. Aiso provides are adenoviral vectors produced by any of the systems described and pharmaceutical acceptable preparations of adenoviral vectors produced by any of the above systems.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a map of the left end of the Adenoviral 5 genome (corresponding to SEQ ID 1 ) and the location of the E1A and E1 B transcripts and coding sequences. E1A gene = 468 ^■ 1632, coding sequence starts at 560; E1 B = 1672-3509, 1672 = start of TATA box; E1 B 19K ends at 2243; plX = 3609- 4031 ; start plX promoter = 3440. Sequence numbering follows the accepted Gen Bank numbering, e.g. AY339865.

Figure 2 shows the configuration of a typical first generation adenoviral vector. In this case the vector is deleted from nucleotides 455 to 3228, and encodes the E.coli beta- galactosidase gene (AdV-RSV-βgal). The Ad5 sequence to the right (3') side of the balactosidase gene is labeled Ad5(Δ3) to signify that the E3 gene is deleted in this segment.

Figure 3 (adapted from Diugou and Young 2005 op. cit.) shows a diagram of the method used to determine the rate of RCA generation in a cell line (here c 24) compared to a standard cell line (here 293 cells). The details of the method are described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

Ail technical and scientific terms used herein have the same meaning as they would to one skilled in the art of the present invention, unless they are specifically defined otherwise. Practitioners are particularly directed to Sambrook et a!., Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Press, Woodbury, N.Y., 2001 and Ausube! FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N. Y. 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

The publications and other materials including ail patents, patent applications, publications (including published patent applications), and database accession numbers referred to in this specification are referred to describe the background of the invention and in particular, cases to provide additional details respecting the practice. The publications and other materials including all patents, patent applications, publications (including published patent applications), and database accession numbers referred to in this specification are incorporated herein by reference to the same extent as if each were specifically and individually indicated to be incorporated by reference in its entirety.

An adenovirus packaging cell (PCL) is a cell that is able to package adenoviral genomes or modified genomes to produce viral particles. It can provide a missing gene product or its equivalent. Thus, packaging cells can provide complementing functions for the genes deleted in an adenoviral genome and are able to package the adenoviral genomes into the adenovirus particle. The production of such particles requires that the genome be replicated and that those proteins necessary for assembling an infectious virus are produced. The particles also can require certain proteins necessary for the maturation of the viral particle. Such proteins can be provided by the vector or by the packaging cell. The packaging cell line is produced by genetically modifying a cell line permissive for adenovirus replication, to comprise adenovirus E1A and/or E1B coding sequences. The adenovirus packaging ceil lines of the present invention provides a packaging cell line that is acceptable for commercial manufacturing, but that retain an overlap of between 5 and 350bp between the vector and the helper genomes in PCL at the 5' ends qf the deletion in the adenoviral vector and between 5 and 600bp at the 3' end. This overlap can be in the E1 region of the vector and helper but can also be in other regions of the adenoviral genome that are deleted and supplied in trans in adenoviral packaging cell lines. Such deletions/complementation's include the plX gene, the E4orf 6 gene and other genes deletions known to those skilled in the art.

A "parent cell" includes an individual cell or ceil culture, which can be or has been a recipient of a viral vector(s) of the invention. Parent cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A parent cell includes a cell transfected or infected in vivo or in vitro with an adenoviral vector of this invention.

As used herein, the term "E1A" refers to all gene products of the adenovirus E1A region, including expression products of the two major RNAs: 13S and 12S. These are translated into polypeptides of 289 and 243 amino acids, respectively. These two proteins differ by 46 amino acids, which are spliced from the 12S mRNA, as described in Chow et al. (1980) Cold Spring Harb Symp Quant Biol. 44 Pt 1 :401 14; and Chow et al. (1979) J. MoI. Biol. 134(2):265 303, and also defined in Genbank listing AY339865 at http://www.ncbi. nlm.nih.gov/entrez/viewer.fcqi?db=nucleotide&val=33465830 ,all herein being specifically incorporated by reference. For the purposes of the invention, the packaging cell line may express the 289 polypeptide, the 243 polypeptide, or both the 289 and the 243 polypeptide. The term E1A is also used herein with reference to partial and variant E1A coding sequences.

As used herein, the term "E1 B" refers to al! gene products of the adenovirus E1 B region, including expression products of the two major RNAs: 13S and 12S as defined in Genbank, http://www.ncbi.nlm.nih, gov/entrez/viewer.fcgi?db=nucleotide&val=33465830 (lisiting number AY339865) herein specifically incorporated by reference. These are translated into polypeptides of 96 amino acids ("19K") and 365 amino acids ("55K"), respectively. For the purposes of the invention, the packaging cell line may express the 19K polypeptide, the 55K polypeptide, or both the 19K and the 55K polypeptide. The term E1 B is also used herein with reference to partial and variant E1B coding sequences.

In the case where the genes complemented are the E1A and E1 B genes, the helper cell line can express these from the natural promoters or from heterologous promoters. The E1A genes and E1B genes can be arranged contiguously on one DNA molecule as they are in the adenovirus or the genes can be can be on separate DNA molecules that are delivered to the parent cell line for construction of a packaging cell line. The E1 A and E1B genes can be delivered by any common methods of gene delivery including: calcium phosphate transfection; use of synthetic delivery lipids for DNA; by other viral vectors such as retroviral vectors, bacuioviral vectors and others; by physical means such as electroporation; and by other means known to those skilled in the art. The cell lines used as parent cells for construction of packaging cells can be human (e.g. HeIa, A549, human embryonic kidney cells, human embryonic retinal cells, KB cells) but also from other species such as dog (e.g. D17 or CF2 ceils), monkey (e.g. Vero cells ) or rodent (e.g. BHK cells). The adenoviral vectors can be derived from any of the mastadenovirus genus, or from the aviadenovirus genus (see T Shenk "Adenoviridiae: the viruses and their replication" in Fields Virology, Fifth Edition, Eds DM Knipe, PM Howley, DE Griffin, RA Lamb, MA Martin, B Roizman, SE Straus; Lippincott Williams & Wilkins, Philadelphia & New York 2007). For example, the vector can be derived from any of subgroups A through F of human adenoviruses, including adenoviruses 2, 5, and 36. As a further example, vectors can be derived from adenoviruses for which the primary host is a species other than human, such as chimpanzee, monkey, sheep, pig, horse, dog, rodent, and bovine. The construction of packaging cell lines and the corresponding vectors is achieved by methods of manipulation of DNA sequences and celi lines by conventional methods of molecular biology, tissue culture and virologicai techniques known to those skilled in the art. The various methods and compositions are described below. Although particular methods are exemplified in the discussion below, it is understood that any of a number of alternative methods are applicable and suitable for use in practicing the invention. It will also be understood that an evaluation of the adenovirus vectors and methods of the invention may be carried out using procedures standard in the art, including the diagnostic and assessment methods described below.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, immunology and virology, which are within the scope of those of skill in the art. Such techniques are explained fuily in the literature, such as, "Molecular Cloning: A Laboratory Manual", Third edition, Cold Spring Harbor Laboratory Press (Sambrook et al., 2001); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et a!., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), and in individual articles in Cold Spring Harbor Protocols, 2006 and 2007, Cold Spring Harbor Press, New York and at http://www.cshprotocols.org/ ), each of which is expressly incorporated by reference herein.

For techniques related to adenovirus, see, inter alia, Feigner and Ringold Nature 337:387 388,1989; Berkner and Sharp (1983) Nucl. Acids Res. 11 :6003 6020, 1983; Graham (1984) EMBO J. 3:2917 2922, 1984; Bett et al. (1993) J. Virology 67:5911 5921 , 1993; Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91 :8802 8806, 1994; Wold and Tollefson "Adenovirus Methods and Protocols" 2^nd Edition, vols 1 & 2 Humana Press, 2006; 'Adenoviral vectors for gene therapy' Eds. D Curiel and JT Douglas, Academic Press, San Diego (2002) each of which is incorporated by reference herein.

EXAMPLES

Example 1. Construction of the E1A and E1 B expression plasmid.

The adenoviral fragment encoding the E1 a and E1b genes that was used to make this line stretches from nucleotide 358 to 3537 (SEQU ID # 2) from Ad 5, and was generated by excision from the plasmid pXC1 ( McKinnon RD, Bacchetti S, Graham FL Tn 5 mutagenesis of the transforming genes of human adenovirus type 5. Gene (Amst), 19: 33-42, 1982. ). by Sst Il/Afill restriction digests followed by isolation of the 3.2 kb. fragment carrying the intact E1 genes from agarose gels. The fragment was blunt-ended by T4 DNA polymerase treatment and ligated into the plasmid pZeoSV (Invitrogen, Carlsbad, CA USA) cut with Pvull so that the genes are expressed under the control of the SV40 early promoter and an SV 40 polyadenylation site. Bacterial transformants from the ligation mix were plated out and selected with zeomycin. Bacterial colonies that grew out were picked and screened by minipreps and restriction analysis for the desired configuration. A plasmid with the correct restriction pattern with Pvull, Hindlll, Xba I ansd Pstl was chosen and designated pXC1/pZeo. This plasmid was prepared at large scale, purified and stored in 1OmM tris 1 mM EDTA pH 8. This configuration of expression vector for E1A and E1B eliminates the adenovirus 5¹ ITR sequence found in the 293 cells. The fragment encodes the complete E1 A and E1 B genes and starts about 110 bases upstream of the start site for RNA transcription of the E1a gene (nt 467). Commonly used adenoviral vectors are deleted from around nt 450 to 3300, so there are only about 100 bases of overlap 5' to the deletion. The 3' end the overlap between the helper sequences and typical vectors is about 200 bp. and maintains the essential E1 b 55k protein. The expression of the E1A and E1B genes was further checked by transfection onto tissue culture cells (see below)

Example 2 Transfection of Parent Cells to generate candidate packaging cell lines

Table 2 lists our initial minimum criteria for a useful candidate cell iine. The frequency of RCA generation is not listed. These are, rather, the criteria that, if met, would justify the effort to characterize, in statistically justifiable way, the frequency of RCA generation in the candidate cell lines.

Table 2: Initial criteria for a cell line to qualify for RCA testing

1. Adenoviral vector production efficiency (burst size) at least 50% that of 293 cells

2. Mean generation time <48 h

3. Stable vector production capacity without antibiotic selection (>20 passages)

4. Sterile: test negative for bacterial and mycoplasma contamination

5. Maximum number of copies of the E1a gene <10 (preferably less than 3, to minimize risk of rearrangement and recombination).

The E1A gene is capable of immortalizing (but not transforming) primary cells, but it can also be quite toxic to the cell. Three types of cells were transfected with the pXC1/pZeo pfasmid and selected with 400ug/ml zeomycin: A549 cells (human lung carcinoma M Lieber, B Smith, A Szakal et al. 'A continuous tumor-ceil line from a human lung carcinoma with properties of type Il alveolar epithelial ceils' Int. J. Cancer 17: 62-70, 1976), MRC9 ceils (human primary fibroblasts JP Jacobs, AJ Garrett and R Merton "Characteristics of a serially propagated human diploid cell designated MRC-9" J Biol Stand 7: 113-122, 1979) and human C3A hepatic carcinoma derived cells (ATCC- CRL-10741), using zeomycin selection. Colonies from the transfection of the A549 cell line grew most reliably so several of these were picked with a Q-tip into 24 well dishes and grown out under zeomycin selection. The colonies were split, with a portion frozen and another passaged for analysis. Six clones that appeared to grow well were tested for: generation-doubling time by time/extent of confluence in 150 cm2 plates after initial inoculums with the same number of cells; for the capability to complement adenoviral vectors by CPE appearance on adenoviral vector transduction; and qualitatively for GFP intensity 48h after infection with a GFP expressing vector. Results are shown in Table 3 with A549 ceils as a negative control and 293 cells as a positive control. All six colonies grew well (generation time under 48 hours) and four clones (C 03, C08, C10 and C 24) appeared to efficiently complement the adenoviral vectors. All the clones were further expanded and frozen down. These were then further tested quantitatively for vector production efficiency in parallel with 293 cells (positive control) and A549 (negative). Quantitative titration on 293 ceils was performed using a dilution series of the test sample, 8 plates per dilution and calculating the titer from the point where only some of the 8 plates were positive (i.e. by endpoint dilution). In this assay the 293 titration plates are incubated for at least 8 days, and the dilution well scored positive or negative for green fluorescence. In experiment A, cells were plated in 60 mm dishes and infected with the GFP adenoviral vector. After 3 days the supernatant (2ml) was harvested and titered quantitatively (Expt A Table 4). In a separate experiment (Expt B Table 4) the same procedure was followed but the infected cells were used to make freeze thaw lysates in 1ml of 10 mM Tris 4% sucrose, 2 mM MgCb pH 8, which were then assayed quantitatively.

Table 3 Initial screening of transfected, zeomycin resistant A549 clones

Cell Line Confluence CPE (%), Act infected GFP staining (uninfected)

C 03 90 70 positive

C 05 80 0 negative

C 08 85 50 positive

C 10 75 50 positive

C 13 100 0 negative

C 24 100 75 positive

A549 100 0 negative

293 100 75 positive

Table 4 Quantitative titration of GFP vector made on different transfected A549 clones

. One clone, designated c24 consistently gave vector yields at least equivalent to those from 293 cells. For clinical use the presence of selection antibiotics such as Zeomycin is undesirable. Thus, to test for stability of the helper function, the c24 cells were serially passaged at 1:5 to 1:10 ratios in batches of > 10 T225 flasks per passage for 23 passages. The cell doubling time was calculated at approximately 24 hrs. After 23 passages, the burst size with various vectors was maintained unchanged (>50,000 per cell) and equaled that of 293 cells. In the absence of antibiotic use the cells continued to test sterile for bacteria and fungus and negative for mycoplasma by the ATTC PCR test. Southern analysis showed one or two copies of the E1 gene was present in the genome, These results qualified the c24 producer tine as a candidate for further testing, per the criteria in Table2.

Example 3 Testing the rate of RCA production in candidate packaging cell lines

The c24 ceils, 293 cells and A549 cells are expanded in MEM, 10% bovine calf serum and harvested and stored at 10e7 cells per via! in medium plus 20% FCS and 5% DMSO in liquid nitrogen. The vector seed stocks that are used in this assay are Ad-RSV-βgal (lot #01298 or equivalent). This is a large vector lot with a titer of 10e12viral particles(vp)/ml prepared from 293 cells (Shabram et al. pp167-2003 in 'Adenoviral vectors for gene therapy' Eds. D Curiel and JT Douglas, Academic Press, San Diego 2002) which has been tested free of RCA at 3x10e10vp. A large purified batch of wtAdδ prepared from the ARM reference stock (ATCC VR-1516 ) by amplification on Heia cells is used as a spike control.

(i) Background to RCA testing

One hurdle in testing for relative frequency of RCA generation is that, even in the worst vector-cell pairings, RCA generation is not so frequent an event at small laboratory scale preparations. It is most often a problem once scale up is attempted. Thus, testing small scale batches for RCA is likely uninformative but making a very large number of large preparations to establish the likelihood of RCA formation is impractical. The generation of RCA is stochastic event, meaning it can happen with equal probability at any time, and this makes it quite amenable to statistical analysis that, in turn, suggests testing strategies that can assign numbers to the RCA generation frequency in a particular circumstance. By holding everything else the same and only varying only the producer cell line it is possible to assign a relative frequency to the two circumstances (293 cells compared to c24 cells) along with the degree of uncertainty. A modified Luria-Delbruck fluctuation test (S Luria and IvI Delbruck "Mutations of bacteria from virus sensitivity to virus resistance" Genetics 28:491 -511 , 1943; Duigou and Young et al. 2005 op.cit.) is used to measure the rate of RCA production in the two or more ceil lines. In this case we compare 293 cells and c24 cells. The wt adenovirus is used as a spike control to monitor the sensitivity of the system, In this assay an adenoviral vector with the E1 sequences deleted from nucleotides 455 -3228 with an RSV LTR promoter driving the E.coli beta-galctosidase gene inserted (Ad-RSV- βgal), is used. This has tested negative for RCA and is slightly bigger than 100% adenoviral genome size and so is expected to have measurable frequencies of RCA generation in this system. As it is straightforward to stain cells for production of this protein with the X-GaI assay that turns positive cells blue, it is simple to stain cells for its presence when caused by RCA assisted vector transfer to primary, secondary or tertiary test plates. A diagram of the procedure is shown in Figure 3. Other test vectors with or without the simple read-outs of the beta-gal vector can also be used in this test to compare two cell lines.

(ii) Testing RCA rates by a modified Luria-Delbruck fluctuation test Twelve 35-mm plates of either 293 cells or c24 cells are infected with the adenoviral vector using 100 to 1 ,000 beta-ga! focus-forming units (BFU)/plate, and adsorbed for 2h in a volume of 0.2 ml per plate. These quantities of vector can be assumed to be free of RCA based on previous RCA testing. After 2 hours, 2.5 ml of medium (MEM plus 10% FCS) are added, cells incubated at 37°C and examined daily. After all cells display CPE, they are washed and subjected to freeze-thawing in 2.5 ml 10 mM Tris 4% sucrose, 2 mM MgCI₂ pH 8 to produce the "original yield" (OY). The original yield is assayed as follows: 0.1 ml - titers by BFU focus assay on 293 cells (corrected for diffusion effects, C Nyberg-Hoffman, P Shabram W Li et al. "Sensitivity and reproducibility in adenoviral infectious titer determination" Nat Med 3:808-81 1 , 1997); 0.2 ml - infect A549 cells grown in 35-mm- diameter plates; the remainder - infect A549 cells grown in 150-mm-diameter plates. The large plate provides a large enough number of A549 cells to avoid "input" cytopathic effect (CPE)from vector uptake and duplicate assays provide greater testing confidence. Infected A549 cells are incubated at 37⁰C in MEM plus 10 % fetal bovine serum. After full cytopathic effect or 10 days of growth, the cells are subjected to three cycles of freeze-thawing and 0.3 ml per sample is used to infect fresh A549 cells grown in 35-mm-diameter plates (passage 2). After 10 to 13 days, the passage 2-infected cells are subjected to freeze-thawing and 0.3 ml used to infect fresh 35-mm-diameter plates of A549 cells. An OY is scored positive when A549 cells display CPE at any step of serial infection. Initial apparent RCA positives are verified by preparing a modified Hirt extract DNA designed for intracellular adenovirus DNA (FC Volkert and CSH Young "The genetic analyses of recombination using adenovirus overlapping terminal DNA fragments" Virology 125: 175-193,1983), from A549 cells passage #3 from each of the positive and at least one negative CPE plate as a control. PCR amplification of the E1 a region as described by Lochmulier et al. 1994 (op.cit) is used to detect RCA genomes. This effect is calibrated by separately spiking both the original 293 or c24 cells and the material put on the passage #1 ceils, with 1 , 3 and 10 ARM RCA and checking the detection frequency by CPE, or on occasion by PCR. This is performed both in the presence and absence of the standard test vector to evaluate the potential effect of competitive inhibition. To calculate the RCA rate/BFU or RCA rate per viral particle (vp), each OY is scored positive or negative and the total number of BFU (or vp) in the total original yield calculated. Then the number of RCA/viral vector otherwise called the RCA rate = -In (number of OY without RCA/total number of OY)/(average BFU/plate). The same calculation with vp substituted for BFU gives the RCA rate per viral particle. Vector particles from crude lysates are determined by HPLC analysis in parallel with standard purified preparations of adenovirus or adenoviral vectors.

The experiment is repeated a minimum of 3 times per condition and Student's T test used to analyze the data and allow determinations of the uncertainty of the measurements. More replicates are performed if required. This comparison allows determination of the RCA rate of the c24 cell line and that of the 293 cells, and shows a level of RCA that is at least 10 fold reduced in c24 cells compared to 293 cells.

(iii) Testing RCA rates by serial passage and modified Lurta-Delbruck fluctuation testing The c24 cell line is also compared to 293 cells by serial passage of the beta gal vector on the packaging ceil lines as described by Hehir et ai.1996 (op.cit.) in 35 mm dishes. The standard fluctuation assay is performed initially at spaced intervals (e.g. passage 4, 8 and 12). Vector yields (BFU) at each passage is measured with 0.1 ml of the cell lysate, 0.1 ml serially passaged onto a new plate and the remainder stored or analyzed by the RCA fluctuation analysis. When a positive RCA readout is obtained, if it is completely positive in all dishes, then the stored OY between the last completely negative assay and the current positive one is assayed to determine when the RCA first showed up, and at what frequency. If the first positive assay has only some positive plates, then RCA generation occurred in the latest passage. The same formula can be used as before (part (ii) above) and the total BFU is the sum of those that are generated in this process and the total number of plates is the sum of al! those that wouid have been used in the standard assay up to the point where RCA appeared. This test also shows that c24 cells have an RCA rate that is at least 10 fold less than that of 293 cells.

The invention is not to be limited in scope by the recombinant expression vectors and cell lines exemplified, which are intended as illustrations of one aspect of the invention, it is to be understood that the above detailed examples and described embodiments are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

SEQUENCE ID LISTING

SEQ ID NO: 1

<110> Advantagene Inc. <120> Production cell lines for adenoviral Manufacturing <130> 107:6-07 <141> 2007-06-26 <160> 1 <210> 1 <211> LENGTH : 4320 <212> TYPE: DNA <213> Adenovirus <220> <223> Left end of adenovirus 5, nucleotides 1-4320, encodes E1a, E1b, proteins see Figure 1

<300> <301> Estuardo Aguilar-Cordova Douglas J. JoNy

<400> 1 1 catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 61 ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 121 gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 181 gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 241 taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 301 agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 361 gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 421 cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 481 tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 541 tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 601 aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 661 tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 721 cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 781 gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 841 cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 901 ccttgtaccg gaggtgatcg atcttacctg ccacgaggct ggctttccac ccagtgacga 961 cgaggatgaa gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg 1021 caggtcttgt cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg 1081 ctatatgagg acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga 1141 tagagtggtg ggtttggtgt ggtaattttt tttttaattt ttacagtttt gtggtttaaa 1201 gaattttgta ttgtgatttt tttaaaaggt cctgtgtctg aacctgagcc tgagcccgag 1261 ccagaaccgg agcctgcaag acctacccgc cgtcctaaaa tggcgcctgc tatcctgaga 1321 cgcccgacat cacctgtgtc tagagaatgc aatagtagta cggatagctg tgactccggt 1381 ccttctaaca cacctcctga gatacacccg gtggtcccgc tgtgccccat taaaccagtt 1441 gccgtgagag ttggtgggcg tcgccaggct gtggaatgta tcgaggactt gcttaacgag 1501 cctgggcaac ctttggactt gagctgtaaa cgccccaggc cataaggtgt aaacctgtga 1561 ttgcgtgtgt ggttaacgcc tttgtttgct gaatgagttg atgtaagttt aataaagggt 1621 gagataatgt ttaacttgca tggcgtgtta aatggggcgg ggcttaaagg gtatataatg 1581 cgccgtgggc taatcttggt tacatctgac ctcatggagg cttgggagtg tttggaagat 1741 ttttctgctg tgcgtaactt gctggaacag agctctaaca gtacctcttg gttttggagg 1801 tttctgtggg gctcatccca ggcaaagtta gtctgcagaa ttaaggagga ttacaagtgg 1861 gaatttgaag agcttttgaa atcctgtggt gagctgtttg attctttgaa tctgggtcac 1921 caggcgcttt tccaagagaa ggtcatcaag actttggatt tttccacacc ggggcgcgct 1981 gcggctgctg ttgctttttt gagttttata aaggataaat ggagcgaaga aacccatctg 2041 agcggggggt acctgctgga ttttctggcc atgcatctgt ggagagcggt tgtgagacac 2101 aagaatcgcc tgctactgtt gtcttccgtc cgcccggcga taataccgac ggaggagcag 2161 cagcagcagc aggaggaagc caggcggcgg cggcaggagc agagcccatg gaacccgaga 2221 gccggcctgg accctcggga atgaatgttg tacaggtggc tgaactgtat ccagaactga 2281 gacgcatttt gacaattaca gaggatgggc aggggctaaa gggggtaaag agggagcggg 2341 gggcttgtga ggctacagag gaggctagga atctagcttt tagcttaatg accagacacc 2401 gtcctgagtg tattactttt caacagatca aggataattg cgctaatgag cttgatctgc 2461 tggcgcagaa gtattccata gagcagctga ccacttactg gctgcagcca ggggatgatt 2521 ttgaggaggc tattagggta tatgcaaagg tggcacttag gccagattgc aagtacaaga 2581 tcagcaaact tgtaaatatc aggaattgtt gctacatttc tgggaacggg gccgaggtgg 2641 agatagatac ggaggatagg gtggccttta gatgtagcat gataaatatg tggccggggg 2701 tgcttggcat ggacggggtg gttattatga atgtaaggtt tactggcccc aattttagcg 2761 gtacggtttt cctggccaat accaacctta tcctacacgg tgtaagcttc tatgggttta 2821 acaatacctg tgtggaagcc tggaccgatg taagggttcg gggctgtgcc ttttactgct 2881 gctggaaggg ggtggtgtgt cgccccaaaa gcagggcttc aattaagaaa tgcctctttg

2941 aaaggtgtac cttgggtatc ctgtctgagg gtaactccag ggtgcgccac aatgtggcct

3001 ccgactgtgg ttgcttcatg ctagtgaaaa gcgtggctgt gattaagcat aacatggtat 3061 gtggcaactg cgaggacagg gcctctcaga tgctgacctg ctcggacggc aactgtcacc 3121 tgctgaagac cattcacgta gccagccact ctcgcaaggc ctggccagtg tttgagcata 3181 acatactgac ccgctgttcc ttgcatttgg gtaacaggag gggggtgttc ctaccttacc 3241 aatgcaattt gagtcacact aagatattgc ttgagcccga gagcatgtcc aaggtgaacc 3301 tgaacggggt gtttgacatg accatgaaga tctggaaggt gctgaggtac gatgagaccc 3361 gcaccaggtg cagaccctgc gagtgtggcg gtaaacatat taggaaccag cctgtgatgc 3421 tggatgtgac cgaggagctg aggcccgatc acttggtgct ggcctgcacc cgcgctgagt 3481 ttggctctag cgatgaagat acagattgag gtactgaaat gtgtgggcgt ggcttaaggg 3541 tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg 3601 ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc 3661 gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc 3721 ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg 3781 agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg 3841 actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg 3901 acaagttgac ggctcttttg gcacaattgg 'attctttgac ccgggaactt aatgtcgttt 3961 ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca 4021 atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt 4081 cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt 4141 cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat 4201 acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg 4261 gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt

Claims

1. An adenoviral packaging system comprising an adenoviral vector deleted in the

E1A and E1 B regions and a packaging cell line (PC) which provides sufficient complementary E1 A and E1 B functions that i) the vector is produced with an efficiency of at least 20% of that produced on

293 cells and ii) with an RCA rate at least 10-fold less than the same vector on 293 ceils

wherein said PC carries a helper gene or genes that encode the adenoviral E1B and

E1 B genes that maintain an overlap with the genome of said vector of between 1 and 350 base pair at the left (5¹) end and an overlap of between 1 to 600bp at the right (3') end.

2. An adenoviral packaging system of claim 1 , where the overlap of the helper genes in the PC with the genome of the vector being produced is between 1 to.200 base pair at the left (5') end and between 1 and 210bp, 300bp, 400bp or 500bp at the right (3¹) end.

3. An adenoviral packaging system of claim 1 , where the overlap of the helper genes in the PC with the genome of the vector being produced is between 1 to 200 base pair at the left (5') end and between 1 and 210bp, 300bp, 400bp or 500bp at the right (3¹) end.

4. An adenoviral packaging system comprising an adenoviral vector deleted in the

E1A and E1B regions and a packaging eel! line (PC) which provides sufficient complementary E1A and E1 B functions that iii) the vector is produced with an efficiency of at least 50% of that produced on 293 cells and iv) with an RCA rate at least 10-fold less than the same vector on 293 cells

wherein said PC carries a helper gene or genes that encode the adenoviral E1B and E1B genes that maintain an overlap with the genome of said vector of between 1 and 200 base pair at the left (5') end and an overlap of between 1 and 210bp, 300bp, 400bp or 500bp at the right (3') end.

5. An adenoviral packaging system of claim 4, where the overlap of the helper genes in the PC with the genome of the vector being produced is between 1 to 100 base pair at the left (5') end and between 1 and 210bp, 300bp, 400bp or 500bp at the right (3¹) end.

6. An adenoviral packaging system of claim 4 where the overlap of the helper genes in the PC with the genome of the vector being produced is between 1 and 97 base pair at the left (5') end and between 1 and 210bp at the right {3') end.

7. An adenoviral vector made with any of the systems in claims 1 through 6.

8. A pharmaceutical preparation of an adenoviral vector prepared using any of the production systems in claims 1-6.

9. A PC used in any of the systems in claims 1 through 6.

10. A PC as in claim 9, where the PC is derived form A549 cells.

11. A c24 cell line.

12. An adenoviral packaging system comprising an adenoviral vector deleted in the E1A and E1B regions and a packaging cell line (PC) which provides complementary

E1 A and E1 B functions wherein said PC carries a helper gene or genes that encode the adenoviral E1B and E1B genes that maintain an overlap with the genome of said vector of between 1 and 350 base pair at the left (5') end and an overlap of between 1 to 600bp at the right (3') end.