Adenovirus E1-complementing cell lines

The present invention relates to adenovirus (Ad) E1-complementing cell lines which significantly reduce the presence of replication competent Ad (RCA) and can serve for the large scale production of infectious E1-deleted adenoviral particles that may be used for the treatment human patients as for example in gene therapy. As well the invention relates to a method for the large scale production of recombinant infectious adenoviral particles harboring an exogenous sequence of interest and to a RCA-free stock of infectious adenoviral particles. The invention further relates to a recombinant vector for transfecting an eukaryotic cell line in order to construct Ad E1-complementing cell lines which significantly reduce the presence of RCA and to a method therefor.

FIELD OF THE INVENTION 
The present invention relates to the introduction of exogenous genetic 
sequences into cells, cell lines or organisms, and to gene transfer and 
gene therapy. The invention further relates to defective adenoviral 
vectors and E1-complementing cell lines. More specifically the present 
invention relates to adenovirus (Ad) E1-complementing cell lines which 
significantly reduce the presence of replication competent Ad (RCA) and 
can serve for the large scale production of E1-deleted Ad vectors that may 
be used for the treatment of human patients. As well the invention relates 
to a method for the large scale production of recombinant Ad harboring an 
exogenous sequence of interest. 
BACKGROUND OF THE INVENTION 
Therapeutic strategies in various discases include: nonspecific measures to 
mitigate or eliminate a cell dysfunction and prevent cell death; 
replacement of a missing or malfunctioning protein; introduction of 
functional nucleic acids (RNA or DNA) into cells to replace a mutated gene 
and introduction of novel genetic constructs to alter a cellular function. 
Advances in DNA technology have had a major impact on each of those 
therapeutic possibilities and nucleic acid transfer into diseased cells 
appears by far the most promising modality (Mulligan 1993, Science 
260:926-932). 
Viral vectors permit the expression of exogenous genes in eukaryotic cells, 
and thereby enable the production of proteins which require 
post-translational modifications unique to animal cells. 
The wealth of information accumulated on adenoviruses over the last 
decades, has promoted them at the forefront of the gene therapy or 
immunization fields. Several features of adonoviruses make them attractive 
as gene transfer tools: (1) the structure of the adenoviral genome is well 
characterized; (2) large portions of viral DNA can be substituted by 
foreign sequences; (3) the recombinant variants are relatively stable, (4) 
the recombinant virus can be grown at high titer; (5) no human malignancy 
is associated with adenovirus; and (6) the use of attenuated wild-type 
adenovirus as a vaccine is safe. 
Ad ate thus considered as very good vector candidates for in vivo gene 
transfer. Generally, such vectors are constructed by inserting the gene of 
interest in place of essential viral sequences such as E1 sequences 
(Berkner 1988 BioTechniques 6:616-629; Graham et al., 1991, Methods in 
Molecular Biology, 7:109-128, Ed: Murcy, The Human Press Inc.). This 
insertion results in an inactivation of the Ad since it can no longer 
replicate, hence the term replication-defective Ad. In order to propagates 
such vectors must be provided with the deleted element, (i.e. E1 
proteins). 
The elucidation of the nucleotide sequence of many Ad subtypes has enabled 
a precise characterization of the genomic organization thereof. The 
nucleotide sequence of human Ad5 is available from GenBank under accession 
number M73260. In simplistic terms adenoviruses comprise: (1) two inverted 
terminal repeats (ITR) at each end (5' and 3') which are essential for 
viral replication; (2) the early region 1 (E1) containing the E1A and E1B 
regions, both indispensible for replication, E1A and E1B are also required 
for complete transformation of various rodent cell lines, and polypeptide 
IX (pIX) which is essential For packaging of full-length viral DNA; and 
(3) the E2, E3 and E4 regions, with E3 being dispensable for replication 
(reviewed in Acsadi et al., 1995, J. Mol. Med. 73:165-180). 
Recently, human Ad serotypes 2 and 5 have been used as vectors for 
efficient introduction of genes into several cell types both in vitro and 
in vivo (reviewed in Trapnell et al., 1994, Current Opinion Biotech. 5: 
617-625; and Acsadi et al., 1995, J. Mol. Med. 73:165-180). Several 
factors need to be taken into consideration during the generation of Ad 
recombinants, among them is the impaired growth characteristics of some of 
them ( Imler et al., 1995 Gene Ther. 2:263-268; Massie et al., 1995 
Bio/Technol. 13:602-608; and Schaack et al., 1995 J. Virol. 69: 3920-3923) 
which complicate the screening, propagation and production of high quality 
recombinant viral stocks with high titers (more than 10.sup.11 pfu/ml). 
Recently, critical issues relating to the characterization of such Ad 
vectors for gene therapy were reviewed in relation to clinical trials of 
the cystic fibrosis gene therapy (Engelhardt et al., 1993 Nature Genetics 
4:27-34; Zabner et al., 1993 Cell 75:207-216; Boucher et al., 1994 Human 
Gene Ther. 5:615-639; Mittereder et al., 1994 Human gene Ther. 5:717-729; 
and Wilmot et al., 1996 Human Gene Ther. 7:301-318). Presently, a number 
of human clinical trials making use of Ad recombinants for the treatment 
of diseases like cystic fibrosis, Duchenne muscular dystrophy, and cancer, 
have started or are being considered (Lochmuller et al., 1994 Hum. Gene 
Ther. 5: 1485-1491). Potential sites for the insertion of a gene of 
interest in the recombinant Ad vectors comprise the E1 or E3 regions (i.e. 
E1+E3-deleted Ad recombinants) or the region between the end of the E4 and 
the beginning of the 3' ITR sequences. The majority of in vivo gene 
transfer experiments and human trials have been carried out using E1- and 
E3-deleted human type 2 or 5 adenoviruses. As alluded to above, E3-deleted 
recombinants are replication competent E1-deleted recombinants however, 
are unable to replicate and the missing E1 gene products are provide in 
trans by the E1-complementing cell line 293 (Lochmuller et al., 1994 Hum. 
Gene Ther. 5: 1485-1491). The 293 cells were established by stable 
transfection of a human embryonic kidney cell with adenoviral (human type 
5) DNA containing the full length E1 region. The maximum deletion of up to 
2.9 kb in the E1 region leaves intact the ITR sequence, the packaging 
signal at the left and of the adenoviral DNA (188-358 bp) and the pIX 
coding region (starting at 3507 bp). A useful E3 deletion was made by 
deletion of a 1.9 kb Xba I fragment (79 and 85 mu). These combined E1 and 
E3 deletions allowed for inserting approximately 7 kb of foreign DNA 
sequences in this first-generation recombinant. Extensions of the deletion 
in the E3 regions further increased the insert capacity to 8 kb, which 
meets the size requirements for most of the gene therapeutics (Bett et 
al., 1994 Proc. Natl. Acad. Sci. 91:8802-8806). 
It is important to note that the recombinant Ad produced for clinical use 
have all been obtained using 293 cells (Graham et al., 1977, J. Gen. 
Virol. 36:59-72). Until the present invention, 293 cell line was the only 
available complementation cell line which efficiently expressed E1A and 
E1B RNAs and proteins. Unfortunately, it hats been documented that 
replication competent, also termed "revertant" virus can appear during 
multiple passages of the E1- and E3-deleted recombinant Ad in 293 cells, 
and eventually outgrow the original recombinant in large scale 
preparations (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). The 
E1 region is acquired from the 293 cells (and its derivatives) by 
homologous recombination at a very low frequency, but the E1-positive 
revertants seem to have a growth advantage with respect to their 
E1-negative counterparts. The presence of these revertants could thus 
jeopardize the safely of human gene therapy trials, especially when one 
considers the number of infectious viral particles required in certain 
applications. Experiments performed with mouse muscle have taught the use 
of of 2.times.10.sup.9 virus particulars to transduce more than 80% of the 
muscle fibers, since a human muscle is 2500 times larger, that would 
translate in the use of approximately 10.sup.12 -10.sup.13 viral 
particulars to inject only one human muscle. Supposing the presence of as 
little as 1/10 particules of E1+ revertants, in the stock, 10.sup.3 
-10.sup.4 replication-competent particules would be injected in the 
muscle. It is clear that such an approach would fail to satisfy regulatory 
agencies. 
Indeed, the 293 cells have been deemed "not suitable for large scale 
production of clinical grade material since batches are frequently 
contaminated with unacceptably high levels of replication competent 
adenovirus (RCA) arising through recombination" (Imler et al., 1996 Gene 
Ther. 3: 75-84). It should be stressed that the same authors have reported 
that numerous attempts to construct stable and efficient E1-complementing 
cell lines have failed and is therefore not a trivial task. In an attempt 
to solve this problem of RCA generation (Imler et al., 1996 Gene Ther. 3: 
75-84) produced an E1-complementing cell line by stably transforming human 
lung A549 cells with E1 sequences containing the E1A, E1B and pIX regions. 
Novel A549 E1-complimenting cell lines were obtained which express high 
levels of E1 RNA and proteins. Strikingly however, the authors were unable 
to detect E1B protein expression in any of the A549 clones analyzed 
whether or not they produce high level of E1B RNA. Thus, the presence of a 
functional genetic unit does not necessarily predict that upon stable 
integration in the host, it will give rise to the expected proteins. It is 
also reported therein that the A549 clones, testing positive for infection 
with E1-deleted Ad vectors, showed a transformed phenotype and that the 
amplification yields therewith arc significantly lower than those obtained 
with 293 cells. Unfortunately, the generation of RCA with these A549 cells 
was not assessed. It should be noted that in the Imler et al., constructs, 
a significant overlap between the complementing element and the defective 
adenoviral vector occurs at the 3' end of the E1 region (approximately 700 
bp). It follows that this overlap significantly increases the 
probabilities of homologous recombination and hence of the production of 
E1+ revertants. A disclosure of defective Ad vectors for the expression of 
exogenous nucleotide sequences in a host cell or organism, as well as 
vectors for the construction of E1-complementing cell lines, along the 
same lines is also found in the French publication to Imler et al., 
WO94/28152. However, this document fails to give an assessment of the 
yield of production of recombinant Ad by the complementation cell line, of 
the expression of the different adenoviral transcripts and proteins by the 
complementing cell line, and very importantly of the presence or absence 
of RCA during the production process leading to the obtention of the stock 
of defective Ad harboring the exogenous sequence of interest. It should be 
noted that WO94/28152 claims to diminish the problem of RCA production by 
deleting the 5' ITR (a non-substantiated declaration). 
There still remains a need for the description of an E1-complementing cell 
line which combines at least one of the following properties: it expresses 
E1A and E1B proteins; it minimizes or abrogates the production of E1+ 
revertants; it is substantially as efficient as 293 or its derivatives in 
producing recombinant Ad; and it does not show a transformed and rounded 
phenotype. It would thus be advantageous to be provided with such 
E1-complementing cell lines which are efficient for the large scale 
production of E1-deleted Ad vectors devoid of RCA. 
All of the above-cited citations are herein incorporated by reference. 
SUMMARY OF THE INVENTION 
An object of one aspect of the present invention is therefore to provide an 
E1-complementing cell line which satisfies at least one of the following 
properties: (1) it expresses functional E1A and E1B proteins; (2) it 
minimizes the production of E1+ revertants; (3) it is substantially as 
efficient as 293 or its derivatives in producing infectious recombinant 
Ad; and (4) it does not show a transformed and rounded phenotype. 
Another object of one aspect of the present invention is to provide a 
method for the large scale production of E1-defective recombinant Ad which 
minimizes the production of RCA. 
An additional object of one aspect of the present invention is to provide a 
recombinant adenovirus construct for the establishment of an 
E1-complementing cell line in accordance with the present invention. 
Yet another object or one aspect of the present invention is to provide a 
therapeutic use of an E1-complimenting cell line in accordance with the 
present invention. 
A further object of one aspect of the present invention is to provide a 
method of treatment by which a therapeutically or prophylactically 
efficacious quantity of a recombinant Ad, produced in an 
E1-complementation cell line in accordance with the present invention, or 
an E1-complementation cell line in accordance with the present invention 
harboring a recombinant Ad is administered to a patient in need of such a 
treatment or prophylaxy. 
More specifically, in accordance with the present invention, there is 
provided an adenovirus (Ad) E1-complementing cell line having a stably 
integrated complementation element comprising a portion of the Ad E1region 
covering the E1A gene and the E1B gene but lacking the 5' inverted 
terminal repeat (ITR), the packaging sequence, and the E1A promoter; the 
E1A gene being under control of a first promoter element and the E1B gene 
being under control of a second promoter element, the stably integrated 
complementation element giving rise to functional E1A and functional E1B 
proteins, whereby the stably integrated complementation element 
complements in trans a defective adenoviral vector and does not generate 
replication competent adenovirus (RCA) produced by homologous 
recombination between said defective adenoviral vector and said 
complementing element at a detectable level. 
In accordance with the invention, there is also provided a method for large 
scale production of infectious E1-defective adenoviral particles 
comprising: a) transfecting an E1-defective adenoviral vector into an 
E1-complementing cell line to obtain plaques; b) screening the plaques to 
identify plaques positive for E1-defective adenovirus (Ad); c) submitting 
the E1-defective Ad of b) to at least two rounds of plaque purification by 
infection into an E1-complementing cell line to obtain substantially pure 
infectious E1-detective adenoviral particles, and d) scaling up production 
of the substantially pure infectious E1-detective particles of c) by 
infecting an E1-complementing cell line and growing the cell line to 
obtain a concentrated stock of infectious E1-defective adenoviral 
particles, wherein in at least one of steps a), b), c), and d), the 
E1-complementing cell line of claim 1 is used, thereby minimizing the 
production of RCA in the concentrated stock of E1-defective Ad infectious 
particles obtained in d). In addition there is provided a RCA-free stock 
of defective adenoviral vector produced in accordance with the 
above-recited method. 
In accordance with the present invention, there is also provided a 
recombinant vector for constructing an Ad E1-complementing cell line in 
accordance with the present invention and a method of producing an Ad 
E1-complementing cell line comprising: a) transfecting a euaryotic cell 
line with a recombinant vector according to the present invention; b) 
selecting a cell having stably integrated the complementation element; and 
c) selecting the cell of b) expressing functional E1A and E1B protein 
selecting the cell of b) expressing functional E1A and E1B proteins and 
complementing an E1-defective adenoviral vector so as to yield of 
infectious E1-defective adenovirus particles while avoiding the generation 
of a detectable level of RCA.

Other objects, advantages and features of the present invention will become 
more apparent upon reading of the following non restrictive description of 
preferred embodiments with reference to the accompanying drawings which 
are exemplary and should not be interpreted as limiting the scope of the 
present invention. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to E1-complementing cell lines which can 
complement E1-defective adenoviral vectors while significantly reducing 
the presence of E1+ revert ants which have become replication competent 
(RCA) through recombination with the adenoviral sequences present in the 
E1-complementing cell line. The minimizations if not the total abrogation 
of RCA, is crucial if the adenoviral vector harboring an exogenous genetic 
sequence is to be used in human therapies such as gene therapy. It should 
be understood that PCR analysis is not sensitive enough to assess the 
level of purity of an infectious viral stock warranted by the regulatory 
agencies. The detection of RCAs is thus generally based on tedious plaque 
assays. The limit of detection of RCA currently available to the skilled 
artisan is approximately between one RCA per 10.sup.7 to 10.sup.8 (perhaps 
10.sup.9) infectious particles. Preferably, the number of RCA in the final 
adenoviral infectious stock should be inferior or equal to approximately 
one RCA per 10.sup.9 infectious particle and especially preferably 
inferior or equal to approximately one RCA per 10.sup.10 infectious 
particles. 
According to the present invention, when relating to adenoviral vectors, 
adenoviral sequences, it should be understood that they can be derived 
from a natural or wild type adenovirus or preferably from a canine, avian 
or human adenovirus, more preferably a human adenovirus of type 2, 3, 4, 5 
or 7 and especially preferably a human adenovirus type 5 (Ad5). In the 
preferred embodiments described herein, Ad5 was used and the nucleotide 
positions referred to are taken from the nuclcotide positions 532-3525 as 
described in GenBank under the reference N.degree. M73260. 
It should also be understood that numerous E1-defective adenoviral vectors 
are encompassed by the scope of the present invention. One of the crux of 
the invention lying in a minimization of the formation of RCA, a 
particular E1-defective adenoviral vector should be chosen so as to 
minimize the presence of homologous sequences between the defective 
adenoviral vector and the complementing element stably integrated into the 
genome of the E1-complementing cell line chosen. It will be understood 
that the complementing cell line should provide the essential elements 
lacking in the Ad-defective vector used. Defective adenoviral vectors 
contemplated within the scope of the present invention include without 
being limited thereto, vectors which comprise in the 5' to 3' direction, 
the 5' ITR, the packaging sequence, the E2 region, the E4 region the 3' 
ITR as well as the Major Late transcript region. Since the E3 region is 
dispensable for replication, that region can be deleted from the Ad vector 
thereby permitting the insertion of a larger exogenous genetic sequence 
therein. In a preferred embodiment, the defective adenoviral vector used 
is Ad5 .DELTA.E1 .DELTA.E3. It is also contemplated that a defective 
adenoviral vector having additional deletions in essential regions (such 
as E2 and/or E4) can also be used with the cell lines of the present 
invention, provided that all defective elements thereof are complemented. 
For example E2 and/or E4 could be supplied in the E1-complementing cell 
lines of the invention by a cotransfection of the defective Ad with a 
vector providing the lacking essential element(s), thereby complementing 
all the replication defects of the chosen defective adenoviral vector. The 
term viral "particle" is well known in the art. 
The terms "deletion or deleted" should be understood to mean the removal of 
at least one nueleotide from the targeted region. As well, this deletion 
can be continued or discontinued. Deletions which remove large portions of 
the targeted regions are preferred over small deletions, since they 
diminish the possibility of homologous recombination between the defective 
adenoviral vector and the complementation element stably integrated in the 
chromosome of the complementing cell line. The deletion can be a partial 
or total deletion of the targeted region. 
The defective adenoviral vectors to be used according to the present 
invention arc incapable of replication but gain the capability of 
replication and encapsidation in the complementing cell line which 
supplies the defective products in trans. This generates an adenoviral 
particle which is still defective, since it is incapable of replicating in 
an autonomous fashion in a cellular host but is nevertheless infectious 
since it can deliver the vector to the host cell it inflects. 
The term "exogenous nucleotide sequence" is meant to cover nucleic acids 
such as coding sequences and regulating sequences which are generally not 
present in the genome of adenoviral viruses. It is also to be understood 
that the exogenous nucleotide sequences should have necessary information 
to be expressed inside the host cell towards which the defective 
adenoviral vector is ultimately targeted. It is to be understood that the 
exogenous sequence can also be expressed in the complementing cell line. 
The exogenous sequences are introduced in the adenoviral vector by the 
classical techniques of genetic engineering between the packaging sequence 
in 5' and the 3' ITR. The exogenous nucleotide sequence can contain one or 
many genetic sequences of interest and preferably the gene(s) of interest 
have therapeutic or prophylactic potential. Such a gene of interest can 
code for an antisense RNA or a mRNA which can be translated into a protein 
of interest. The gene of interest can be a genomic copy, a cDNA or a 
chimera of both. It can code for a mature protein, a precursor thereof, a 
protein chimera, a fusion protein, mutant or modified versions of all such 
proteins. The mutant protein can be obtained by way of mutation, deletion, 
substitution and/or addition of the nucleotide sequence encoding the 
initial protein. The exogenous sequence can be natural, genetically 
engineered, synthesized chemically or combinations thereof. 
The gene of interest can be placed under the control of appropriate control 
elements ensuring the expression thereof in the host cell. The appropriate 
control elements comprise transcription elements, generally known as 
promoter elements and enhancer elements (promoter/enhancer elements), and 
the translation elements. Herein, the terms promoter element and 
promoter/enhancer elements are used interchangeably in the broad sense as 
control elements. The promoter/enhancer element controlling the 
transcription can be either a constitutive or a regulatable or inducible 
promoter of eukaryotic or viral origin. It can also be the normal control 
element for the gene of interest Eukaryotic promoter/enhancer elements are 
well known to the skilled artisan and can be readily inserted by standard 
genetic engineering practices in front of the gene of interest or modified 
to suit the proper need thereof. The promoter/enhancer element can also be 
tissue specific. The promoter/enhancer element include but are not limited 
to the SV40 early promoter region, the RSV promoter (in the 3' LTR), the 
TK HSV promoter, the regulatory sequence of the metallothionine gene, tile 
insuline control region which is active in pancreatic B cells, 
immunoglobulin gene control region which is active in lymphoid cells, 
mouse mammary tumor virus control region which is active in testicular, 
breast, lymphoid and mast cells, and human beta actin promoter. Preferably 
the promoter/enhancer element controlling E1A is a strong promoter. 
Genes of interest which are encompassed by the scope of the present 
invention are in essence illimited since a skilled artisan can adapt by 
conventional method the teachings of the present invention to the 
expression of his own favorite gene of interest. It should be understood 
that a limiting factor is the packaging limit of the defective adenoviral 
vector. In any event, without being limited thereto gene of interest 
includes: growth factors, receptors, for such growth factors or for other 
molecules as well as for pathogens, suicide genes, factors involved in 
blood coagulation, dystrophin, insulin, genes involved in cellular 
transport such as the cyctic fibrosis transmembrane conductance regulator, 
or the natural resistance associated macrophage protein gene, genes coding 
for antisense or inhibitors of pathogenic organisms, inhibitors of 
defective metabolic processes or inhibitors of pathogens, cancer 
suppressor genes, genes expressing transdominant proteins, genes encoding 
antigenic epitopes or variable regions from specific antibodies and 
imnunomodulator genes. 
It should be understood that the adenoviral vectors of the present 
invention need not contain only genes or nucleotide sequences having 
therapeutic or prophylactic potential. Nevertheless the non-limitating 
applications of the present invention to gene therapy include a targetting 
of the following tissues: lung, muscles, liver, kidney, spleen, the 
nervous system and macrophage. Non-limitative examples of diseases for 
gene therapy include cystic fibrosis, hemophilia and cancer. 
The host cell which is to be chosen to eventually become an 
E1-complementation cell line in accordance with the present invention can 
be chosen among a variety of eukaryotic host cells by a skilled artisan. 
Advantageously, it will be a mammal cell line or preferably a human cell 
line. 
The complenentatioLi cell line according to the present invention can be 
derived from an immortalized cell line or a primary cell line. In 
accordance with the present invention, one of the crux of which is to 
minimize the extent of homologous region between the complementation 
element and the adenoviral sequences in the defective adenoviral vector, 
the Eukaryotic E1-complementing cell line according to the invention or 
their derivatives should minimize the formation of E1+ revert ants or RCA. 
RCA refers to replication competent adenoviruses which are no longer 
defective for replication and packaging and can therefore infect cells and 
lead to toxicity and deleterious immunological reactions. Preferably, the 
E1-complementing cell lines according to the present invention do not 
yield detectable RCA by PCR analysis and/or plaque assay on 
non-complementing cell lines such as A549. More preferably, the 
E1-complementing cell lines according to the present invention yield a 
number of RCA per number of infectious viral particles which is 
insufficient to pose a hazard to a patient when a therapeutic or 
prophylactic dose of adenoviral infectious particles is administered 
thereto. Especially preferably, the E1-complementing cell lines of the 
present invention give rise to no RCA. 
The cell line to be chosen to become the E1-complementing cell line of the 
invention should be a pharmaceutically acceptable cell line. The term 
pharmaceutically acceptable cell line is meant to refer to the fact that 
this cell line has been characterized (in terms of history and origin) 
and/or has been used for the production of products destined for human use 
(production of material for clinical assays or material destined for 
sale). Such cell lines are available in depositories such as the ATCC. 
Without being limited thereto, such cell lines include human carcinoma 
cells A549, human pulmonary cell line MRC5, human pulmonary cell W138, KB 
cells, Hela cells and 143 cells. Most preferably, the chosen cell line is 
the A549 cell line. The A549 and 293 cell lines for example, grow in 
monolayer. Other cell lines are able to grow in suspension which permits 
an easier scaling up of production since much larger volumes of cells can 
be grown. The derivative of the 293 E1-complementing cell line, 293S, 
possesses this advantageous property of growing in suspension instead of 
on a solid support. 
The method used to stably integrate the complementation element can be 
performed by standard genetic engineering procedures (Graham et al., 1991, 
Methods in Molecular Biology, 7: 109-128, Ed.: Murey, The Human Press 
Inc.) and as described by the present invention. The "complementation 
element" as used herein refers to a nucleic acid element which can in 
trans, complement tie replication defect of the defective adenoviral 
vector used. The E1-complementation cell line is thus capable of producing 
the protein(s) which is necessary for the replication and packaging of the 
E1-defective adenoviral vector. It should be understood that the 
complementation element could be mutated by deletion and/or addition of 
nucleotides, as long as these modifications do not alter the 
complementation capacity thereof. 
In accordance with the present invention, the complementing element, is 
expressible, and gives rise to tie E1A and E1B mRNAs as well as to the 
functional E1A proteins (289aa and 243aa) and E1B proteins (19 KDa and 55 
KDa). In accordance with a preferred embodiment of the present invention, 
the E1A promoter enhancer element has been replaced by the human 
beta-actin promoter, while the E1B enhancer promoter element is the 
natural E1B promoter. Of course, these elements can be substituted by 
other types of enhancer promoter elements which are well known to the 
skilled artisan. In addition, they could be mutated or modified so as to 
adapt the expression of E1A and E1B to a particular situation. 
Advantageously, the complementing element comprises a transcription 
termination signal and a polyadenylation signal, and preferably those of 
SV40. In a preferred embodiment of the present invention, the 
complementation element comprises the nucleotide sequence between 
nucleotides 532 to 3525 of human Ad type 5 as disclosed in Genbank under 
reference M73260. 
The vector of the present invention enabling the construction of the 
E1-complementing cell line, should comprise the complementing element with 
the necessary control elements, as well as a selection marker permitting 
an assessment of the stable integration of the complementation element 
into the genome of the host cell. Such selection markers include but are 
not limited to neomycin resistance (G418), hygromycin resistance, 
phleomycin resistance and puromycin resistance. In another embodiment, the 
selectable markers could be supplied by a co-transfected vector. It is 
also possible to synthesize by way of PCR or chemically the nucleotide 
sequences required to construct an E1-complementing cell line in 
accordance with the invention. Since it is a preferred embodiment to 
transfect a cell line with a linear fragment comprising the 
E1-complementing element, the E1-complementing element (or cassette) need 
not necessarily be on a vector. Such a cassette could be for example 
co-transfected with a vector providing a eukaryotic selectable marker 
which enables stable integration of the complementing cassette in the 
genome of the transfected cell. This cassette preferably comprises non-E1 
region nucleic acid sequences favoring the integration of a functional 
E1-complementing element. 
In a preferred embodiment of the present invention, the selectable marker 
is SV2-neo. Preferably, the vector used to construct the E1-complementing 
cell line will also contain a selectable marker and an origin of 
replication enabling replication and selection in a microorganism. 
Selectable markers and origins of replication for microorganisms such as 
bacteria and lower cukaryotes are well known in the art. The former 
include without being limited thereto antibiotic resistance, auxotrophic 
markers and the killer gene system. Non-limitative examples of origins of 
replication include the standard ColE1 type for bacteria and the 2.mu. for 
yeast. 
It is also within the scope of the invention to use the complementing cell 
line harboring a defective adenoviral vector which comprises an exogenous 
sequence of interest, directly by implantation into a patient. For such an 
embodiment an E1-complementing cell line could be derived from cells taken 
from the patient, transfected with the defective adenoviral vector 
containing the exogenous sequence of interest and implanted back into the 
patient. Thus, the present invention also encompasses a therapeutic or 
prophylactic use of a vector containing the complementing element, for 
deriving an E1-complementing cell line, and the E1-complementing cell 
lines themselves. In addition, E1-complementing cell lines of the present 
invention can be used in a method for the preparation of the infectious 
adenoviral particles which can then be administered to a patient. The 
administration of such infectious particles for a therapy or prophylaxy, 
are known to those skilled in the art, since such technologies have been 
used in a clinical trial for the treatment of cyclic fibrosis for example. 
The method of preparation of infectious adenoviral particles according to 
the present invention, is also based on one of the crux of the invention, 
the minimization of formation or RCA particles. It will thus become 
apparent, that the cell lines of the present invention offer a significant 
advantage over the available complementing cell lines which give rise to a 
significant number of RCA. Since RCA can outgrow the E1-defective 
adenoviruses, appearance of RCA early in the course of production of the 
infectious adenoviral particles could negate the using of a stock of these 
infectious particles for therapy or prophylaxy. It is of course known that 
in human gone therapy trial, safety issues are of paramount importance. 
One of the key requirements is the stable purity of the therapeutic Ad 
recombinant stocks. Thus, the use of E1-complementing cell lines according 
to the present invention during the course of production of the viral 
stock, can be of critical importance for the obtention of a stock of 
infectious particles which can be administered to patients. It will be 
understood that the protocol for the production of these infectious 
particles can be adapted in a variety of ways, by using for example only 
E1-complementing cell lines according to the present invention, or using 
other available E1-complementing cell lines in different phases of the 
scaling up procedure as long as the number of passages in a complementing 
cell line which gives rise to RCA is minimized. 
The therapeutic maid prophylactic uses which are envisioned as falling 
within the scope of the present invention are related to the type of 
exogenous sequence which is inserted into the defective adenoviral vectors 
described above. Pharmaceutical compositions in accordance with the 
present invention can be manufactured by conventional method. In 
particular, a therapeutically efficacious quantity of a defective 
adenoviral particle produced in accordance with the present invention or 
of an E1-complementing cell line harboring such an adenoviral particle 
will be mixed with a suitable support or carrier. Compositions encompassed 
by the present invention can be administered by way of aerosol or any 
other conventional fashion known in the art, in particular by oral, 
sub-cutaneous, intramuscular, intravenous, intra-peritoneal, 
intra-pulmonary or intra-tracheal routes. The administration can be in 
unit dose or repetitive doses with varying intervals in between doses. The 
administration of the appropriate dose will vary in accordance with 
different parameters including the individual to be treated, the disease, 
the type of exogenous sequence harbored by the adenoviral particle and the 
type of exogenous sequence harbored by the defective adenoviral particle. 
As a general rule, the health practitioner will adapt the dosage in 
accordance with those and other parameters. 
The emergence of replication-competent E1+ revert ants in stocks of 
replication-defective Ad recombinants (.DELTA.E1+.DELTA.E3) which has been 
demonstrated (Lochmuller et al., 1994 Hum. Gene Ther. 5:1485-1491), is 
most likely due to a recombinational event, which occurs at very low 
frequency, between the complementing element and the defective Ad. 
Although the population of replication-competent Ad is found at very low 
level in early passages, this population dramatically increases during the 
cycles of amplification required to produce large Ad stocks for gene 
therapy experiments (Lochmuller et al., 1994 Hum. Gene Ther. 5: 
1485-1491). 
The present invention aims at solving this E1+ revertant problem. 
MATERIAL AND METHODS 
Cells and viruses 
293 E1-transformed human embryonic kidney cells (Graham et al., 1991, 
Methods in Molecular Biology, 7: 109-128, Ed: Murey, The Human Press 
Inc.), A549, and Hela S3 cells were purchased from ATCC and grown at 
37.degree. C. in Dulbecco's modified Eagle's medium (Gibco) supplemented 
with 10% fetal bovine serum (Hyclone) and 2 mM glutamine (Gibco). 293 and 
BMAdE1 clones were infected with AdCMVlacZ (Acsadi et al., 1994 Hum. Mol. 
Gen. 3:579-584) and AdGFP (a recombinant adenovirus expressing green 
fluorescent protein) at a MOI of 5-10. 
Protein analysis 
Cells were harvested, washed in PBS and lysed in Laemmli buffer (10% 
glycerol, 80 mM tris pH 6.8, 2% SDS). Protein concentration was determined 
by a Lowry's modified method using the De kit.TM. (Bio-Rad). Proteins were 
their separated by SDS-PAGE on a NOVEX.TM. 10 or 12% precast gel (Helixx). 
Western blot hybridization 
Proteins were transferred to a Hybond.TM.-C nitrocellulose membrane 
(Amersham) in a Bio-Rad apparatus. Membranes were blocked overnight using 
5% milk in TBS, and hybridized with the appropriate antibodies. Washes 
were carried out with 0.1% Tween-20.TM. in TBS. Revelation was made by 
chemiluminescence using the ECL.TM. kit (Amersham). 
In vitro plaque assay with 293 and BMAdE1 cells 
Different dilutions of virus (AdCMVIacZ or AdGFP) were plated on 
5.times.10.sup.5 cells in a 60-mm dish and overlayed with 1% 
sea-plaque.TM. agarose (FMC). Plaques were observed between 7 to 14 days 
after infection, overlayed with Bluo-gal.TM. (1% sea-plaque.TM. agarose, 
0.3% NP40.TM., and 0.2% Bluo-gal.TM. from Gibco) if needed, and counted. 
The plaques were observed using an inverted fluorescence microscope for 
the AdGFP infections. 
Indirect plaque assay by beta-gal expression in Hela S3 cells 
Hela S3 were infected with virus stocks obtained by infecting BMAdE1and 293 
cells with AdCMVlacZ. Different volumes of virus were used in order to 
obtain a value in the linear range of the beta-gal assay. A standard curve 
was niade with the AdCMVlacZ stock used for tie stock infections. 
Beta-gal assay 
Beta-galactosidase assays were performed by a chemiluminescent detection 
technique using the Galacto-Light.TM. kit (Tropix). Cells were harvested, 
washed twice with PBS and resuspended in lysis buffer (100 mM KPO.sub.4 pH 
7.8, 0.2% Triton X-100 .TM., 1 mM fresh DTT) at a concentration of 
1.times.10.sup.6 cells in 100 .mu.L. Reaction buffer (70 .mu.L of 
Galacton.TM. in 100 mM NaPO.sub.4 pH 8.0, 1 mM MgCl.sub.2) was then added 
to cell extracts (10 .mu.L) in a luminometer cuvette and the cuvette was 
placed in the luminometer (Berthold) after 1 hour of incubation. 100 .mu.L 
of Accelerator (10% Emerald enhancer in 0.2M NaOH) were then injected and 
the sample was counted for 10 seconds. The positive control consisted of 1 
.mu.L of beta-galactosidase from Sigma (in 01M NaPO.sub.4 pH 7.0, 1% BSA) 
added Lo a mock cell extract while The negative control was a mock cell 
extract. 
CONSTRUCTION OF pH.beta.E1AEIB 
As a first step in the production of E1-complementing cell lines of the 
invention, an expression vector containing Ad sequences was constructed. 
The genetic engineering methods used for the construction of the vectors 
and cell lines of the present invention are well known methods in the 
arts. Restriction endonucleases and other DNA modifying enzymes were used 
according to the supplier's recommendations or according to standard 
protocols such as that of Sambrook et al., (1989 CSH press; the contents 
of which is heroin incorporated by reference). Transformation of bacteria, 
purification of plasmids, transfection, and other molecular biology assays 
were also performed in accordance with known methods such as found in 
(Sambrook et al., 1989 CSII press). Escherichia coli DH5 strain was made 
competent and transformed plasmid DNA was prepared by the alkaline lysis 
method and purified by CsCl-ethidium bromide density gradient 
centrifugation. 
Briefly, the plasmid pH.beta.E1AE1B was constructed by subcloning the 3.0 
kb genomic DNA of Ad5 E1region (532-3525) as a Sall-BamH1 fragment into 
the Sall-BamH1 cloning sites of the pH.beta.Apr-1-neo expression vector. 
These restriction sites were introduced by site-directed mutagenesis in 
the plasmid pXC38 which contains the Ad5 E1 region from nt 1-5788 (Xhol) 
subcloned in pBR322 between EcoRI and Sall (a generous gift of Dr. Phil 
Branton, McGill University, Montreal). The site-directed mutagenesis was 
performed lining the Transformer.TM. site-directed mutagenesis kit from 
Clonetech Laboratories Inc. (Gunning et al. 1987, Proc. Natl. Acad. Sci. 
USA 82:4831-4835). pH.beta.E1AEIB (FIG. 1A) thus contains as a 
complementing element the human Ad5 coding region spanning nucleotides 532 
to 3525 (Genebank M73260), which consists in the E1A gene, the E1B 
promotor, and the E1B gene. The expression of E1A is not controlled by the 
natural E1A promoter but by the strong constitutive human .beta.-actin 
promotor. As further shown in FIG. 1A pH.beta.E1AE1B only harbors less 
than 200 bp overlap with the parental adenoviral genome (3334-3525). Since 
it does not contain the packaging and Ori sequences (the 5' ITR: 0-350), a 
recombination, although probabilistically infrequent, between the minimal 
overlap thereof and the Ad genome of the complementing element in the 
E1complementing cell line should not give rise to a viable particle, 
thereby eliminating the problem due to the presence of Ad E1+ revert ants 
or RCA. 
ENGINEERING OF THE BMAdE1 COMPLEMENTING CELL LINES 
To construct the BMAdE1 complementing cell lines the A549 (human lung 
epithelial cells) were transfected with pH.beta.E1AE1B (FIG.1A). 
A total of 10 .mu.g of purified pH.beta.E1AE1, previously cut with ScaI, 
were transfected onto A549 cells, a human lung carcinoma cell line (ATCC, 
CCL185) using the standard calcium phosphate precipitation technique. It 
should be understood that various methods of transfection arc well known 
in the art and that the present invention is not limited to transfection 
by the calcium phosphate procedure. For example, the vector could be 
lipofected or electroplated. 
INITIAL CHARACTERIZATION OF BMAdE1 CLONES 
To determine whether or not the A549 cell lines expressed Ad E1A and E1B 
functions, after transfection with pH.beta.E1AE1B, they were infected with 
AdCMVlacZ tat a multiplicity of infection of 5-10 pfu/cell. Seventy-two 
hours later, cytopathy was apparent and the non-infected controls of the 
positive cell lines were harvested, lysed, and analyzed on a Western blot 
(FIG. 1B). 
Results show that the expression of the E1B 19K and 55K proteins was not as 
high in the A549 cell lines as in 293 cells. However, in one of the A549 
cell lines there was more E1A proteins than in 293 cells. It is 
conceivable that the cell lines which were less infected by AdCMVlacZ 
expressed less E1A and E1B. In other words that there is a correlation 
between the extent of infection and the expression of E1A and E1B protein 
from the complementing element. 
The expression of adenovirus E1A and E1B products had already been shown in 
KB cells (Babiss et al., 1983 J. Virol. 46: 454-465). However the KB 
complementing cell lines of Babiss et al., are similar to 293 cells since 
the whole E1region, including the 5' ITR and packaging sequences are 
present in the complementing cell line and therefore does not diminish the 
RCA problem encountered with 293 cells. In addition, the yields of 
infectious particles in these cells is generally lower than in that of the 
reference complementing cell line, the 293 cell line. Consequently, the 
complementation exerted by the KB complementing cell lines is only partial 
as compared to 293 cells. The present studies differ from the previous 
work on two points. Firstly, the expression of E1A products in the present 
construct is controlled by the stronger human beta-actin constitutive 
promotor, instead of the SV40 early promotor. This seems to ensure a 
better expression of the Ad E1proteins. Secondly, no cell lines were 
identified which only expressed E1A proteins. 
VIRAL PROGENY IN SELECTED BMAdE1CLONES 
To assess the virus yield of the BMAdE1 clones, different clones were 
infected with AdCMVlacZ at a multiplicity of infection of 5-10 pfu/cell. 
The virus stock obtained with this infection was indirectly titered by a 
beta-gal chemiluminescent assay, the expression of lacZ being considered 
proportional to the amount of infectious virus (Table 1). 
TABLE 1 
______________________________________ 
Ad5CMVlacZ progeny comparison between BMAdE1 
clones and 293 cells 
production 
Ad5CMVlacZ (PFU.sup.a per cell) 
in cell line: 
______________________________________ 
293 BMAdE1 78-42 
BMAdE1 220-8 
800 200 800 
______________________________________ 
.sup.a Number of PFU was determined indirectly by gal expression in Hcla 
S3 cells after 48 hours of infection with Ad5CMVlacZ stocks done on 293 
and BMAdE1 clones. 
The result show that the expression of lacZ, which reflects the expression 
of the virus, in the BMAdE1 clones is delayed when comnpared to 293 cells 
(FIG. 2). Although the viral progeny of the BMAdE1 78-42 clone is four 
times inferior to that of the 293 cells, the BMAdE1 220-8 clone gives the 
same amount of infectious viral particules. Thus, in relative terms, the 
BMAdE1 220-8 complementing cell line complements the E1-defect to the same 
extent as the 293 complementation cell line. 
VIRAL TITRATION WITH THE SELECTED BMAdE1 CLONES 
To determine the capacity of the BMAdE1 clones to plaque efficiently, 
dilutions or an AdGFP stock were plated on BMAdE1 78-42, BMAdE1 220-8, and 
293 cells. The plaquing efficiency assessed the quality of the plaques and 
how easy it is to distinguish and count them following a productive 
infection. The results are presented in Table 2. 
TABLE 2 
______________________________________ 
Viral titers of a AdGFP stock.sup.a of BMAdE1 
clones compared to 293 cells 
AdGFP titer (PFU/ml) in cell line: 
______________________________________ 
293 BMAdE1 78-42 
BMAdE1 220-8 
7 .times. 10.sup.9 
2 .times. 10.sup.9 
3 .times. 10.sup.9 
______________________________________ 
.sup.a AdGFP stock was made by infecting 5X10.sup.8 293 cells. Harvesting 
was done at 48 hpi, and the pellet was resuspended in 50 ml of medium and 
frozen/thawed three times. 
Titers three and two fold inferior to that obtained with 293 cells were 
observed with the -78 and -220 clones, respectively. 
For the AdGFP infection, the plaques were observed with an inverted 
fluorescence microscope, thereby allowing a visualization of at least 
twice the amount of plaques observable with the naked eye. This was 
confirmed by using bluo-gal.TM. for AdCMVlacZ infection. 
The fact that BMAdE1 220-8 grows more in clusters as compared to BMAdE1 
78-42 does not allow the easiest visualization of the plaques with the 
naked eyes. Nevertheless, BMAdE1 clones can still be used to plaque-purify 
viruses after co-transfections, since one of the main goals of the present 
invention is to get rid of the RCAs in virus stocks. In a preferred 
embodiment, 293 cells could be used for the initial transfection. The 
scaling up of the production of infectious E1-defective particles would be 
performed using the complementing cell lines of the present invention, 
preferably BMAdE1 220, since it produces infectious particles at the same 
level as 293 cells. The 293 cells could also be used for titering purposes 
in view of their good plaquing efficiency. A property which is somewhat 
shared by BMAdE1 78. When the RCA problem is not so acute, the 293S 
derivative, which grows in suspension, can be used for later stages of the 
preparation of the infectious particles. The conventional 293 cell line as 
well as the BMAdE1 cell lines of the present invention all grow in 
monolayer and hence do not provide the ease of scaling up of cells growing 
in suspension. When both BMAdE1 and 293 cell lines are used for the 
preparation of infectious particles it is preferable to use the latter for 
the late passages, thereby avoiding the expansion of RCAs. 
DEPOSITS 
The E1-complementing cell lines BMAdE1-78-42 and BMAdE1 220-8 have been 
deposited at the American Type Culture Collection (ATCC) 10801 University 
Blvd., Manassas, Va. 20110-2209 under accession numbers CRL-12408 and 
CRL-12407, respectively. 
CONCLUSION 
In summary the present invention provides in particular, E1-complementing 
cell lines which overcome the problem of RCA production and a recombinant 
vector for constructing such E1-complementing cell lines. These cell lines 
avoid the emergence of E1+ revert ants during multiple passages and 
amplification of Ad helper-independent defective vectors. It was herein 
demonstrated that the BMAdE1-220 cell lines can complement 
.DELTA.E1.DELTA.3Ad recombinants at the same level as 293 cells. This cell 
line allows the production of approximately 1000 infectious particles of 
Ad5CMVlacZ per cell, a value which is comparable to that obtained in 293 
cells (Table 1). This is in contrast to the E1-complementing cell lines 
obtained by Imler et al., (1996 Gene Ther. 3:75-84), which "are able to 
support replication of E1-deleted adenoviruses, although not as 
efficiently as 293 cells (Table 1)". Indeed, the best complementing cell 
line obtained thereby yields approximately 5 fold less infectious 
particles per cell, and in one case as low as 100 fold less (Imler et al., 
1996 Gene Ther. 3:75-84). 
The BMAdE1-78 E1-complementing cell line while not being as good a producer 
of infectious particles as BMAdE1-220 or 293 (producing about 4 fold less 
per cell), provides however the advantage of showing a less transformed, 
rounded phenotype than BMAdE1-220, making it a better cell line for plaque 
purification. 
Importantly, the cell lines of the present invention have not been shown to 
generate RCA during multiple passages. By providing a region of homology 
of less than 200 bp between the complementing cell lines of the present 
invention and the E1-defective adenoviral vectors, which is less than 
previously disclosed cell lines used, the likelyhood of RCA emergence is 
expected to be lower than that of previously disclosed complementing cell 
lines. In fact, no RCA is expected to emerge during the production of the 
stocks of infectious Ad particles using the complementing cell lines of 
the invention. 
Finally, the expression of functional E1B proteins in the complementing 
cell lines of the present invention is thought to favor expression of 
viral proteins and lead to superior yields of infectious virus particles 
per cell. In addition E1B protein expression might diminish the known 
toxic effects that accompany E1A expression. 
The present invention is not to be limited in scope by the recombinant 
constructs and cell lines exemplified or deposited which are intended as 
but single illustrations of one aspect of the invention. Indeed, various 
modifications of the invention in addition to those shown and described 
herein will become apparent to those skilled in the art from the foregoing 
description and accompanying figures. Such modifications are intended to 
fall within the scope of the appended claims.