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Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirusRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal Cell, The Polynucleotide Is Encapsidated Within A Virus Or Viral CoatCell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060003451, Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of U.S. patent application Ser. No. 10/053,194, filed Jan. 16, 2002, which is a continuation of U.S. patent application Ser. No. 09/659,203, filed Sep. 11, 2000, which claims the benefit under 35 USC 119(e) of U.S. Patent Application No. 60/156,644, filed Sep. 29, 1999. BACKGROUND OF THE INVENTION [0003] This invention relates generally to the field of constructs and methods for producing viral vectors, and more particularly, for the production of adenoviruses. [0004] Recombinant adenoviruses have been described as useful for delivery of transgenes to cells for a variety of purposes, including both therapeutic and prophylactic (vaccine) uses. However, successful commercialization of E1-deleted adenoviruses will require suitable manufacturing processes, which have yet to be developed. Infection of an E1 trans-complementing cell line with the vector and purification of the resulting lysate is a simple and scalable process that yields sufficient quantities of product. Unfortunately, production of E1-deleted adenovirus vectors for gene therapy has been plagued by emergence of replication competent adenovirus (RCA) caused by homologous recombination between the vector and transfected E1 gene. [0005] Several strategies have been described to avoid RCA. However, to date none of these approaches has resulted in an E1-complementing cell line which is stable and produces high yields of E1-defective adenoviruses in the absence of detectable RCA. J.-L. Imler et al., Gene Ther., 3:75-84 (1996) describes an A549 cell stably transfected with E1a and E1b open reading frames (ORFs) and contiguous pIX gene. [0006] The E1a was driven by phosphoglycerate kinase promoter and RCA was reportedly eliminated. However, more recent publications describing this system reveal that Imler was unable to detect E1b protein expression. See, Introgene, WO 97/00326, published Jan. 3, 1997. [0007] This Introgene application described an alternative system to that of Imler, cited above. This application describes cell lines derived from certain human diploid cells with E1a and E1b expressed, but no pIX (ECACC NO. 96022940). The cells were produced by transfection of human embryonic retinoblast (HER) cells with a vector containing nt 459-3510 of Ad, which corresponds to E1a, E1b, but excludes the E1a promoter, a portion of the E1b gene encoding the E1b 8.3 kb protein, and any pIX sequences. [0008] Another system for avoiding RCA is described in Massie, U.S. Pat. No. 5,891,690. The patent describes an Ad E1-complementing cell line having a stably integrated complementation element comprising a portion of the Ad E1 region covering the E1 a gene and the E1b gene, but lacking the 5' ITR, the packaging sequence, and the E1a promoter. Further, the E1a gene is under control of a first promoter element and the E2b gene is under control of a second promoter. A specific cell line described and claimed by Massie contains nt 532-3525 of Ad5, which includes E1a, the E1b promoter, and a portion of the E1b gene. This cell line does not contain the carboxy terminus of the E1b gene, which encodes the 8.3 kb product, nor does it contain pIX gene sequences. [0009] What is needed in the art is a stable E1-complementing cell line, which expresses all adenoviral E1a and E1b gene products, and which produces high yields of E1-defective adenoviruses in the absence of detectable RCA. SUMMARY OF THE INVENTION [0010] Advantageously, the present invention provides E1 expressing cell lines that are stable, can be adapted to suspension culture in serum free medium, and yields high quantity of vector. Significantly, the cell lines allow isolation and subculture of E1-deleted recombinant adenoviruses in an environment free of replication competent adenovirus (RCA). Further, the cell lines of the invention effectively plaque vector to allow isolation and subculture of new recombinants in an environment free of RCA. [0011] Thus, in one aspect, the invention provides an E1-complementing cell line useful for production of recombinant E1-defective adenoviruses in the absence of detectable replication-competent adenovirus. The E1-complementing cell line contains an aneuploid cell line stably transformed with a nucleic acid molecule comprising nucleic acid sequences encoding adenovirus E1a and adenovirus E1b under the control of a phosphoglycerate kinase (PGK) promoter. Suitably, the nucleic acid molecule lacks adenovirus sequences 5' to the sequences encoding adenovirus E1a. [0012] In another aspect, the invention provides a method for packaging of E1-defective adenoviral particles in the absence of replication competent adenovirus. The method involves introducing a vector into cells from the E1-complementing cell line of the invention, where the vector contains a defect in the adenovirus E1 region, adenovirus 5' and 3' cis-elements necessary for replication and packaging, adenovirus pIX, and regulatory sequences necessary for expression of the adenoviral genes and transgene. [0013] In another aspect, the invention provides a method of producing E1-defective adenoviral particles in the absence of detectable replication competent adenovirus. The method involves infecting cells from the E1-complementing cell line of the invention with an E1-defective adenovirus and culturing under conditions which permit the cell to express the E1a and E1b proteins. [0014] Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG. 1 is a schematic representation of the structures of the E1-deleted recombinant adenoviral vector, Ad5 DNA sequence in 293 cells and PGK Ad5E1 fragment in the new E1 cell line. [0016] FIG. 2A is a graph of the growth kinetics of an E1-deleted recombinant adenovirus, H5.CBLacZ, in 293 and new E1 cell lines. See Example 2C for details of the study. The yield of H5.CBLacZ virus in each cell line is shown on the y axis in a log scale. The time points are shown on the x axis. [0017] FIG. 2B is a bar chart showing the relative plaquing efficiency (RPE) for H5.CBLacZ virus on new E1 cell lines which were compared with 293 cells. See Example 2D for details of the study. RPEs were computed as the percentage of the titer of H5.CBLacZ virus. Solid bars, the mean RPE of each cell line from three different experiments; error bars represent standard deviations. DETAILED DESCRIPTION OF THE INVENTION [0018] The present invention provides a method of producing E1-deleted adenoviruses in the absence of detectable replication-competent adenovirus (RCA), as well as cell lines and vectors useful in this method. The resulting E1-deleted adenoviruses are particularly well suited for use in delivering genes to a mammal, because these adenoviruses are substantially free of contaminating RCA. [0019] In one desirable embodiment, the invention provides HeLa based cell lines that stably expresses the E1 locus from a promoter derived from the phosphoglycerate kinase (PGK) gene. These cell lines have no adenoviral sequences 5' to the E1 open reading frame (ORF) and reduced (or no) homology 3' to E1. These cell line supports plaquing and amplification of E1-deleted vectors at levels equal to or better than 293 cells without the emergence of RCA. [0020] One example of such a cell line is the GH329 cell line, which has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA on Sep. 29, 1999, and has been assigned accession number PTA-803. This deposit has been made pursuant to the provisions of the Budapest Treaty and in conformity with the requirements of 37 CFR .sctn..sctn. 1.801 et seq. The GH329 cells been found to complement (and allow the replication of) E1-deleted viral vectors in the absence of detectable replication competent adenoviruses (RCA) over at least 20 passages. Currently, GH329 is believed to express a single copy of each the E1a and E1b proteins. However, yields obtained using the GH329 cell line are at least equivalent to those obtained in 293 cells, in which RCA is observed following 5 to 10 passages. Continue reading about Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus... Full patent description for Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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