U.S. patent application number 11/075385 was filed with the patent office on 2006-01-05 for cell lines and constructs useful in production of e1-deleted adenoviruses in absence of replication competent adenovirus.
Invention is credited to Guangping Gao, James M. Wilson.
Application Number | 20060003451 11/075385 |
Document ID | / |
Family ID | 22560427 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003451 |
Kind Code |
A1 |
Gao; Guangping ; et
al. |
January 5, 2006 |
Cell lines and constructs useful in production of E1-deleted
adenoviruses in absence of replication competent adenovirus
Abstract
Novel cell lines useful for trans-complementing E1-deleted
adenoviral vectors are described. The cell lines are capable of
providing high yields of E1-deleted adenoviral vectors in the
absence of replication-competent adenovirus over multiple
passages.
Inventors: |
Gao; Guangping; (Rosemont,
PA) ; Wilson; James M.; (Gladwyne, PA) |
Correspondence
Address: |
HOWSON AND HOWSON;ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Family ID: |
22560427 |
Appl. No.: |
11/075385 |
Filed: |
March 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10053194 |
Jan 16, 2002 |
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11075385 |
Mar 8, 2005 |
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09659203 |
Sep 11, 2000 |
6365394 |
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10053194 |
Jan 16, 2002 |
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60156644 |
Sep 29, 1999 |
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Current U.S.
Class: |
435/456 ;
435/235.1; 435/367 |
Current CPC
Class: |
C12N 7/00 20130101; C07K
14/005 20130101; A61K 48/00 20130101; C12N 2710/10322 20130101;
C12N 15/86 20130101; A61P 31/20 20180101; C12N 2710/10343 20130101;
C12N 2710/10352 20130101 |
Class at
Publication: |
435/456 ;
435/367; 435/235.1 |
International
Class: |
C12N 15/861 20060101
C12N015/861; C12N 7/00 20060101 C12N007/00; C12N 5/08 20060101
C12N005/08 |
Goverment Interests
[0002] This work was supported by NIDDK (DK47757-06 &
DK49136-05), NHLBI (HL49040-08) and NICHD (HD32649-05) of the
National Institutes of Health. The US government may have certain
rights in this invention.
Claims
1. An E1-complementing cell line useful for production of
recombinant E1-defective adenoviruses in the absence of detectable
replication-competent adenovirus, said E1-complementing cell line
comprising 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, and wherein the nucleic acid sequences
further comprise a deletion of adenovirus sequences 5' to the
sequences encoding adenovirus E1a.
2. The E1-complementing cell line according to claim 1, wherein the
aneuploid cell line is a HeLa cell line.
3. The E1-complementing cell line according to claim 1, wherein the
nucleic acid sequences further comprise nucleic acid sequences of
the pIX gene region.
4. The E1-complementing cell line according to claim 1, wherein the
nucleic acid molecule is a plasmid vector.
5. The E1-complementing cell line according to claim 1, wherein the
nucleic acid molecule comprises multiple copies of the sequences
encoding adenovirus E1a and adenovirus E1b.
6. The E1-complementing cell line according to claim 1, wherein the
E1-complementing cell line comprises multiple copies of said
nucleic acid molecule.
7. The E1-complementing cell line according to claim 1, wherein the
sequences encoding adenovirus E1a and the sequences encoding E1b
are independently selected from adenovirus type 5.
8. The E1-complementing cell line according to claim 1, wherein the
cell line is selected from the group consisting of GH364 and
GH354.
9. An adenovirus E1-complementing cell line designated GH329,
deposited with the ATCC under accession number PTA-803.
10. A method for packaging of E1-defective adenoviral particles in
the absence of replication competent adenovirus, said method
comprising the steps of: (a) providing cells from an
E1-complementing cell line comprising an aneuploid cell line stably
transformed with a nucleic acid molecule comprising nucleic acid
sequences encoding adenovirus E1 a and adenovirus E1 b under the
control of a phosphoglycerate kinase (PGK) promoter, wherein the
nucleic acid sequences further comprise a deletion of adenovirus
sequences 5' to the sequences encoding adenovirus E1 a; (b)
transfecting said cells with a recombinant vector comprising, from
5' to 3', adenovirus 5' inverted terminal repeat sequences (ITRs),
nucleic acid sequences encoding adenovirus pIX under the control of
sequences which direct expression of adenovirus pIX in said cells,
and a defect in the adenovirus E1 region, and adenovirus 3' ITRs;
and (c) culturing said transfected cells under conditions which
permit packaging of the E1-defective vector into a recombinant
E1-defective adenoviral particle.
11. The method according to claim 10, wherein said recombinant
vector further comprises a selected transgene.
12. The method according to claim 11, wherein said transgene is
located between the 5' and 3' ITRs.
13. The method according to claim 10, further comprising the step
of transfecting said cells with a second recombinant vector
comprising adenovirus sequences encoding at least one adenoviral
gene and a defect in the adenovirus E1 region.
14. The method according to claim 13, wherein said second
recombinant vector encodes adenovirus E2a.
15. The method according to claim 13, wherein said second
recombinant vector encodes adenovirus E4 or a functional fragment
thereof.
16. The method according to claim 15, wherein the functional
fragment is E4 ORF6.
17. The method according to claim 10, wherein the E1-complementing
cell line is selected from the group consisting of GH329, ATCC
PTA-803; GH364 and GH354.
18. A method of amplifying E1-defective adenoviral particles in the
absence of replication competent adenovirus, the method comprising
the steps of: (a) infecting an E1-complementing cell line with
E1-defective adenoviruses, wherein said cell line comprises 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, and wherein the nucleic acid sequences further comprise a
deletion of adenovirus sequences 5' to the sequences encoding
adenovirus E1a; (b) passaging the E1-defective adenoviral particles
on the E1-complementing cell line for 2 to 20 passages, and (c)
collecting the E1-defective adenoviral particles.
19. The method according to claim 18, wherein the E1-defective
adenoviruses of (a) are prepared by the steps comprising: (i)
providing cells from an E1-complementing cell line comprising 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, wherein the nucleic acid sequences further comprise a
deletion of adenovirus sequences 5' to the sequences encoding
adenovirus E1a; (ii) transfecting said cells with a recombinant
vector comprising adenovirus 5' and 3' inverted terminal repeat
sequences (ITRs), nucleic acid sequences encoding adenovirus pIX
under the control of sequences which direct expression of
adenovirus pIX in said cells, and a defect in the adenovirus E1
region; (iii) culturing said transfected cells under conditions
which permit packaging of the E1-defective vector into a
recombinant E1-defective adenoviral particle; and (iv) purifying
the recombinant E1-defective adenoviral particle from substantially
all cellular debris.
Description
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.
[0021] Another suitable cell line is the GH364 cell line, which
expresses between 5 to 10 copies of the E1a and E1b proteins. Yet
another cell line of the invention is the GH354 cell line. The
GH354 cells have been found to complement (and allow replication
of) E1-deleted viral vectors in the absence of detectable RCA over
at least 20 passages. Further, as with the GH329 cell line, yields
obtained using the GH354 cell line are at least equivalent to those
obtained in 293 cells.
[0022] The cell lines of the invention are particularly well suited
for production of E1-deleted adenovirus for preclinical and
clinical use, as they are readily adapted to growth in serum free
media. The cell lines of the invention, e.g., GH329, may be adapted
to growth in serum-free media using techniques well known those of
skill in the art. These serum-free-media adapted cell lines are
encompassed by the present invention.
[0023] Optionally, other useful cell lines may be derived from a
cell line of the invention. For example, the GH329 (or GH354 or
GH364) cell line may be modified to stably express another desired
protein(s) using the techniques described herein, as well as
techniques known in the art. In one desirable embodiment, a
derivative of the GH329 cell line may be produced by stably
transforming GH329 cells such that they contain one or more
sequences expressing adenoviral proteins (or functional fragments
thereof) required for replication of the E1-deleted virus. In
addition to the adenoviral E1a and E1b functions provided by the
cell line, the required adenoviral functions include E2a and E4
ORF6. Thus, in one embodiment, a GH329 derivative cell line may be
produced which expresses the required functions of the E2 region or
E4 region, or combinations thereof. Suitably, the nucleic acid
molecule(s) used to produce the GH329 derivative cell line contains
no adenoviral sequences 5' to the E1 coding region and only the
minimal adenoviral sequences required to express the desired
functional proteins in the host cell. Given this information, one
of skill in the art may readily engineer other GH329 derivative
cell lines.
I. E1-Complementing Cell Line
[0024] A. Target Cells
[0025] The vector used to transform HeLa cells and produce the
GH329 cell line of the invention may be used to develop other
E1-trans-complementing cell lines. Preferably, such other cell
lines are derived from HeLa cells, an aneuploid epithelial-like
cell derived from cervical carcinoma [ATCC CCL2]. However, using
the vector described herein, another mammalian host cell may be
selected from any mammalian species, such as human cell types,
including without limitation, cells such as Vero cells, A549 and
HKB cells. Other mammalian species cells are also useful, for
example, primate cells, rodent cells or other cells commonly used
in biological laboratories. The selection of the mammalian species
providing the cells is not a limitation of this invention; nor is
the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor
cell, etc.
[0026] B. Transforming DNA Molecule
[0027] Suitably, the target cells are transformed with a DNA
molecule carrying, at a minimum, DNA sequences encoding adenovirus
E1a and E1b under the control of a PGK promoter. This molecule
lacks adenoviral sequences 5' of the E1 region, preferably
excluding the native E1a promoter and contains minimal sequences 3'
to the E1 region (i.e., optionally contains partial pIX
sequences).
[0028] The DNA sequences encoding the adenovirus E1a and E1b genes
useful in this invention may be selected from among any known
adenovirus type, including the presently identified 41 human types.
Similarly, adenoviruses known to infect other animals may supply
the gene sequences. The selection of the adenovirus type for each
E1a and E1b gene sequence does not limit this invention. The
sequences for a number of adenovirus serotypes, including that of
serotype Ad5, are available from Genbank. A variety of adenovirus
strains are available from the ATCC, or are available by request
from a variety of commercial and institutional sources. In the
following exemplary embodiment the E1a and E1b gene sequences are
those from adenovirus serotype 5 (Ad5).
[0029] By "adenoviral DNA which expresses the E1a gene product", it
is meant any adenovirus gene encoding E1a or any functional E1a
portion. Similarly included are any alleles or other modifications
of the E1a gene or functional portion. Such modifications may be
deliberately introduced by resort to conventional genetic
engineering or mutagenic techniques to enhance the E1a expression
or function in some manner, as well as naturally occurring allelic
variants thereof.
[0030] By "adenoviral DNA which expresses the E1b gene product", it
is meant any adenovirus gene encoding E1b or any functional E1b
portion. Similarly included are any alleles or other modifications
of the E1b gene or functional portion. Such modifications may be
deliberately introduced by resort to conventional genetic
engineering or mutagenic techniques to enhance the E1b expression
or function in some manner, as well as naturally occurring allelic
variants thereof.
[0031] The nucleic acid molecule carrying the Ad E1a and Ad E1b may
be in any form which transfers these components to the host cell.
Most suitably, these sequences are contained within a vector. A
"vector" includes, without limitation, any genetic element, such as
a plasmid, phage, transposon, cosmid, chromosome, virus, virion,
etc. In one particularly suitable embodiment, the nucleic acid
molecule is a plasmid carrying Ad E1a, Ad E1b, partial pIX
sequences, and the PGK promoter.
[0032] The nucleic acid molecule may contain other non-viral
sequences, such as those encoding certain selectable reporters or
marker genes, e.g., sequences encoding hygromycin or purimycin, or
the neomycin resistance gene for G418 selection, among others. The
molecule may further contain other components.
[0033] Conventional techniques may be utilized for construction of
the nucleic acid molecules of the invention. See, generally,
Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratories, Cold Spring Harbor, N.Y.
[0034] Once the desired nucleic acid molecule is engineered, it may
be transferred to the target cell by any suitable method. Such
methods include, for example, transfection, electroporation,
liposome delivery, membrane fusion techniques, high velocity
DNA-coated pellets, viral infection and protoplast fusion.
Thereafter, cells are cultured according to standard methods and,
optionally, seeded in media containing an antibiotic to select for
cells containing the cells expressing the resistance gene. After a
period of selection, the resistant colonies are isolated, expanded,
and screened for E1 expression. See, Sambrook et al, cited
above.
II. Use of E1-Complementing Cells in Production E1-Deleted
Adenovirus
[0035] The E1-complementing cells of the invention are useful for a
variety of purposes. Most suitably, the cells (e.g., GH329) are
used in packaging recombinant virus (i.e., viral particles) from
E1-defective vectors and in high yield production of E1-defective
adenoviruses in the absence of detectable RCA.
[0036] The cells of the invention which express Ad5 E1a and E1b are
suitable for use in packaging recombinant virus from E1-defective
vectors (e.g., plasmids) containing sequences of Ad5 and Ad2.
Further, these cells are anticipated to be useful in producing
recombinant virus from other Ad serotypes, which are known to those
of skill in the art.
[0037] A. Packaging of E1-Defective Vectors
[0038] In a preferred embodiment, this method of the invention
involves packaging of an E1-deleted vector which contains a
transgene into an E1-deleted adenoviral particle useful for
delivery of the transgene to a host cell. In a preferred
embodiment, the E1-deleted vector contains all adenoviral genes
necessary to produce and package an infectious adenoviral particle
which replicates only in the presence of complementing E1 proteins,
e.g., such as are supplied by cell line of the invention. The
vector contains defects in both the E1a and E1b sequences, and most
desirably, is deleted of all or most of the sequences encoding
these proteins.
[0039] At a minimum, the E1-deleted vector to be packaged contains
adenoviral 5' and 3' cis-elements necessary for replication and
packaging, a transgene, and a pIX gene or a functional fragment
thereof. The vector further contains regulatory sequences which
permit expression of the encoded transgene product in a host cell,
which regulatory sequences are operably linked to the transgene. As
used herein, "operably linked" sequences include both expression
control sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest. Also included in the vector are
regulatory sequences operably linked to other gene products, e.g.,
the pIX gene, carried by the vector.
[0040] 1. Adenoviral Elements
[0041] The E1-defective vector to be packaged includes, at a
minimum, adenovirus cis-acting 5' and 3' inverted terminal repeat
(ITR) sequences of an adenovirus (which function as origins of
replication) and the native 5' packaging/enhancer domain. These are
5' and 3' cis-elements necessary for packaging linear Ad genomes
and further contain the enhancer elements for the E1 promoter.
[0042] The E1-deleted vector to be packaged into a viral particle
is further engineered so that it expresses the pIX gene product.
Most suitably, the pIX gene is intact, containing the native
promoter and encoding the full length protein. However, were
desired, the native pIX promoter may be substituted by another
desired promoter. Alternatively, sequences encoding a functional
fragment of pIX may be selected for use in the vector. In yet
another alternative, the native sequences encoding pIX or a
functional fragment thereof may be modified to enhance expression.
For example, the native sequences may be modified, e.g., by
site-directed mutagenesis or another suitable technique, to insert
preference codons to enhance expression in a selected host cell.
Optionally, the pIX may be supplied to the E1-complementing cell
line on a separate molecule.
[0043] An exemplary vector containing only the minimal adenoviral
sequences is termed the Ad.DELTA.E1-E4 vector, and lacks all
functional adenoviral genes including E1, E2, E3, E4, intermediate
gene IXa and late genes L1, L2, L2, L4 and L5) with the exception
of intermediate gene IX which is present. However, in a preferred
embodiment, the E1-deleted vector contains, in addition to the
minimal adenoviral sequences described above, functional adenoviral
E2 and E4 regions. In another suitable embodiment, the adenoviral
sequences in the E1-deleted vector include the 5' and 3'
cis-elements, functional E2 and E4 regions, intermediate genes IX
and IXa, and late genes L1 through L5. However, the E1-deleted
vector may be readily engineered by one of skill in the art, taking
into consideration the minimum sequences required, and is not
limited to these exemplary embodiments.
[0044] The vector is constructed such that the transgene and the
sequences encoding pIX are located downstream of the 5' ITRs and
upstream of the 3' ITRs. The transgene is a nucleic acid sequence,
heterologous to the adenovirus sequence, which encodes a
polypeptide, protein, or other product, of interest. The transgene
is operatively linked to regulatory components in a manner which
permits transgene transcription.
[0045] 2. Transgene
[0046] The composition of the transgene sequence will depend upon
the use to which the resulting virus will be put. For example, one
type of transgene sequence includes a reporter sequence, which upon
expression produces a detectable signal. Such reporter sequences
include without limitation, DNA sequences encoding
.beta.-lactamase, .beta.-galactosidase (LacZ), alkaline
phosphatase, thymidine kinase, green fluorescent protein (GFP),
chloramphenicol acetyltransferase (CAT), luciferase, membrane bound
proteins including, for example, CD2, CD4, CD8, the influenza
hemagglutinin protein, and others well known in the art to which
high affinity antibodies directed thereto exist or can be produced
by conventional means, and fusion proteins comprising a membrane
bound protein appropriately fused to an antigen tag domain from,
among others, hemagglutinin or Myc.
[0047] However, desirably, the transgene is a non-marker sequence
encoding a product which is useful in biology and medicine, such as
proteins, peptides, anti-sense nucleic acids (e.g., RNAs), enzymes,
or catalytic RNAs. The transgene may be used to correct or
ameliorate gene deficiencies, which may include deficiencies in
which normal genes are expressed at less than normal levels or
deficiencies in which the functional gene product is not expressed.
One desirable type of transgene sequence encodes a therapeutic
protein or polypeptide which is expressed in a host cell. The
invention further includes using multiple transgenes, e.g., to
correct or ameliorate a gene defect caused by a multi-subunit
protein. In certain situations, a different transgene may be used
to encode each subunit of a protein, or to encode different
peptides or proteins. This is desirable when the size of the DNA
encoding the protein subunit is large, e.g., for an immunoglobulin,
the platelet-derived growth factor, or a dystrophin protein. In
order for the cell to produce the multi-subunit protein, a cell is
infected with the recombinant virus containing each of the
different subunits. Alternatively, different subunits of a protein
may be encoded by the same transgene. In this case, a single
transgene includes the DNA encoding each of the subunits, with the
DNA for each subunit separated by an internal ribozyme entry site
(IRES). This is desirable when the size of the DNA encoding each of
the subunits is small, e.g., total of the DNA encoding the subunits
and the IRES is less than five kilobases. Other useful gene
products include, molecules which induce an immune response,
non-naturally occurring polypeptides, such as chimeric or hybrid
polypeptides having a non-naturally occurring amino acid sequence
containing insertions, deletions or amino acid substitutions. For
example, single-chain engineered immunoglobulins could be useful in
certain immunocompromised patients. Other types of non-naturally
occurring gene sequences include antisense molecules and catalytic
nucleic acids, such as ribozymes, which could be used to reduce
overexpression of a gene. However, the selected transgene may
encode any product desirable for delivery to a host or desirable
for study. The selection of the transgene sequence is not a
limitation of this invention.
[0048] 3. Regulatory Sequences
[0049] In addition to the major elements identified above for the
vector, (e.g, the adenovirus sequences and the transgene), the
vector also includes conventional control elements necessary to
drive expression of the transgene in a host cell containing with
the transgene. Thus the vector contains a selected promoter which
is linked to the transgene and located, with the transgene, between
the viral sequences of the vector. Suitable promoters may be
readily selected from among constitutive and inducible promoters.
Selection of these and other common vector elements are
conventional and many such sequences are available [see, e.g.,
Sambrook et al, and references cited therein].
[0050] Examples of constitutive promoters include, without
limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter
(optionally with the RSV enhancer), the cytomegalovirus (CMV)
promoter (optionally with the CMV enhancer) [see, e.g., Boshart et
al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate
reductase promoter, the .beta.-actin promoter, the phosphoglycerol
kinase (PGK) promoter, and the EF1 a promoter [Invitrogen].
Inducible promoters are regulated by exogenously supplied
compounds, including, the zinc-inducible sheep metallothionine (MT)
promoter, the dexamethasone (Dex)-inducible mouse mammary tumor
virus (MMTV) promoter, the T7 polymerase promoter system [WO
98/10088]; the ecdysone insect promoter [No et al, Proc. Natl.
Acad. Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible
system [Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551
(1992)], the tetracycline-inducible system [Gossen et al, Science,
268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem.
Biol., 2:512-518 (1998)], the RU486-inducible system [Wang et al,
Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther.,
4:432-441 (1997)] and the rapamycin-inducible system [Magari et al,
J. Clin. Invest., 100:2865-2872 (1997)].
[0051] 4. Other Vector Elements
[0052] The vector carrying the Ad ITRs flanking the transgene and
regulatory sequences (e.g., promoters, polyA sequences, etc.) may
be in any form which transfers these components to the host cell.
Preferably, the vector is in the form of a plasmid. Preferably to
avoid homologous recombination, the plasmid does not contain any
adenovirus sequences in the E1 region or the region 5' to the E1
region. It may contain non-viral sequences, such as those encoding
certain selectable reporters or marker genes, e.g., sequences
encoding hygromycin or purimycin, among others. Other components of
the plasmid may include an origin of replication and an amplicon,
such as the amplicon system, employing the Epstein Barr virus
nuclear antigen, for example, the vector components in pCEP4
(Invitrogen). See, also, J. Horvath et al, Virology, 184:141-148
(1991). This amplicon system or similar amplicon components permit
high copy episomal replication in the cells.
[0053] Other heterologous nucleic acid sequences optionally present
in this vector include sequences providing signals required for
efficient polyadenylation of the RNA transcript, and introns with
functional splice donor and acceptor sites. A common poly-A
sequence which is employed in the vectors useful in this invention
is that derived from the papovavirus SV-40. The poly-A sequence
generally is inserted following the transgene sequences and before
the 3' AAV ITR sequence. A vector useful in the present invention
may also contain an intron, desirably located between the
promoter/enhancer sequence and the transgene. One possible intron
sequence is also derived from SV-40, and is referred to as the
SV-40 T intron sequence. Selection of these and other common vector
elements are conventional and many such sequences are available
[see, e.g., Sambrook et al, and references cited therein at, for
example, pages 3.18-3.26 and 16.17-16.27].
[0054] 5. Co-Transfection of Adenoviral Sequences
[0055] Preferably, the E1-deleted vector contains all functional
adenoviral sequences required for packaging and replication in the
presence of the E1-complementing cell line of the invention. In
addition to the E1a and E1b functions supplied by the
trans-complementing cell line, functional adenoviral E2a and E4 ORF
6 region are required. However, where the required functions are
lacking from the E1-deleted vector (i.e., the E1-deleted vector
further contains functional deletions in E2a and/or E4 ORF6), these
functions may be supplied by other sources. In one embodiment,
these functions may be supplied by co-transfection of the
E1-complementing cell line with one or more nucleic acid molecules
capable of directing expression of the required adenoviral
function. Alternatively, a modified GH329 cell line of the
invention which has been transformed to supply the required
adenoviral functions may be utilized.
[0056] For example, a vector deleted of E1 and having a defective
E2 region may be complemented in GH329 cells of the invention by
transfecting the cells with a nucleic acid molecule (e.g., a
plasmid) expressing required E2 functions (e.g., E2a). As another
example, a vector lacking E1 through E4 functions may be
complemented in GH329 cells by transfecting the cells with a
nucleic acid molecule expressing functional E2, E3 and E4 (e.g., E4
ORF6). Where a nucleic acid molecule is co-transfected into the
cells of the invention, such a nucleic acid molecule contains no
adenoviral E1 sequences; nor does it contain any sequences 5' to
the E1 region. Construction of these nucleic acid molecules is
within the skill of those in the art.
[0057] Suitably, a selected recombinant vector, as described above,
is introduced into E1-complementing cells from a cell line of the
invention using conventional techniques, such as the transfection
techniques known in the art [see, K. Kozarsky et al, Som. Cell and
Molec. Genet., 19(5):449-458 (1993)]. Thereafter, recombinant
E1-deleted adenoviruses are isolated and purified following
transfection. Purification methods are well known to those of skill
in the art and may be readily selected. For example, the viruses
may be subjected to plaque purification and the lysates subjected
to cesium chloride centrifugation to obtain purified virus.
[0058] B. Amplification of E1-Deleted Adenoviruses
[0059] The E1-trans-complementing cell line of the invention (or a
derivative thereof) may be used to amplify an E1-defective
adenovirus. Suitably, the E1-defective adenovirus will have been
isolated and purified from cellular debris and other viral
materials prior to use in this method. This is particularly
desirable where the E1-defective adenovirus to be amplified is
produced by methods other than those of the present invention.
Suitable purification methods, e.g., plaque purification, are well
known to those of skill in the art.
[0060] A culture, or preferably, a suspension of cells from an
E1-trans-complementing cell line of the invention, e.g., GH329, are
infected with the E1-defective adenovirus using conventional
methods. A suitable multiplicity of infection (MOI) may be readily
selected. However, an MOI in the range of about 0.1 to about 100,
about 0.5 to about 20, and/or about 1 to about 5, is desirable. The
cells are then cultured under conditions which permits cell growth
and replication of the E1-defective adenovirus in the presence of
the E1 expressed by the cell line of the invention. Suitably, the
viruses are subjected to continuous passages for up to 5, 10, or 20
passages. However, where desired, the viruses may be subjected to
fewer, or more passages.
[0061] The cells are subjected to two to three rounds of
freeze-thawing, the resulting lysate is then subjected to
centrifugation for collection, and the supernatant is collected.
Conventional purification techniques such as chloride gradient
centrifugation or column chromatography are used to concentrate the
rAd-AE1 from the cellular proteins in the lysate. Advantageously,
however, the method of the invention through use of the cell lines
of the invention avoid the problems of contaminating RCA which
plague conventional production techniques.
III. E1-Deleted Ad Produced by Method of Invention
[0062] The E1-deleted adenoviruses produced according to the
present invention are suitable for a variety of uses and are
particularly suitable for in vivo uses, as the present invention
permits these adenoviruses to be produced in serum-free media, and
in the absence of detectable RCA. Thus, the E1-deleted adenoviruses
produced according to the invention are substantially free of
contamination with RCA.
[0063] In one embodiment, E1-deleted viruses have been deemed
suitable for applications in which transient transgene expression
is therapeutic (e.g., p53 gene transfer in cancer and VEGF gene
transfer in heart diseases). However, the E1-deleted adenoviruses
are not limited to use where transient transgene expression is
desired. The E1-deleted adenoviruses are useful for a variety of
situations in which delivery and expression of a selected transgene
is desired.
[0064] Suitable doses of E1-deleted adenoviruses may be readily
determined by one of skill in the art, depending upon the condition
being treated, the health, age and weight of the veterinary or
human patient, and other related factors. However, generally, a
suitable dose may be in the range of 10.sup.10 to 10.sup.18, and
preferably about 10.sup.14 to 10.sup.16 viral particles per dose,
for an adult human having weight of about 80 kg. This dose may be
suspended in about 0.01 mL to about 1 mL of a physiologically
compatible carrier and delivered by any suitable means. The dose
may be repeated, as needed or desired, daily, weekly, monthly, or
at other selected intervals.
[0065] The following examples are provided to illustrate the
production of the exemplary cell lines of the invention and their
use in producing E1-deleted adenovirus which are free of detectable
RCA over 20 passages. These examples do not limit the scope of the
invention. One skilled in the art will appreciate that although
specific reagents and conditions are outlined in the following
examples, modifications can be made which are meant to be
encompassed by the spirit and scope of the invention.
EXAMPLE 1
Production of E1-Complementing Cell Lines
[0066] As described in this example, Vero, A549 and HeLa cells were
stably transfected with plasmid constructs carrying a 3.4 kb DNA
fragment of Ad 5 genome spanning 511 to 3924 bp (E1a and E1b open
reading frames and part of the pIX gene). FIG. 1 is provides a
schematic representation of the relevant constructs. In these
constructs, the E1a native promoter was replaced with either
sequences from the cytomegalovirus early gene (CMV) or human
phosphoglycerate kinase gene (PGK). There is no overlap with the 5'
region of the E1-deleted vector (0-360 bp) described below and
reduced overlap at the 3' region (vector begins at 3312 bp while
the adenovirus sequence in 293 extends to 4300 bp).
[0067] A. Construction of PGKE1 Plasmids
[0068] Ad5 E1 region (m.u.1.42-10.9) was cloned into pBluescript
SK(-) vector. The 3.4 kb E1 fragment was further subcloned into a
plasmid which allowed expression from promoters derived from the
phosphoglycerate kinase gene [PGK] [Adra et al., Gene, 60:65-74
(1987)] or the immediate early gene of cytomegalovirus [CMV]
[Thomsen et al., Proc. Natl. Acad. Sci. USA, 81:659-663 (1984)].
Both contained the neomycin resistance gene for G418 selection.
[0069] B. Transfections and Selection of G418-Resistant Clone
[0070] HeLa, A549 and Vero cells were obtained from ATCC and
maintained as monolayers in Dulbecco modified Eagle's minimal
essential medium (DMEM) supplemented with 10% fetal bovine serum.
Plasmids were transfected by calcium phosphate precipitation onto
the cells seeded in 100 mm plates, using 10 .mu.g of plasmid DNA
for each plate. Twenty-four hours post-transfection, cells were
trypsinized and seeded in G418-containing media at various
dilutions ranging from 1:5 to 1:40. After 2 weeks of selection,
G418-resistant colonies were isolated, expanded and screened for E1
expression.
[0071] Only one stable clone formed from A549 transfectants, while
over 70 clones from HeLa and 50 clones from Vero cell transfectants
were isolated (data not shown).
[0072] C. Screening Procedure for New E1 Lines
[0073] Stable G418-resistant clones were first screened with a blue
comet formation assay in which 1.times.10.sup.6 cells in 6 well
plates were infected with 200 LacZ Forming Units (LFU) of H5.CBLacZ
[an E1-deleted adenovirus expression LacZ from a .beta.-actin
promoter] [Gao et al., J. Virol., 70:8934-8943 (1996)]. Six days
post-infection cells were histochemically stained with
5-Bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal) and
comets of blue cells in each well were scored. Subsequently, the
strongly positive clones were seeded into 4 well glass chamber
slides for 24 hours. Expression level of E1a and E1b proteins in
the cells was assessed by immunofluorescent staining, using mouse
monoclonal antibodies (Oncogene Science).
[0074] G418 resistant clones were expanded and initially screened
for their ability to support propagation of an E1-deleted
adenovirus vector harboring the LacZ transgene; the only clones
capable of sustaining E1-deleted adenovirus replication were HeLa
derived.
EXAMPLE 2
Characterization of New E1 Complementing Cell Lines
[0075] A. Genetic Constitution
[0076] Total genomic DNAs were isolated from each E1 complementing
clone, digested with appropriate restriction endonucleases to
release an internal E1-containing plasmid fragment, and evaluated
by DNA hybridization after electrophoresis. More particularly, DNAs
were fractionated on 1% agarose gels, transferred to nylon filters
and hybridized with a 1.1 kb E1 Hind III/Sma I fragment.
[0077] All cell lines tested have at least one copy of the E1 gene
integrated into HeLa genome. One PGK-E1 clone (GH364) and one
CMV-E1 cell line (GH414) harbor 5-10 copies of the E1 gene.
[0078] B. Production of E1 proteins Cell pellets of each clone were
harvested from 60 mm plates and resuspended in 200 .mu.l of lysis
buffer (20 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% NP-40 v/v, 1 mM
PMSF, 1 .mu.g/ml each leupeptin, antipain, chymostatin, soybean
trypsin inhibitor). Lysates were incubated on ice for one hour and
spun in a microcentrifuge at 14,000 rpm for 30 minutes at 4.degree.
C. Supernatants were collected and total protein concentrations
determined by Lowry's method. Samples (50 .mu.g) were fractionated
on 10% SDS-PAGE gels and electrotransferred to nitrocellulose
membranes. E1a and E1b proteins were detected using the enhanced
chemiluminescence (ECL) system (Amersham Life Science, Arlington
Heights, Ill.) with a mouse polyclonal antibody (PharmMingen, San
Diego, Calif.) and rat monoclonal antibodies (Oncogene Science)
respectively. Total cellular proteins from 293 cells were used as
controls.
[0079] Western blot analysis revealed variable E1 protein
expression profiles among different clones, in terms of total
expression and the ratios of E1a and E1b proteins. The anti-Ad5
antibody identified the E1a protein as a doublet at approximately
35-46 kDa.
[0080] C. Growth Kinetics of an E1-Deleted Recombinant Virus H5.
CBLacZ on the Novel E1 Complementing Cell Lines
[0081] HeLa, 293 and the cells of new E1 cell lines were infected
with H5.CBLacZ at multiplicity of infection (MOI) equal to 0.5.
Infected cells were harvested at 24, 48, 72, 96 and 120 hours
post-infection. Cells were lysed in the infection medium by 3
rounds of freeze/thaw and the titer of virus was determined by
serial dilution infections on 293 cells followed by histochemical
staining with X-gal. Cells were histochemically stained with X-gal
after 20 hours, and blue cells were counted. Titers are expressed
as LacZ forming units (LFU/ml) where one LFU is defined as the
quantity of virus sufficient to cause visually detectable LacZ
expression in one cell at 24 hour post-infection.
[0082] The yield of H5.CBLacZ virus in each cell line is shown on
FIG. 2A, where the y axis is a log scale and the time points are
shown on the x axis. Two cell lines, GH329 and GH354, were
equivalent if not better than 293 cells in terms of production
(FIG. 2A) of E1-deleted virus.
[0083] D. Relative Plaquing Efficiencies (RPES) for H5. CBLacZ
Virus on New E1 Cell Lines
[0084] The new E1 cell lines were compared with 293 cells in their
abilities to support plaque formation of H5.CBLacZ virus. Cells
were infected with H5.CBLacZ with a range of serial dilutions and
overlaid with top agar after 20 hours. Plaques were detected by
staining with neutral red on day 10 post infection. RPEs were
computed as the percentage of the titer of H5.CBLacZ virus as
compared to that on 293 cells. Cells were infected with H5.CBLacZ
over a range of serial dilutions and overlaid with top agar after
20 h.
[0085] Plaques were detected by staining with neutral red on day 10
post infection. 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, standard deviations. Two
cell lines, GH329 and GH354, were equivalent if not better than 293
cells in terms of plaquing efficacy (FIG. 2B) of E1-deleted
virus.
EXAMPLE 3
Detection of RCAs in the E1-Deleted Recombinant Virus Preps After
Multiple Passages in Either 293 or GH329 Cells
[0086] The propensity to generate RCA was studied by serially
passaging an E1-deleted LacZ virus (initially isolated on GH329)
and both GH329 and 293 cells. A portion of each lysate was used to
infect a non-E1 expressing cell line (A549) to assess for RCA,
which presented itself as cytopathology on serial passage and was
confirmed by DNA hybridization analysis, as follows. However, since
crude Hirt DNAs were used for the Southern blot analysis, it would
be difficult to use the assay to quantify the amount of RCAs in
each sample.
[0087] H5.CBLacZ virus was plaque-purified twice on GH329 cells
following the standard protocol (Gao et al., J. Virol.,
70:8934-8943 1996)]. The blue plaques identified by X-gal
histochemical staining were selected, expanded to a large prep in
GH329 cells and purified by CsCl gradient centrifugation. The
purified H5.CBLacZ virus was designated as passage 0 (P0) and used
for continuous passages on 293 and GH329 cells simultaneously for
up to 20 passages. Large-preparation viruses grown up in each cell
line were CsCl gradient-purified from passages 5, 10, 15 and 20.
A549 cells were obtained from ATCC and cultured in F-12K medium
supplemented with 10% FBS. For the RCA assay, cells were seeded at
a density of 1.times.10.sup.7 cells per an 150 mm plate 24 hours
prior to the virus infection. A total of 1.times.10.sup.8 PFU each
of testing viruses were diluted in 80 ml of F-12K medium with 2%
fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) and
added to four 150 mm plates of A549 cells after removal of the
growth medium from each plate. At 24 hours post infection, 1.6 mls
of FBS were added into each plate. As a positive control, 1 PFU of
Ad5 wild type virus was spiked into each of 1.times.10.sup.8 PFU
testing articles for the infection procedure described above.
Negative control plates were also analyzed in parallel. Infected
cells from each plate were harvested at 14 days later and lysed in
infection medium by three rounds of freezing-thawing. Twenty
percent of total cell lysate from each plate were used to infect
one plate of A549 cells following the protocol described above.
Seven days post infection, the plates were examined under a light
microscope for cytopathic effects. The RCA assay used in this study
can detect 1 PFU of RCA in 10.sup.8 PFUs of recombinant viruses.
The infected cells from each plate were harvested with the medium
and spun down for collection of the cell pellet. Each cell pellet
was resuspended in 0.5 ml of 10 mM Tris-Cl, pH 8.0 and lysed by
three cycles of freezing-thawing. After centrifugation in a
Sorvall-26 at 3,200 rpm for 15 min, the supernatant of each sample
was collected. One-third of each supernatant was mixed with an
equal volume of 2.times. pronase solution (2 mg/ml pronase, 100 mM
Tris-Cl, pH 7.6, 2 mM EDTA, 1% SDS, incubate the solution at 37 C
for 45 min), incubated at 37.degree. C. for 4 hours, extracted with
phenol-chloroform and ethanol precipitated. The crude viral DNA
samples were resuspended in equal volume of TE buffer and subjected
to Nsi I endonuclease digestion and Southern blot analysis. Blots
were hybridized with a 420 bp E1-Xba I/Cla I DNA probe.
[0088] The results showed that significant RCA emerged between
passage 5 and 10 on 293 cells whereas no RCA was detected after 20
passages on GH329. The sensitivity of the RCA assay was confirmed
by spiking a zero passage GH329 cells in the presence of vector
with 1 pfu of wild type Ad.
[0089] All publications cited in this specification are
incorporated herein by reference. While the invention has been
described with reference to a particularly preferred embodiment, it
will be appreciated that modifications can be made without
departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the appended claims.
* * * * *