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Definitions

• This invention relates to novel cells and methods for use in propagating El- deleted adeno viruses.
• adenoviral vectors used in gene therapy applications were designed to have deletions in the El region of the adenovirus 5 (Ad5) genome.
• the El region, not including region IX, consists of 9% of the left end of Ad5 (1.2 - 9.8 map units), and is subdivided into two regions, E1A and E1B, each one coding for several proteins.
• E1A/E1B is required for virus replication and for expression of all other Ad5 proteins (E2-E4, Late Proteins; Ginsberg, H.S. The Adeno viruses. Plenum Press, New York. p.46-67(1984).
• El deficient adenoviral vectors are propagated in an Ad5 helper cell line called 293 (Graham, F.L. and Smiley, J, J. Gen. Virol. 36, p.54-72 (1977).
• 293 cells were derived by transforming human embryonic kidney cells with sheared fragments of Ad5 DNA. Genomic analysis revealed that 293 cells contain four to five copies per cell of the left 12% of the viral genome (including the entire El region) and approximately one copy per cell of 9% of the right end, the E4 region (Aiello,L.,et al, Virology 94, p.460-469 (1979).
• RCA in 293 cells can present severe ramifications for the safety of human gene therapy trials (Lochmuller, H., et al., Human Gene Therapy 5 . , p. 1485-1491 (1994).
• recombination in 293 cells can also cause deletions and rearrangements that affect transgene expression, thereby decreasing the titer of functional adenovirus particles.
• This invention encompasses a series of helper cell lines for the complementation, amplification, and controlled attenuation of El-deleted adenovirus. These cell lines are advantageous because they can complement adenovirus El gene deletions without production of replication competent adenovirus (RCA).
• a preferred embodiment is an A549E1 cell line that contains only the Ad5 El DNA sequences sufficient for complementation of El -deleted adenoviral vectors without sequences that overlap with the adenovirus vector.
• the El DNA sequences comprise El A and E1B genes.
• the present invention embodies methods for selectively propagating mini-adenovirus without generating RCA, by transfecting an A549E1 cell line with DNA sequences that encode a polypeptide sufficient for packaging attenuation of El -deleted helper virus.
• the polypeptide comprises Cre recombinase.
• the polypeptide comprises TetR-KRAB.
• Figure 1 is a diagram indicating the structure of adenovirus sequences in a typical El- deleted Ad helper virus (top line), in 293 cells (ref.5), and in other El -containing cell lines, including 911 cells (ref.8) and A549E1-68 cells (this invention); and how recombination between homologous adenovirus sequences occurs to generate a replication-competent adenovirus (RCA).
• Figure 2 is a diagram of the CMV-E1 mammalian expression vector.
• Figure 3 is a Southern blot analysis of G418 r A549E1 clones.
• Figure 4A is a Western blot analysis of El A protein expression in A549 cells, 293 Cells, and A549El-68.
• Figure 4B is the metabolic 35 S labeling and immunoprecipitation of EIB proteins in
• Figure 5 is a representation of the El -deleted adenovirus vector, Ad5-CA-GFP.
• Figure 6 is an agarose gel analysis of 40 PCR reactions using ElA-specific primers for detection of RCA.
• Figure 7 is a diagram of a system for the attenuation of helper virus with a loxP -modified packaging signal.
• Figure 8 is a diagram of the pCMV-Cre-Puro vector.
• Figure 9 is a diagram of the pBS/loxP-stop/MCLpA vector.
• Figure 10 is a bar graph depicting luciferase expression in both control cells (A549E1-
• This invention provides cell lines that can complement El -deleted adenovirus without the disadvantage of undesirable recombination and RCA. These cell lines are obtained by cloning and expressing in the A549 cell line only those sequences that are required for El complementation and excluding from the cell line all other Ad5 sequences that have homology to the vector and could cause recombination to produce RCA.
• FIG. 1 shows the structure of adenovirus sequences in 293 cells versus other El -containing cell lines (including A549E1 cells), and how recombination between homologous adenovirus sequences occurs to generate a replication-competent adenovirus (RCA).
• RCA replication-competent adenovirus
• A549E1-68 contains no sequence overlap with sequences present in the El -deleted Ad helper virus and thus, recombination to produce RCA is not possible (FIG. 1). Characterization of this A549E1 cell line demonstrated the production of El A and EIB proteins, high infectivity with adenovirus vectors, complementation of El- deleted adenovirus to produce high-titer virus stocks, as well as, the lack of production of replication-competent adenovirus (RCA).
• A549-E1 complementing cell lines which, in addition to producing El, also produce proteins required for further manipulation of adenoviral vectors, thus providing a novel series of RCA-free adenovirus helper cell lines, as tools for novel virus production.
• the first example is an A549E1 cell line which expresses the Cre recombinase.
• This invention provides a novel El -deleted helper virus whose packaging signal is flanked by loxP sites, and when this helper virus is propagated in an A549E1 cell line expressing Cre, the packaging signal is deleted by excision, thus attenuating helper virus packaging and enriching for packaging of mini-adenovirus (Ad5 virus which is devoid of all viral protein-coding sequences).
• This is advantageous because during the production of helper-dependent, mini-adenovirus it becomes necessary to attenuate helper virus packaging in order to enrich for the mini-virus.
• 293-Cre cells have been generated for this purpose (Parks, R. , et al, P.N.A.S. 93, p.13565-13570 (1996), however, A549E1- Cre cells have an advantage in that they would perform this task in an RCA-free environment.
• a further embodiment of the present invention includes an A549-E1 complementing cell line which expresses the TetR-KRAB fusion protein, which would be used to amplify, and control the packaging efficiency of an El -deleted helper virus whose packaging signal has been modified to contain multiple tetracycline operator (tetO) sites.
• tetO tetracycline operator
• Packaging of the helper virus can be restored by growing the cells and virus in the presence of tetracycline, which binds to the tet-KRAB repressor causing its dissociation from the tetO/packaging signal and a reversal of packaging repression.
• tetracycline which binds to the tet-KRAB repressor causing its dissociation from the tetO/packaging signal and a reversal of packaging repression.
• Example 1 Construction of the E1A/E1B vector To generate an expression vector which harbors only the E1A/E1B sequences required for complementation, a 3.1 kb DNA fragment coding for Ad5 El A and EIB genes was cloned in two pieces, sequentially, into the superlinker vector, pSL301 (Invitrogen). First, an 881 bp Afl III to Xbal fragment (Ad5 base pairs 462-1343) was cloned from pBRXad5KpnICl (a subclone of pJM17) into pSL301 (Afl III/XbaI).
• a contiguous 2194 bp Xbal to Afl II (Ad5 base pairs 1343-3537) was cloned from pBRXad5XhoIClinto the same vector.
• the resultant 3075 bp El fragment (in pSL301) contains the TATA box and RNA cap site for El A, El A coding sequence, complete EIB promoter, and EIB coding sequence, including the stop codon for EIB p55 protein, but not including region IX.
• This CMV-El expression plasmid (FIG. 2), was transfected using Lipofectamine (Gibco BRL) into A549 human lung carcinoma cells (ATCC CRL 185) and G418 R colonies were isolated. Single-cell clones were screened for functional E1A/E1B expression.
• An El -deleted adenovirus containing a green florescence protein (GFP) expression cassette, Ad5 CA-GFP was used to infect the A549-E1 clones. Three days post-infection, clones were screened for production of El -complemented Ad5 CA-GFP adenovirus by visual examination for cytopathic effect (CPE).
• CPE cytopathic effect
• A549E1-68 displayed 100% CPE in 3 days, similar to that observed for 293 cells.
• the clear area in the center of the plaque is evidence of CPE caused by El -complemented virus amplification.
• This clone also showed high infectivity, in that virtually 100% of the cells fluoresced green 24 hours post-infection.
• the high infection rate and rapid generation of CPE induced in this cell line is strong evidence that functional E1A/E1B proteins are being produced that are capable of promoting replication and amplification of the El- deleted Ad5-CA-GFP virus.
• the A459E1 cell was deposited at the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty on January 15, 1998 as ATCC Designation CRL-12458 (viability confirmed January 20, 1998).
• FIG. 3 shows a Southern blot using an El sequence-specific DNA probe. This assay demonstrated the presence of the CMV-El transgene in A549E1-68 (Lane 4), and a subclone of A549E1-68 (El-68.3), but not in the parental A549 cell line (Lane 2). Sequences hybridizing with the El -specific probe were also observed in 293 cells as expected since they complement El-deleted adenovirus (Lane 3). The morphology of the El-transfected cells was significantly different from the parental A549 cell line.
• A549 cells at sub-confluent density grow as distinct single cells with an elongated, fibroblast- like morphology, whereas the El cell line A549E1-68 grows as colonies of cells with a more cuboidal morphology.
• A549E1-68 was also compared with 293 cells for production of El -deleted adenovirus (Ad5 CA-GFP) by plaque assay and found to produce an equivalent titer of complemented virus (7 x 10 9 pfu for A549E1-68 vs. 9 x 10 9 pfu for 293).
• FIG. 4A shows a Western blot analysis using an EIA specific antibody (M73, Oncogene Science). This antibody detected two ElA-specific bands with apparent molecular weights of 46kd and 42kd in the A549E1-68 cell line (lane 3), corresponding to products expected from EIA 13S and 12S mRNAs (Ginsberg, 1984), and identical in size to those observed in 293 cells (lane 2). These El A-specific bands were not detected in parental A549 cells (lane 1).
• FIG. 4B shows the immunoprecipitation of metabolically-radiolabeled proteins by a monoclonal antibody specific for EIB p55.
• A549E1-68 produced an immunoreactive band of approximately 55 kd (lane 3) that was not detected in parental A549 cells. This 55 kd, ElB-specific band, as well as secondary background bands, were observed in 293 cells also (lane 2). Extra "background" bands found in both experimental and control lanes have been observed by other authors and are attributed to co-immunoprecipitation of a variety of proteins including, cyclins, p53, and Rb. It is clear that A549E1-68 not only expresses EIA and EIB, but that they are functional, since this cell line can complement for production of high titer, El -deleted, recombinant adenovirus.
• Example 3 El-deleted adenovirus produced in A549E1 cells is RCA-free
• Ad5 helper cell line can complement without production of RCA
• a series of PCR RCA assays were performed following amplification in A549E1 cells of the E 1 -deleted Ad5 -CA-GFP adenovirus vector.
• the Ad5-CA-GFP vector is illustrated in FIG. 5. It contains a transcriptional control element consisting of the CMV
• Ad5-CA-GFP virus was serially propagated through 20 passages on A549E1-68 cells. Following serial propagation and virus amplification, Ad5-CA-GFP virus DNA was isolated by freeze-thaw lysis, and PCR was performed using primers specific for either the EIA region or the E2B region. Amplification of an 880 bp E2B product serves as a PCR positive control, while the presence of a 1086 bp El A-specific product is evidence that an El (+) replication-competent adenovirus (RCA) has been produced during amplification of the El (-) Ad5 -CA-GFP.
• RCA replication-competent adenovirus
• Ad5-CA- GFP virus DNA (equivalent to 1 x 10 10 virus particles), obtained from amplification in A549E1 cells, was divided into 40 PCR reactions and tested for RCA using the EIA primers (FIG. 6). For both top and bottom panels of FIG.
• lane 1 contains 1 kb DNA markers
• lane 2 contains wild type Ad5 virus DNA
• lane 3 consists of PCR of Ad5-CA- GFP virus DNA (E1-) isolated from 20+ passages on A549E1-68 cells using EIA and E2B specific primers (positive control)
• lanes 4-20 consist of PCR of Ad5-CA-GFP virus DNA (E1-) isolated from 20+ passages on A549E1-68 cells, using ElA-specific primers only. No 1086 bp El region specific PCR fragments were detected in any of the reactions indicating that no RCA was present in the virus prep.
• a second, CPE-based RCA assay was performed by amplifying El -deleted adenovirus (Ad5-CA-GFP) on A549E1-68 cells and testing the amplified virus by passaging, on normal A549 cells (don't make El) for production of El -containing RCA. Plaque formation (CPE) on a monolayer of normal A549 cells would provide evidence for the production of wild-type (El +) virus during amplification on the El helper cell line, A549E1-68. 2 x 10 10 10 El (-) virus particles (amplified using A549E1-68) were used to infect each of five 150mm plates of normal A549 cells (1 x 10" particles total).
• a 2nd generation El -complementing cell line was generated using the A549E1- 68.3 clonal line for transfection with Cre recombinase.
• This cell line will both complement El -deleted adenovirus vectors and mediate the excision of sequences surrounded by loxP sites.
• Our primary use for this cell line is to further attenuate packaging of an Ad5 helper virus, whose packaging signal is flanked by two loxP sites (FIG. 7), in order to enrich for packaging of the desired El -deficient, mini-adenovirus vector.
• 293 cells expressing the Cre recombinase were generated for a similar purpose by Parks et al. (P.N.A.S.
• the A549E1-Cre cell line described in this invention will not only attenuate helper virus packaging in a similar fashion, it also has the advantage that any adenovirus produced will be free of deleterious RCA.
• a Cre expression vector was constructed as a first step towards the production of the A549E1-Cre cell line.
• a 1440 bp SV40 promo ter-puromycin cassette (for selection in Neo R A549E1 cells) was cloned into a unique EcoRI site of the CMV-Cre vector (pBS185, Gibco/BRL) to generate pCMV-Cre-Puro (FIG. 8).
• the pCMV-Cre- Puro vector was transfected by electroporation into A549E1-68 cells, and puromycin R (“puro R ”) clones were isolated. These puro R clones were then screened for expression of functional Cre recombinase.
• the plasmid pBS/loxP-stop/MCLpA contains a lacZ cassette that is non-functional due to the presence of a stop codon (FIG. 9). This stop codon is surrounded by loxP sites, such that the propagation of this vector in a cell line producing Cre would excise the stop signal and activate the lacZ gene.
• the pBS/loxP- stop/MCLpA vector was transiently transfected into each of the A549E1-Cre clones, and after 24 hours, the transfected cells were fixed and stained with X-Gal. LacZ expression of parental A549E1-68 cells (no Cre) was compared to lacZ expression in seven different puro r A549E1-Cre clones.
• lacZ due to expression of Cre was observed as blue cells, at a frequency ranging from 1% to 50% in 20/26 puro R clones. This range of LacZ-expressing cells is most likely a reflection of the transient transfection efficiency of the different puro R clones with the pBS/loxPstop-MCLpA vector, although it could also reflect variations in Cre recombinase expression in different cell lines.
• Western blot analysis using an anti-Cre antibody (Pharmingen) confirmed the presence of the 35 kd Cre protein in these cell lines.
• KRAB Kruppel-associated box
• the TetR-KRAB repressor binds to tetO sequences present in a transcriptional control region and represses transcription of genes placed as far as 3 kb downstream.
• the present invention describes a system for tetracycline-controlled inhibition of helper virus packaging, comprising multiple tetO sequence in the helper virus packaging signal sequence, and an El helper cell line that constitutively expresses the TetR-KRAB protein.
• the helper virus is still capable of replicating and providing all the necessary proteins, in trans, required for replication of the mini Ad vector, however, its packaging is attenuated due to binding of the TetR-KRAB protein to the tetO sites in the packaging signal.
• the overall goal is to hinder or repress helper virus packaging, thus enriching for vector virus packaging.
• This packaging repression is reversible, since in the presence of tetracycline, the TetR-KRAB repressor dissociates from the tetO sequences, and packaging is restored. Details of this tetO-controlled helper virus were presented in an earlier patent application (Serial No. 08/658,961, filed May 31, 1996).
• the TetR-KRAB expressing cell line was derived using the A549E1-68 helper cell line described in Example 2.
• A549E1-68 cells were transfected with a TetR-KRAB gene under control of the CMV promoter (see Deuschle et al., Mol. Cell. Biol. L5_ p. 1907-1914 (1995).
• the TetR-KRAB vector also contains a hygromycin resistance gene for selection in mammalian cells.
• a test vector see Deuschle, et al., Mol. and Cell. Biol. 15.
• Hygromycin-resistant A549E1 -TetR-KRAB clones were transfected with pTetO-CMV-L by electroporation and each clone was split into two wells of a 6-well plate. 24 hours post-transfection, cells from one duplicate well were refed with medium containing tetracycline, and the other duplicate well in medium without tetracycline. After another 24 hours, cells were lysed and assayed for luciferase expression using a Promega Luciferase Assay Kit.
• Two hygro R A549E1 clones (TKE-9 and TKE-12) demonstrated a 4 to 6 fold repression of luciferase reporter activity when grown in the absence of tetracycline versus cells grown in media containing Tet, indicating expression of the TetR-KRAB repressor protein in the cells (FIG. 10).
• These A549E1 -TetR-KRAB cell lines will be used to test attenuation of the TetO-controlled Ad helper virus.

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