U.S. patent application number 10/155649 was filed with the patent office on 2003-02-13 for novel adenovirus gene therapy vehicle and cell line.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Gao, Guang-Ping, Wilson, James M..
Application Number | 20030032613 10/155649 |
Document ID | / |
Family ID | 27040203 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032613 |
Kind Code |
A1 |
Gao, Guang-Ping ; et
al. |
February 13, 2003 |
Novel adenovirus gene therapy vehicle and cell line
Abstract
A novel adenovirus E1/E4 expressing packaging cell line is
provided, which permits the generation of recombinant adenoviruses
deleted in both gene regions. The E1/E4 deleted recombinant
adenovirus is capable of expressing a selected transgene product in
cells in vivo or in vitro. This recombinant virus is useful in the
treatment of genetic disorders.
Inventors: |
Gao, Guang-Ping; (Havertown,
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
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
|
Family ID: |
27040203 |
Appl. No.: |
10/155649 |
Filed: |
May 23, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10155649 |
May 23, 2002 |
|
|
|
09650594 |
Aug 30, 2000 |
|
|
|
09650594 |
Aug 30, 2000 |
|
|
|
08549489 |
Oct 27, 1995 |
|
|
|
6281010 |
|
|
|
|
08549489 |
Oct 27, 1995 |
|
|
|
08462014 |
Jun 5, 1995 |
|
|
|
5756283 |
|
|
|
|
Current U.S.
Class: |
514/44R ;
424/93.2; 435/235.1; 435/366 |
Current CPC
Class: |
C12N 2750/14143
20130101; Y10S 977/802 20130101; C12N 15/86 20130101; A61K 48/00
20130101; C12N 2710/10343 20130101; Y10S 977/907 20130101 |
Class at
Publication: |
514/44 ; 435/366;
424/93.2; 435/235.1 |
International
Class: |
A61K 048/00; C12N
005/08; C12N 007/00 |
Goverment Interests
[0002] This invention was supported by the National Institute of
Health Grant Nos. HD32649-01 and DK49136. The United States
government has rights in this invention.
Claims
What is claimed is:
1. A packaging cell line which expresses an adenovirus E1 gene and
an adenovirus E4 gene or a functional fragment of said E1 or E4
genes.
2. A stable packaging cell which comprises: (a) a first adenovirus
DNA under the regulatory control of a first promoter, wherein said
first adenovirus DNA encodes an adenovirus E1 protein; and (b) a
second adenovirus DNA under the regulatory control of an inducible
promoter, wherein said second adenovirus DNA encodes an adenovirus
E4 ORF6 protein, wherein said packaging cell contains no other
adenovirus E4 ORF DNA; and whereby, in the presence of an inducing
agent, said E1 and E4ORF6 proteins are expressed in amounts
sufficient to permit infection by, and recovery of, an adenovirus
having functional deletions in its E1 and E4 genes.
3. The cell of claim 2, which is a human cell.
4. The cell of claim 2 in which the inducing agent is a metal.
5. The cell of claim 2 in which the metal is zinc.
6. The cell of claim 2 in which the inducing agent is a
glucocorticoid.
7. The cell of claim 2 in which the inducing agent is
dexamethasone.
8. The cell of claim 2 in which the first adenovirus DNA and the
second adenovirus DNA are within the same nucleic acid
molecule.
9. The cell of claim 2 in which the nucleic acid expressing the
adenovirus E4ORF6 protein is pMMTVE4ORF6.
10. The cell of claim 2 in which the nucleic acid expressing the
adenovirus E4ORF6 protein is pMTE4ORF6.
11. The cell of claim 2 which is 293 (MT-ORF6).
12. The cell of claim 2 which is HeLa (MT-ORF6).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
09/650,594, filed Aug. 30, 2000, which is a continuation of U.S.
patent application Ser. No. 08/549,489, filed Oct. 27, 1995, now
U.S. Pat. No. 6,281,010, which is a continuation-in-part of U.S.
patent application Ser. No. 08/462,014, filed Jun. 5, 1995, now
U.S. Pat. No. 5,756,283. The disclosure of parent application Ser.
No. 08/462,014 is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to the field of somatic gene
therapy and the treatment of genetic disorders.
[0004] Adenoviruses are eukaryotic DNA viruses that can be modified
to efficiently deliver a therapeutic or reporter transgene to a
variety of cell types. Human adenoviruses are comprised of a
linear, approximately 36 kb double-stranded DNA genome, which is
divided into 100 map units (m.u.), each of which is 360 bp in
length. The DNA contains short inverted terminal repeats (ITR) at
each end of the genome that are required for viral DNA replication.
The gene products are organized into early (E1 through E4) and late
(L1 through L5) regions, based on expression before or after the
initiation of viral DNA synthesis [see, e.g., M. S. Horwitz et al,
"Adenoviridae and Their Replication", Virology, second edition, pp.
1712, ed. B. N. Fields et al, Raven Press Ltd., New York (1990)].
The adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), are not
associated with human malignancies.
[0005] Recombinant adenoviruses are capable of providing extremely
high levels of transgene delivery to virtually all cell types,
regardless of the mitotic state. The efficacy of this system in
delivering a therapeutic transgene in vivo that complements a
genetic imbalance has been demonstrated in animal models of various
disorders [K. F. Kozarsky et al, Somatic Cell Mol. Genet.,
19:449-458 (1993) ("Kozarsky et al I"); K. F. Kozarsky et al, J.
Biol. Chem., 269:13695-13702 (1994) ("Kozarsky et al II); Y.
Watanabe, Atherosclerosis, 36:261-268 (1986); K. Tanzawa et al,
FEBS Letters, 118(1):81-84 (1980); J. L. Golasten et al, New Engl.
J. Med., 309(11983) :288-296 (1983); S. Ishibashi et al, J. Clin.
Invest., 92:883-893 (1993); and S. Ishibashi et al, J. Clin.
Invest., 93:1885-1893 (1994)]. The use of recombinant adenoviruses
in the transduction of genes into hepatocytes in vivo has
previously been demonstrated in rodents and rabbits [see, e.g.,
Kozarsky II, cited above, and S. Ishibashi et al, J. Clin. Invest.,
92:883-893 (1993)].
[0006] The first-generation recombinant, replication-deficient
adenoviruses which have been developed for gene therapy contain
deletions of the entire E1a and part of the E1b regions. This
replication-defective virus is grown on an adenovirus-transformed,
complementation human embryonic kidney cell line containing a
functional adenovirus E1a gene which provides a transacting E1a
protein, the 293 cell [ATCC CRL1573]. E1-deleted viruses are
capable of replicating and producing infectious virus in the 293
cells, which provide E1a and E1b region gene products in trans. The
resulting virus is capable of infecting many cell types and can
express the introduced gene (providing it carries its own
promoter), but cannot replicate in a cell that does not carry the
El region DNA unless the cell is infected at a very high
multiplicity of infection.
[0007] However, in vivo studies revealed transgene expression in
these E1 deleted vectors was transient and invariably associated
with the development of severe inflammation at the site of vector
targeting [S. Ishibashi et al, J. Clin. Invest., 93:1885-1893
(1994); J. M. Wilson et al, Proc. Natl. Acad. Sci., USA, 85:4421-
4424 (1988); J. M. Wilson et al, Clin. Bio., 3:21-26 (1991); M.
Grossman et al, Som. Cell. and Mol. Gen., 17:601-607 (1991)].
Antigenic targets for immune mediated clearance are viral proteins
expressed from the recombinant viral genome and/or the product of
the transgene [Y. Yang et al, Proc. Natl. Acad. Sci., USA,
91:4407-4411 (May 1994); Y. Yang et al, Immun., 1:433-442 (August
1994)].
[0008] There remains a need in the art for additional recombinant
adenoviruses, therapeutic compositions and methods which enable
effective treatment of disorders and diseases by gene therapy.
SUMMARY OF THE INVENTION
[0009] In one aspect of this invention, a novel packaging cell line
is provided which expresses adenovirus genes E1a, E1b and E4, or
functional fragments thereof. In one embodiment, the E4 gene
fragment is open reading frame (ORF) 6 under the control of an
inducible promoter.
[0010] In another aspect, the invention provides a recombinant
adenovirus comprising the DNA of, or corresponding to, at least a
portion of the genome of an adenovirus having functional deletions
of the E1 and E4 gene regions; a suitable gene operatively linked
to regulatory sequences directing its expression, and an adenovirus
capsid, the recombinant virus capable of infecting a mammalian cell
and expressing the gene product in the cell in vivo or in vitro. In
a preferred embodiment, the cell is a muscle cell.
[0011] In another aspect, the invention provides a mammalian cell
infected with the recombinant virus described above.
[0012] In still another aspect, the invention provides a
recombinant adenovirus shuttle vector comprising the DNA of, or
corresponding to, at least a portion of the genome of an adenovirus
having functional deletions of the E1 and E4 gene regions; a
suitable gene operatively linked to regulatory sequences capable of
directing its expression; and plasmid sequences.
[0013] In still a further aspect, the invention provides a method
for delivering and stably integrating a selected gene into a
mammalian cell comprising introducing into said cell an effective
amount of a recombinant virus described above.
[0014] In another aspect, the invention provides a method for
producing the above-described recombinant Ad virus by
co-transfecting the shuttle vector described above and a helper
adenovirus into the packaging cell line described above, wherein
the transfected cell generates the recombinant adenovirus. The
recombinant adenovirus is subsequently isolated and purified
therefrom.
[0015] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic drawing of an exemplary plasmid useful
for the construction of a packaging cell line of this invention.
Plasmid pMMTVE4ORF6 or pMTE4ORF6, which contains a mouse mammary
tumor virus promoter (MMTV promoter) or a sheep metallothionine
promoter (MT promoter), respectively, in control of a human E4 ORF
6 gene sequence, a growth hormone gene terminator sequence (GH), an
SV40 origin of replication, plasmid sequences from a pBR322-based
plasmid including a neomycin resistance gene, an SV40
polyadenylation site and an ampicillin resistance gene.
[0017] FIGS. 2A through 2F provides the continuous DNA sequence
[SEQ ID NO: 1] of the minigene containing the MMTV promoter in
operative control of the adenovirus serotype 5 E4 gene open reading
frame 6. Nucleotides 1-1506 provide the MMTV promoter. Nucleotides
1523-2408 span E4 ORF6 and the amino acid sequence of ORF 6 [SEQ ID
NO: 2] is indicated under the ORF DNA sequence. Nucleotides
2409-3654 span the growth hormone gene (GH) terminator sequences,
which provide the polyadenylation site.
[0018] FIG. 3 is a schematic map of recombinant adenovirus
H5.001CBLacZ, with indicated restriction endonuclease enzyme sites.
The striated bar represents the CBLacZ minigene; the black bar
represents Ad5 viral backbone, the crosshatched bar represents Ad
E4 deletion.
[0019] FIGS. 4A through 4CC provide the DNA sequence [SEQ ID NO: 3]
of recombinant adenovirus H5.001CBLacZ in which nucleotides 1-330
span Ad map units 0-1; nucleotides 370-928 span the CMV
enhancer/chicken .beta.-actin promoter (CB);nucleotides 945-4429
encode E. coli .beta.-galactosidase, nucleotides 4429-4628 span the
polyadenylation sequence; and nucleotides 4671-35408 span Ad5
sequences m.u. 9.2 to about m.u. 92.1 and from about m.u. 97.3 to
m.u. 100 (containing a substantial deletion of the E4 gene between
m.u. 92 through 97.2).
[0020] FIG. 5 is a graph plotting LacZ forming units/ml vs time
(hours) for E4 complementing cell lines infected with
H5.001CBLacZ.
[0021] FIG. 6A is a graph of the induction, ORF6 expression and
viral production in 293-27-18 packaging cells plotting yield at 24
hours post-infection in LacZ forming units (LFU)/ml and ORF6
protein (abs.mm) vs. concentration of the inducer, dexamethasone
(.mu.M). The unit reference, abs.mm, indicates the intensity of the
size of the protein band on a Western blot and reflects absorbence
and protein size in mm.sup.2. The square represents the yield at 24
hours post infection. The diamond represents ORF6 protein detected
at 24 hours post-infection.
[0022] FIG. 6B is a graph of the induction, ORF6 expression and
viral production in 293-10-3 packaging cells plotting yield at 24
hours post-infection in LFU/ml and ORF6 protein (abs.mm) vs.
concentration of the inducer, zinc (.mu.M). The symbols are as
described for FIG. 6A.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides novel packaging cell lines,
which enable the production of recombinant adenoviruses
functionally deleted in both the E1 and E4 genes, and methods which
enable the therapeutic treatment of disorders with such recombinant
adenoviruses.
[0024] To increase the transgene capacity and decrease immune
response of recombinant adenoviral vectors, as many viral genes as
possible should be deleted to inactivate the adenovirus. However,
it is crucial to generate complementing cell lines for construction
and propagation of such deleted adenoviral vectors. The method and
compositions of the present invention overcome several problems
previously identified in the gene therapy for first generation E1
deleted adenoviruses and display advantages in administration
particularly to muscle tissue.
I. Novel Packaging Cell Lines
[0025] Early region 4 (E4) of adenovirus serotype 5 consists of 7
open reading frames (ORFs) believed to be involved in viral DNA
replication, host cell shut-off, and late mRNA accumulation. To
generate recombinant adenoviruses (Ad) deleted in E4, the function
of the E4 region must be supplied to the recombinant virus by a
helper virus or packaging cell line. However, useful packaging cell
lines have not been available because normally the continuous
expression of functioning Ad E1 and functional E4 in a single cell
line are toxic to the cell. Such cells are therefore not useful for
the growth and replication of recombinant adenoviruses. Further,
the DNA encoding the functional Ad E1 and Ad E4 genes, when present
in a packaging cell line, can increase the chances of recombination
with a recombinant Ad virus to cause the virus to revert to a
wildtype Ad virus.
[0026] The present invention avoids these problems by providing a
packaging cell line which contains the Ad5 E1 gene and only the ORF
6 of the Ad5 E4 gene. ORF6 of E4 alone can provide the requirements
for E4 in the viral life cycle. According to this invention, the
ORF6 is further preferably under the transcriptional control of an
inducible promoter, such as the sheep metallothionine promoter,
inducible by zinc, or the mouse mammary tumor virus (MMTV)
promoter, inducible by a glucocorticoid, particularly,
dexamethasone. This packaging cell line permits one to control the
development of toxicity by regulating the expression of the E4 ORF6
gene. After the desired shuttle vector containing the adenoviral
sequences is transfected into the cell line, expression of the E4
ORF6 can be induced by the appropriate inducer. The packaging cell
is thus able to provide both Ad E1 and Ad E4 ORF6 gene products to
the recombinant virus for a sufficient period to allow productive
infection and recovery of the recombinant virus, before the cell
becomes toxic. At present, the time period before the cell
experiences toxicity is about 10 days.
[0027] In its most preferred form, the packaging cell line is a
human embryonic kidney (HEK) 293 E1 expressing cell line into which
is introduced the E4 ORF 6 sequence under the control of an
inducible promoter. The MMTV promoter with its glucocorticoid
inducer is presently preferred, because the zinc sulfate inducer of
the MT promoter can itself be toxic to the cells. However, other
inducible promoters, such as those identified in International
patent application WO95/13392, published May 18, 1995, and
incorporated by reference herein may also be used in the production
of packaging cell lines according to this invention. Constitutive
promoters in control of the expression of ORF6 may be employed,
such as the constitutive Ad5 E4 region promoter, LTR, but are less
preferred.
[0028] It should be understood by one of skill in the art that
another parent cell line may be selected for the generation of a
novel cell line expressing the E1a, E1b, and E4 ORF6 genes of a
selected adenovirus serotype. Among such parent cell lines may be
included HeLa [CCL 2], A549 [CCL 185], KB [CCL 17], Detroit [e.g.,
Detroit 510, CCL 72] and WI-38 [ATCC CCL 75] cells. These cell
lines are all available from the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209. Other
suitable parent cell lines may be obtained from other sources. If
such parent cell lines were selected for modification, the cell
line would need to be further supplied with the E1a and E1b gene
functions, e.g., such as by transfection with a plasmid containing
these genes or functional fragments thereof under a suitable
promoter, as well as with the ORF6 gene as described herein.
[0029] Example 1 below provides specific teaching of the
construction of packaging cell lines containing only the ORF 6 of
Ad5 E4 region or, for functional comparisons, the entire E4 region.
Briefly described, the entire E4 region and an ORF6 sequence of Ad
5 E4 gene were obtained by known techniques [see, e.g., Sambrook et
al., "Molecular Cloning. A Laboratory Manual.", 2d edit., Cold
Spring Harbor Laboratory, New York (1989) and references cited
therein]. To isolate the ORF6 region, the anchored polymerase chain
reaction technique was used to amplify the ORF6 sequence from its
initiation codon to its termination codon. Primers selected from
the published sequence of ORF6 were used to amplify the ORF
sequence and insert restriction sites onto the end of the sequence.
The E4 ORF6 sequence itself is reproduced as nucleotides 1523
through 2408 of SEQ ID NO: 1 in FIG. 2. The entire E4 gene sequence
is published in the Genbank sequence of Ad5 [Genbank Accession No.
M73260].
[0030] A minigene was constructed that placed the ORF6 sequence
under the control of a selected promoter. By "minigene" as used
here is meant the combination of the ORF6 sequence and the other
regulatory elements necessary to transcribe the sequence and
express the gene product in a cell containing that minigene. The
ORF6 sequence gene is operatively linked to regulatory components
in a manner which permits its transcription. Such components
include conventional regulatory elements, such as a promoter to
drive ORF6 expression. One inducible promoter was an Zn.sup.+2
inducible sheep metallothionine (MT) promoter [M. G. Peterson et
al, Eur. J. Biochem., 174:417-424 (1988)]. The second promoter,
i.e, the promoter exemplified in FIG. 2, is the
dexamethasone-inducible mouse mammary tumor virus (MMTV) promoter.
The DNA sequence of the MMTV promoter spans nucleotides 1-1506 of
SEQ ID NO: 1 in FIG. 2.
[0031] The minigene also contains nucleic acid sequences
heterologous to the ORF6 viral sequence, including sequences
providing signals required for efficient polyadenylation of the
transcript (poly-A or pA). A common poly-A sequence which is
employed in this invention is that derived from the growth hormone
(GH) gene terminator sequence. The poly-A sequence generally is
inserted in the minigene following the ORF6 sequence. The polyA
sequence employed in the MMTV-ORF6 minigene described in Example 1
and FIG. 2 is supplied by the growth hormone gene terminator, which
spans nucleotides 2409-3654 of SEQ ID NO: 1 in FIG. 2 and an SV40
origin of replication. A similar minigene differing in promoter
sequence, polyA sequence and/or SV40 origin of replication sequence
can also be designed by one of skill in the art to transfer the E4
ORF6 sequence to a shuttle plasmid. Selection of these and other
common vector elements are conventional [see, e.g., Sambrook et al,
"Molecular Cloning. A Laboratory Manual.", 2d edit., Cold Spring
Harbor Laboratory, New York (1989) and references cited therein]
and many such sequences are available from commercial and
industrial sources as well as from Genbank.
[0032] The ORF6-containing minigene was subcloned into a
pBR322-based shuttle plasmid that contained a neomycin resistance
gene, resulting in the shuttle vector depicted in FIG. 1. Any of
the many known bacterial shuttle vectors may be employed to carry
the minigene, providing that the vector contains a reporter gene or
selectable marker of which many, e.g., neo, amp or purimycin, are
known in the art. It is expected that one of skill in the art can
develop other suitable shuttle vectors using other plasmid
components which are similarly capable of transferring the ORF6
minigene into the chromosome of a cell transfected with the
plasmid.
[0033] As further described in Example 1, other shuttle vectors
were designed for comparative purposes, which contain the complete
or substantially complete Ad5 E4 region under the control of the
constitutive retroviral MLV LTR sequence in the presence or absence
of the endogenous E4 promoter. The shuttle plasmid carrying the
ORF6 minigene (or the entire E4 region) was introduced into HEK 293
cells which express the Ad E1 gene products. Complementing cell
lines were generated that express these Ad E4 or ORF6 genes from
either their endogenous promoters or heterologous inducible
promoters. These cell lines are further characterized by their
genetic constitution, E4 protein synthesis, recombinant AAV helper
function, relative plaque efficiency of H5dl1004 virus, and growth
kinetics of recombinant E1/E4 deleted adenovirus. These
characteristics of exemplary E1/E4 expressing packaging cell lines
are discussed in detail in the following examples.
[0034] The E1/E4 ORF6 expressing packaging cell lines are useful in
the generation of recombinant E1/E4 deleted adenoviruses. These
recombinant adenoviruses are useful in transferring a selected
transgene to a selected cell. In in vivo experiments with the
recombinant virus grown in the packaging cell lines, the E1/E4
deleted recombinant virus demonstrated utility particularly in
transferring a transgene to a muscle cell.
II. Recombinant Adenovirus
[0035] The novel E1/E4 expressing cell line is useful in further
constructing E1/E4 deleted recombinant adenoviruses containing any
selected transgene. The recombinant adenoviruses of this invention
are capable of delivering a suitable gene to mammalian cells and
tissues. These recombinant adenoviruses are functionally deleted in
at least the E1a, E1b and E4 Ad gene regions. By the term
"functionally deleted" is meant that a sufficient amount of the
gene region is removed or otherwise damaged, e.g., by mutation or
modification, so that the gene region is no longer capable of
producing the products of gene expression. If desired, the entire
gene region may be removed.
[0036] The adenovirus sequences used in the construction of the
shuttle vectors, helper viruses, if needed, and recombinant
viruses, and other components and sequences employed in the
construction of the vectors and viruses described herein may be
readily obtained from commercial or academic sources based on
previously published and described sequences. Viral materials may
also be obtained from an individual patient. The viral sequences
and vector components may be generated by resort to the teachings
and references contained herein, coupled with standard recombinant
molecular cloning techniques known and practiced by those skilled
in the art. Modifications of existing nucleic acid sequences
forming the vectors, including sequence deletions, insertions, and
other mutations taught by this specification may be generated using
standard techniques. Similarly, the methods employed for the
selection of viral sequences useful in a vector, the cloning and
construction of the "minigene" and its insertion into a desired
viral shuttle vector and the production of a recombinant infectious
virus are within the skill in the art given the teachings provided
herein.
A. Construction of the Transgene containing "Minigene"
[0037] A "minigene" in this context is defined as above, except
that the components of this minigene are designed to express the
gene product in vivo. Such components include conventional
regulatory elements necessary to drive expression of the transgene
in a cell transfected with the recombinant virus. For this
minigene, a selected promoter is operatively linked to the
transgene and located, with other regulatory elements, within the
selected viral sequences of the recombinant vector. Selection of
the promoter is a routine matter and is not a limitation of this
invention. Useful promoters may be constitutive promoters or
regulated (inducible) promoters, which will enable control of the
amount of the transgene to be expressed. For example, a desirable
promoter is that of the cytomegalovirus (CMV) immediate early
promoter/enhancer [see, e.g., Boshart et al, Cell, 41:521-530
(1985)]. Another desirable promoter includes the Rous sarcoma virus
LTR promoter/enhancer. Still another promoter/enhancer sequence is
the chicken cytoplasmic .beta.-actin (CB) promoter [T. A. Kost et
al, Nucl. Acids Res., 11(23) :8287 (1983)]. Other suitable
promoters may be selected by one of skill in the art.
[0038] The minigene may also desirably contain nucleic acid
sequences heterologous to the viral vector sequences including
poly-A sequences and introns with functional splice donor and
acceptor sites, as described above. The poly-A sequence generally
is inserted in the minigene following the transgene sequences and
before the 3' adenovirus sequences. A minigene of the present
invention may also contain an intron, desirably located between the
promoter/enhancer sequence and the transgene. Selection of these
and other common vector elements are conventional as described
above and many such sequences are available from commercial and
industrial sources as well as from Genbank.
[0039] As above stated, the minigene is located in the site of any
selected deletion in the recombinant adenovirus. In the exemplary
E1/E4 deleted recombinant adenovirus H5.001CBLacZ, the transgene is
located in the deleted E1 gene region. However, the transgene may
be located elsewhere in the adenovirus sequence, as desired.
B. Production of Recombinant Adenovirus
[0040] Adenovirus sequences useful in this invention may include
the DNA sequences of a number of adenovirus types, which are
available from Genbank, including type Ad5 [Genbank Accession No.
M73260]. The adenovirus sequences may be obtained from any known
adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and
further including any of the presently identified 41 human types
[see, e.g., Horwitz, cited above]. Similarly, adenoviruses known to
infect other animals may also be employed in the vector constructs
of this invention. The selection of the adenovirus type is not
anticipated to limit the following invention. A variety of
adenovirus strains are available from the American Type Culture
Collection, Manassas, Virginia, or available by request from a
variety of commercial and institutional sources. In the following
exemplary embodiment an adenovirus, type 5 (Ad5) is used for
convenience.
[0041] However, it is desirable to obtain a variety of adenovirus
shuttle vectors based on different human adenovirus serotypes. It
is anticipated that a library of such plasmids and the resulting
recombinant adenoviruses would be useful in a therapeutic regimen
to evade cellular, and possibly humoral, immunity, and lengthen the
duration of transgene expression, as well as improve the success of
repeat therapeutic treatments. Additionally the use of various
serotypes is believed to produce recombinant viruses with different
tissue targeting specificities. Additionally, the absence of
adenoviral genes E1 and E4 in the recombinant adenovirus of this
invention should reduce or eliminate adverse CTL responses which
normally cause destruction of recombinant adenoviruses deleted of
only the E1 gene.
[0042] Recombinant adenoviruses of this invention are recombinant,
defective adenoviruses (i.e., E1 deleted) which are also deleted
completely or functionally of the E4 gene region. Functional
deletions of E4 gene regions may be assessed by assays of Examples
2 and 3, among other assays. Recombinant adenoviruses of useful in
this invention may optionally bear other mutations, e.g.,
temperature sensitive mutations in the E2a gene region, and
deletions in the E3 gene regions.
[0043] An adenovirus of this invention contains a functional
deletion of the adenoviral early immediate early gene E1a (which
spans mu 1.3 to 4.5) and delayed early gene E1b (which spans mu 4.6
to 11.2). Similarly the adenovirus has a functional deletion of the
E4 region (which spans mu 92 to 97.2), or at least of ORF6 of the
E4 region.
[0044] Gene regions which may be optionally deleted in the E1/E4
deleted recombinant viruses of this invention include all or a
portion of the adenovirus delayed early gene E3 (which spans mu
76.6 to 86.2). The function of E3 is irrelevant to the function and
production of the recombinant virus particle.
[0045] The recombinant adenovirus of this invention may also have a
mutation which results in reduced expression of adenoviral protein
and/or reduced viral replication. For example, a
temperature-sensitive mutation may be introduced into the
adenovirus delayed early gene E2a (which spans mu 67.9 to 61.5).
Among such mutations include the incorporation of the missense
temperature-sensitive (ts) mutation in the (DBP) E2a region found
in the Ad5 H5ts125 strain [P. Vander Vliet et al, J. Virol.,
15:348-354 (1975)] at 62.5 mu. A single amino acid substitution
(62.5 mu) at the carboxy end of the 72 kd protein produced from the
E2a gene in this strain produces a protein product which is a
single-stranded DNA binding protein and is involved in the
replication of adenoviral genomic DNA. At permissive temperatures
(approximately 32.degree. C. ) the ts strain is capable of full
life cycle growth on HeLa cells, while at non-permissive
temperatures (approximately 38.degree. C. ) no replication of
adenoviral DNA is seen. In addition, at non-permissive
temperatures, decreased immunoreactive 72 kd protein is seen in
HeLa cells. See, e.g., J. F. Engelhardt et al, Hum. Gene Ther.,
5:1217-1229 (1994); J. F. Engelhardt et al, Proc. Natl. Acad. Sci.,
USA, 91:6196-6200 (1994) and International patent application Ser.
No. WO95/13392, published May 18, 1995, incorporated by reference
herein.
[0046] However, it should be understood that other deletions in the
adenovirus genome as previously described in the art or otherwise
may also occur in the recombinant viruses of this invention. One
minimal type of recombinant adenovirus can contain adenovirus
genomic sequences from which all viral genes are deleted. More
specifically, the adenovirus sequences may be only the 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, that contains sequences necessary for
packaging linear Ad genomes and enhancer elements for the E1
promoter. The adenovirus 5' sequence containing the 5' ITR and
packaging/enhancer region (Ad5 mu 0-1 or bp 1-360) can be employed
as the 5' adenovirus sequence in recombinant adenoviruses of this
invention. The 3' adenovirus sequences including the right terminal
(3') ITR sequence of the adenoviral genome spanning about bp
35,353--end of the adenovirus genome, or map units
.sup..about.98.4-100 may be desirably employed as the 3' sequence
of the recombinant adenovirus. These sequences, which are clearly
devoid of the E1 and E4 genes, can flank, or be operatively
associated with the minigene in a recombinant virus. Any other
necessary Ad gene products will then be supplied by helper viruses
and the E1/E4 ORF6 expressing packaging cell of this invention.
[0047] Exemplary recombinant adenoviruses for use in this
invention, for example, may be obtained by homologous recombination
of desired fragments from various recombinant adenoviruses, a
technique which has been commonly employed to generate other
recombinant adenoviruses for gene therapy use. In the examples
below, a representative recombinant adenovirus, H5.001CBLacZ, is
constructed by homologous recombination between the adenovirus
dl1004 (also H5dl1004) viral backbone and pAdCBLacZ minigene DNA.
H5dl1004 is an Ad5 virus deleted of from about map unit 92.1
through map unit 98, i.e, substantially the entire E4 gene. The
dl1004 virus is described in Bridge and Ketner, J. Virol., 632(2)
:631-638 (Feb. 1989), incorporated by reference herein.
[0048] The pAdCBLacZ vector is a cDNA plasmid containing Ad m.u.
0-1, an E1 deletion into which is inserted a bacterial
.beta.-galactosidase gene under the control of a chicken
.beta.-actin promoter, with other regulatory elements as described
below, and flanked by Ad m.u. 9-16 and plasmid sequence.
[0049] The production of the E1/E4 recombinant adenovirus of this
invention in the novel packaging cell line of this invention
utilizes conventional techniques. Such techniques include
conventional cloning techniques of cDNA such as those described in
texts [Sambrook et al, cited above], use of overlapping
oligonucleotide sequences of the adenovirus genomes, polymerase
chain reaction, and any suitable method which provides the desired
nucleotide sequence. Standard transfection and co-transfection
techniques are employed, e.g., CaPO.sub.4 transfection techniques
using the complementation 293 cell line. Other conventional methods
employed include homologous recombination of the viral genomes,
plaquing of viruses in agar overlay, methods of measuring signal
generation, and the like.
[0050] For example, following the construction and assembly of the
desired minigene-containing plasmid vector pAdCBLacZ, the E1/E4
expressing packaging cell line of this invention is infected with
the helper virus H5dl1004. The infected cell line is then
subsequently transfected with the an adenovirus plasmid vector by
conventional methods. Homologous recombination occurs between the
E4-deleted H5dl1004 helper and the pAdCBLacZ vector, which permits
the adenovirus-transgene sequences in the vector to be replicated
and packaged into virion capsids, resulting in the recombinant
virus. About 30 or more hours post-transfection, the cells are
harvested, an extract prepared and the recombinant virus containing
the LacZ transgene is purified by buoyant density
ultracentrifugation in a CsCl gradient.
III. Use of the Recombinant Virus in Gene Therapy
[0051] The resulting recombinant adenovirus containing the
transgene produced by cooperation of the adenovirus vector and E4
deleted helper virus and packaging cell line, as described above,
thus provides an efficient gene transfer vehicle which can deliver
the transgene to a patient in vivo or ex vivo and provide for
integration of the gene into a mammalian cell.
[0052] The above-described recombinant viruses are administered to
humans in a conventional manner for gene therapy and serve as an
alternative or supplemental gene therapy for the disorder to which
the transgene is directed. A recombinant adenovirus of this
invention may be administered to a patient, preferably suspended in
a biologically compatible solution or pharmaceutically acceptable
delivery vehicle. A suitable vehicle includes sterile saline. Other
aqueous and non-aqueous isotonic sterile injection solutions and
aqueous and non-aqueous sterile suspensions known to be
pharmaceutically acceptable carriers and well known to those of
skill in the art may be employed for this purpose.
[0053] The recombinant viruses are administered in sufficient
amounts to transfect the desired target cells, e.g., muscle, liver,
epithelial, etc. and provide sufficient levels of transfer and
expression of the transgene to provide a therapeutic benefit
without undue adverse or with medically acceptable physiological
effects which can be determined by those skilled in the medical
arts. Conventional and pharmaceutically acceptable routes of
administration include direct delivery to the muscle or other
selected cell, intranasal, intravenous, intramuscular,
subcutaneous, intradermal, oral and other parental routes of
administration. Routes of administration may be combined, if
desired.
[0054] Dosages of recombinant virus will depend primarily on
factors such as the condition being treated, the age, weight and
health of the patient, and may thus vary among patients. For
example, a therapeutically effective human dose of the recombinant
adenovirus is generally in the range of from about 20 to about 100
ml of saline solution containing concentrations of from about
1.times.10.sup.9 to 1.times.10.sup.11 pfu/ml virus. A preferred
human dose is estimated to be about 50 ml saline solution at
2.times.10.sup.10 pfu/ml. The dose will be adjusted to balance the
therapeutic benefit against any side effects. The levels of
expression of the transgene can be monitored to determine the
frequency of administration.
[0055] An optional method step involves the co-administration to
the patient, either concurrently with, or before or after
administration of the recombinant virus of a suitable amount of a
short acting immune modulator. The selected immune modulator is
defined herein as an agent capable of inhibiting the formation of
neutralizing antibodies directed against the recombinant vector of
this invention or capable of inhibiting or substantially delaying
cytolytic T lymphocyte (CTL) elimination of the vector. Among
desirable immune modulators are interleukin-12 [European Patent
Application No. 441,900]; gamma interferon [S. C. Morris et al, J.
Immunol., 152:1047 (1994)]; interleukin-4 [U.S. Pat. No.
5,017,691]; antibody to the CD4 protein, such as anti-OKT 3+ [see,
e.g., U.S. Pat. No. 4,658,019] or antibody GK1.5 (ATCC Accession
No. TIB207); a soluble CD40 molecule or an antibody to CD40 ligand
(Bristol-Myers Squibb Co) [European patent application 555,880,
published Aug. 18, 1993]; a soluble form of B7 or an antibody to
CD28 or CTLA4 [CTLA4-Ig (Bristol-Myers Squibb Co), European patent
application 606,217, published Jul. 20, 1994], or agents such as
cyclosporin A or cyclophosphamide.
[0056] Thus, the compositions and methods of this invention provide
a desirable gene therapy treatment.
[0057] The following examples illustrate the construction and
testing of the novel packaging cell lines, the E1/E4 deleted
recombinant adenovirus of the present invention and the use
thereof. These examples are illustrative only, and do not limit the
scope of the present invention.
EXAMPLE 1
Novel E1a/E1b and E4 Expressing Packaging Cell Lines
A. Construction of E4 ORF 6 Expressing Plasmids
[0058] 1. pMTE4ORF6
[0059] One exemplary plasmid useful for the construction of a
packaging cell line of this invention is pMTE4ORF6, which contains
a sheep metallothionine promoter (MT promoter) [M. G. Peterson et
al, cited above] in control of the transcription of a human E4 ORF
6 gene sequence (nucleotides 1521 to 2406 of SEQ ID NO: 1 in FIG.
2), a growth hormone terminator (GH), an SV40 origin of
replication, plasmid sequences from pBR322-based plasmid including
a neomycin resistance gene, an SV40 polyadenylation site and an
ampicillin resistance gene.
[0060] The various functional fragments of this plasmid may be
readily replaced with other conventionally used sequences and are
not critical to the design of the plasmid.
[0061] 2. pMMTVE4ORF6
[0062] Another exemplary plasmid useful for the construction of a
packaging cell line of this invention is pMMTVE4ORF6, which
contains a mouse mammary tumor virus promoter (MMTV) (nucleotides
1-1506 of SEQ ID NO:1 in FIG. 2) in transcriptional control of a
human E4 ORF 6 gene sequence (nucleotides 1523-2408 of SEQ ID NO: 1
in FIG. 2), a growth hormone terminator (GH) (nucleotides 2409-3654
of SEQ ID NO: 1 in FIG. 2), an SV40 origin of replication, plasmid
sequences from plasmid pBR322, including a neomycin resistance
gene, and an ampicillin resistance gene. The various functional
fragments of this plasmid may be readily replaced with other
conventionally used sequences and are not critical to the design of
the plasmid.
[0063] 3. pLTR.E4(-) Endogenous E4 Promoter
[0064] A plasmid used as a control for the construction of a
packaging cell line of this invention is pLTR.E4(-). This plasmid
contains the constitutive retroviral MLV LTR and most of the Ad E4
gene region except that the endogenous E4 promoter and a portion of
E4 ORF1 are missing. The other plasmid sequences remain the same as
described above.
[0065] 4. pLTR.E4(+) Endogenous E4 Promoter
[0066] Still another plasmid useful for the study of the methods of
this invention is pLTR.E4, which contains the constitutive MLV LTR
and endogenous E4 promoter and an intact E4 gene. The other plasmid
sequences remain the same as described above.
B. Transfections and Selection of Clones
[0067] Each of the above-described plasmids was transfected by the
calcium phosphate precipitation technique into the human embryonic
kidney cell line 293 [ATCC CRL1573] which expresses the product of
the adenovirus E1 genes, seeded on 100 mm plates (10 .mu.g
plasmid/plate). Twenty four hours post-transfection, cells were
harvested and seeded at varying dilutions (1:10-1:100) in 100 mm
plates for about 10 days. Seeding media contain G418 (Geneticin,
BRL) at 1 mg/ml. Resistant colonies that developed were selected
using the following assays and expanded. Preliminary analysis of
clones was based on enhanced transduction efficiency of a
recombinant adeno-associated virus, AV.CMVLacZ, and
immunofluorescence localization of Ad E4 protein as described in
the following examples.
EXAMPLE 2
AV-CMVLacZ Transduction Enhancement Assay
[0068] E1 and E4 Ad gene products are needed for recombinant
adeno-associated virus (AAV) function. This primary assay involves
seeding the packaging cell lines of Example 1 in 96 well 35 mm
culture plates (2.times.10.sup.6 cells/well) and infecting the
cells with purified, heat-treated AV.CMVLacZ at an MOI of 1000
virus particles/cell.
A. Preparation of AV.CMVLacZ
[0069] A recombinant AAV virus was prepared by conventional genetic
engineering techniques for the purposes of this experiment.
Recombinant AAV was generated by plasmid transfections in the
presence of helper adenovirus [Samulski et al, J. Virol.,
63:3822-3828 (1989)]. A cis-acting plasmid pAV.CMVLacZ was derived
from psub201 [Samulski et al, J. Virol., 61:3096-3101 (1987)] and
contains an E. coil .beta. galactosidase minigene in place of AAV
Rep and Cap genes. The 5' to 3' organization of the recombinant
AV.CMVLacZ genome (4.9 kb) includes
[0070] (a) the 5' AAV ITR (bp 1-173) was obtained by PCR using pAV2
[C. A. Laughlin et al, Gene, 23: 65-73 (1983)] as template;
[0071] (b) a CMV immediate early enhancer/promoter [Boshart et al,
Cell, 41:521-530 (1985)];
[0072] (c) an SV40 intron;
[0073] (d) E. coli beta-galactosidase cDNA;
[0074] (e) an SV40 polyadenylation signal (a 237 Bam HI-BclI
restriction fragment containing the cleavage/poly-A signals from
both the early and late transcription units; and
[0075] (f) 3' AAV ITR, obtained from pAV2 as a SnaBI-BglII
fragment.
[0076] Rep and Cap genes were provided by a trans-acting plasmid
pAAV/Ad [Samulski et al, cited above].
[0077] Monolayers of 293 cells grown to 90% confluency in 150 mm
culture dishes (5.times.10.sup.7 cells/plate) were infected with
H5.CBALP at an MOI of 10. H5.CBALP (also called H5.010ALP) is a
recombinant adenovirus that contains an alkaline phosphatase
minigene in place of adenovirus E1a and E1b gene sequences (map
units 1-9.2 of the Ad5 sequence of GenBank [Accession No. M73260]).
The alkaline phosphatase cDNA is under the transcriptional control
of a CMV-enhanced .beta.-actin promoter in this virus. This helper
virus is described in Goldman et al, Hum. Gene Ther., 6:839-851
(July, 1995); Engelhardt et al, Hum. Gene Ther., 5:1217-1229
(October, 1994); and references cited therein.
[0078] Infections were done in Dulbecco's Modified Eagles Media
(DMEM) supplemented with 2% fetal bovine serum (FBS) at 20 ml
media/150 mm plate. Two hours post-infection, 50 .mu.g plasmid DNA
(37.5 .mu.g trans-acting and 12.5 .mu.g cis-acting) in 2.5 ml of
transfection cocktail was added to each plate and evenly
distributed. Transfections were calcium phosphate based as
described [B. Cullen, Meth. Enzymol., 152:684-704 (1987)]. Cells
were left in this condition for 10-14 hours after which the
infection/transfection media was replaced with 20 ml fresh DMEM/2%
FBS. Forty to fifty hours post-transfection, cells were harvested,
suspended in 10 mM Tris-Cl (pH 8.0) buffer (0.5 ml/150 mm plate)
and a lysate prepared by sonication. The lysate was brought to 10
mM manganese chloride, after which bovine pancreatic DNase I
(20,000 units) and RNase (0.2 mg/ml final concentration) were
added, and the reaction incubated at 37.degree. C. for 30 minutes.
Sodium deoxycholate was added to a final concentration of 1% and
incubated at 37.degree. C. for an additional 10 minutes.
[0079] The treated lysate was chilled on ice for 10 minutes and
solid CsCl added to a final density of 1.3 g/ml. The lysate was
brought to a final volume of 60 ml with 1.3 g/ml CsCl solution in
10 mM Tris-Cl (pH 8.0) and divided into three equal aliquots. Each
20 ml sample was layered onto a CsCl step gradient composed of two
9.0 ml tiers with densities 1.45 g/ml and 1.60 g/ml.
[0080] Centrifugation was performed at 25,000 rpm in a Beckman
SW-28 rotor for 24 hours at 4.degree. C.
[0081] Fractions containing peak titers of functional AV.CMVLacZ
virus were combined and subjected to three sequential rounds of
equilibrium sedimentation in CsCl. Rotor selection included a
Beckman NVT-90 (80,000 rpm for 4 hours) and SW-41 (35,000 rpm for
20 hours). At equilibrium, AV.CMVLacZ appeared as an opalescent
band at 1.40-1.41 g/ml CsCl. Densities were calculated from
refractive index measurements. Purified vector was exchanged to 20
mM HEPES buffer (pH7.8) containing 150 mM NaCl (HBS) by dialysis
and stored frozen at -80.degree. C. in the presence of 10% glycerol
or as a liquid stock at -20.degree. C. in HBS/40% glycerol.
[0082] Purified virus was tested for contaminating H5.CBALP helper
virus and AV.CMVLacZ titers. Helper virus was monitored by
histochemical staining for reporter alkaline phosphatase activity.
A sample of purified virus representing 1.0% of the final product
was added to a growing monolayer of 293 cells seeded in a 60 mm
plate. Forty-eight hours later, cells were fixed in 0.5%
glutaraldehyde/phosphate buffered saline (PBS) for 10 minutes at
room temperature, washed in PBS (3.times.10 minutes) and incubated
at 65.degree. C. for 40 minutes to inactivate endogenous alkaline
phosphatase activity. The monolayer was allowed to cool to room
temperature, rinsed once briefly in 100 mM Tris-Cl (pH9.5)/100 mM
NaCl/5 mM MgCl, and incubated at 37.degree. C. for 30 minutes in
the same buffer containing 0.33 mg/ml nitroblue tetrazolium
chloride (NBT) and 0.165 mg/ml 5-bromo-4-choro-3-indolphosphate
p-toluidine salt (BCIP). Color development was stopped by washing
the monolayer in 10 mM Tris-Cl (pH 8.0)/5 mM EDTA. Routinely the
purification scheme described above removed all detectable H5.CBALP
helper virus by the third round of buoyant density
ultracentrifugation.
[0083] AV.CMVLacZ titers were measured according to genome copy
number (virus particles/ml), absorbance at 260 nm (A.sub.260
particles/ml) and LacZ Forming Units (LFU/ml). Virus particle
concentrations were based on Southern blotting. Briefly, a sample
of purified AV.CMVLacZ was treated with capsid digestion buffer (50
mM Tris-Cl, pH 8.0/1.0 mM EDTA, pH 8.0/0.5% SDS/Proteinase K 1.0
mg/ml) at 50.degree. C. for one hour to release virus DNA. The
reactions were allowed to cool to room temperature, loading dye was
added and electrophoresed through a 1.2% agarose gel. Standard
quantities of ds AV.CMVLacZ genome were also resolved on the
gel.
[0084] DNAs were electroblotted onto a nylon membrane, hybridized
with a .sup.32P random primer labeled restriction fragment, and the
resulting blot scanned on a PhosphorImager 445 SI (Molecular
Dynamics). A standard curve was generated from the duplex forms and
used to extrapolate the number of virus genomes in the sample. LFU
titers were generated by infecting indicator cells with limiting
dilutions of virus sample. Indicator cells included HeLa and 293.
Twenty-four hours later, cells were fixed in glutaraldehyde and
cells were histochemically stained for E. coli .beta.-galactosidase
(LacZ) activity as described in J. M. Wilson et al, Proc. Natl.
Acad. Sci. USA, 85:3014-3018 (1988). One LFU is described as the
quantity of virus that is sufficient to cause visually detectable
.beta.-galactosidase expression in one cell 24 hours
post-infection.
B. Induction of ORF6 Expression
[0085] Induction of ORF6 expression with 10 .mu.M dexamethasone or
150 .mu.M zinc sulfate (for negative control, no inducer used) was
initiated 2 hours before the addition of virus and continued
throughout the duration of the experiment. Twenty-four hours after
the addition of virus, cells were harvested, lysates were generated
by sonication and analyzed for the .beta.-galactosidase expression
(i.e., .beta.-galactosidase activity) and virus DNA as described
above. Hirt extracts were prepared from low molecular weight DNA
from cell extracts. The preparation of the Hirt extracts and
subsequent analysis by Southern hybridization were performed by
resort to conventional procedures known to one of skill in the
art.
[0086] In the absence of the inducers, the packaging cell lines
generate lower levels of .beta.-galactosidase in rAAV infected
cells. Induction of ORF6 expression with the inducer dexamethasone
results in a concomitant rise in AV.CMVLacZ cell transduction to a
level that was much greater than the parent 293 line. Expression of
E1 alone was insufficient to have an effect in the adenovirus
mediated augmentation of rAAV transduction.
[0087] Results are demonstrated for certain positive clones in the
Table I below (see Example 4). However, for 30 cell lines having an
MMTV promoter and ORF6 sequence, 4 demonstrated over 90% blue cells
illustrative of LacZ production in the presence of dexamethasone,
i.e., 293-27-6, 293-27-17, 293-27-18 and 293-27-28.
EXAMPLE 3
Immunofluorescence Localization of Ad5 Late Protein
[0088] Positive clones from the assay of Example 2 were infected
with the recombinant E4 deleted adenovirus H5dl1004 and screened
for E4 complementation using an immunofluorescence assay for late
gene expression. The H5dl1004 virus was obtained from Dr. Ketner of
Johns Hopkins University and is described in Bridge and Ketner, J.
Virol., 632(2) :631-638 (Feb. 1989), incorporated by reference
herein. Because ORF6 of E4 complements late Ad gene expression,
specifically in the formation of the hexon and penton fibers of the
adenovirus, cell lines containing ORF6 are able to bind with
antibody against these proteins.
[0089] Each cell line of Example 1 is infected with E4 deleted
virus H5dl1004 virus at an MOI of 0.1. The cells were treated with
mouse anti-adenovirus FITC-labeled monoclonal antibody to either
the hexon or penton fibers in a 1:10 dilution (Chemicon
International Inc., Temecula, Calif. ). Positive clones were
identified by reaction with the antibody.
EXAMPLE 4
Relative Plaguing Efficiency
[0090] The cell lines of Example 1 demonstrating with strong
complementation ability in Example 3 were screened for relative
plaquing efficiency of H5dl1004 as compared to W162 cells (an
E4-complementing Vero cell line which does not express E1)
[Weinberg and Ketner, Proc. Natl. Acad. Sci, USA, 80(17) :5383-5386
(1983)]. In Table II below, RPE%, i.e., relative plaguing
efficiency, represents the titer of H5dl1004 on tested cell
lines/titer of H5dl1004 on W162 cells. For example, the RPE of 293
cells is 0.
[0091] The positive cell lines selected by all criteria are
identified in Table I below, with the results of the assays of
Examples 2, 3 and 4.
1TABLE I E1/E4 Double Complementing Cell Lines Cell Trans- Pro-
AV.CMV Line Gene moter IF/LP LacZ RPE % 293-10-3 ORF6 MT ++++ ++++
246 293-39-11 ORF6 LTR ++++ +++ 52 293-84-31 E4- LTR ++++ ++++ 179
293-12-31 whole LTR + ++++ ++++ 174 E4 E4 293-27-6 ORF6 MMTV +++++
327 293-27-17 ORF6 MMTV ++++ 313 293-27-18 ORF6 MMTV +++++ 339
293-27-28 ORF6 MMTV ++++ 261
EXAMPLE 5
Construction and Purification of H5.001CBLacZ
[0092] The plasmid pAd.CBLacZ was constructed as described in
detail in K. Kozarsky et al, Som. Cell Mol. Genet., 19(5): 449-458
(1993), incorporated by reference herein. This plasmid contained a
minigene comprising a 5' flanking NheI restriction site, followed
by Ad5 sequence m.u. 0-1, followed by an E1 deletion into which is
inserted a CMV enhancer/chicken .beta.-actin promoter sequence [T.
A. Kost et al, Nucl. Acids Res., 11(23) :8287 (1983)], which
controls the transcription of the following bacterial
.beta.-galactosidase, followed by a poly A sequence and flanked 3'
by Ad m.u. 9-16, and another NheI site. In the plasmid, the
minigene was flanked on both sides by plasmid sequence containing
drug resistance markers.
[0093] The plasmid pAd.CBLacZ was linearized with NheI and
co-transfected by the calcium phosphate co-transfection method into
the novel packaging cell line of Example 1 with ClaI digested
H5dl1004 (an Ad5 sequence deleted of from about map unit 92.1
through map unit 98, corresponding to substantially the entire E4
gene).
[0094] Homologous recombination occurs in the cell line between
these two viral constructs between Ad map units 9-16, resulting in
recombinant adenovirus, designated H5.001CBLacZ (FIGS. 3 and 4 ).
This recombinant adenovirus contains the sequence from about
nucleotide 1 to about 4628 from pAd.CBLacZ and Ad5 map units 9-92.1
and 97.3 to 100 from H5dl1004. This recombinant adenovirus is
thereby functionally deleted, and substantially structurally
deleted, of the Ad E1 and E4 genes.
[0095] Viral plaques were selected and screened by the
.beta.-galactosidase assay [Wilson (1988), cited above] and
H5.001CBLacZ was isolated following three rounds of plaque
purification. The purified virus was also subjected to cesium
chloride density centrifugation and large scale production. For the
following mouse experiments, virus was used after column
purification and glycerol was added to a final concentration of 10%
(v/v). Virus was stored at -70.degree. C. until use.
EXAMPLE 6
Growth Kinetics of H5.001CBLacZ in Packaging Cell Lines
[0096] The cell lines reported in Example 1 were infected with
recombinant H5.001CBLacZ at an MOI of 0.5. The growth kinetics of
this virus in the E4 complementing cell lines are shown in FIG.
5.
[0097] Maximum viral yield is reported as LFU/ml in Table II
below.
2 TABLE II Cell Line Maximum Viral Yield 293-10-3 2.8 .times.
10.sup.10 293-39-11 9.5 .times. 10.sup.8 293-84-31 1.1 .times.
10.sup.9 293-12-31 4.5 .times. 10.sup.8 293-27-6 2.8 .times.
10.sup.10 293-27-17 2.5 .times. 10.sup.10 293-27-18 2.9 .times.
10.sup.10 293-27-28 1.2 .times. 10.sup.10
[0098] When grown in 293-27-18 cells (the E4 ORF6 cell line with
MMTV promoter inducible by dexamethasone) the maximum yield of this
virus is 2.9.times.10.sup.10 LFU/ml. Several of the cell lines were
passaged between 5 and 20 times and the viral production of the
passages remained stable. However, RPE did fall following repeated
passages of cells.
EXAMPLE 7
Other Recombinant Adenoviruses
[0099] Other related recombinant adenoviruses were prepared
similarly to H5.001CBLacZ by homologous recombination between
pAdCBLacZ and other helper viruses.
[0100] As one example, H5.000CBLacZ is a recombinant E1 deleted Ad5
which contains the same minigene as H5.001CBLacZ, but has an intact
E4 gene. This recombinant virus was prepared as described by
homologous recombination between pAdCBLacZ and a wild-type Ad5.
[0101] As another example, H5.010CBLacZ contains the adenovirus map
units 0-1, followed by a CMV enhanced, chicken cytoplasmic
.beta.-actin promoter, the E. Coli beta-galactosidase gene (lacZ),
a polyadenylation signal (pA), and adenovirus type 5 map units
9-100, with a small deletion in the E3 gene (the Ad 5 sub360
backbone). This recombinant virus may be prepared by homologous
recombination between the pAdCBLacZ vector and Ad5 virus sub360,
which contains a 150 bp deletion within the 14.6 kD protein of the
E3 gene. See, e.g., J. F. Engelhardt et al, Proc. Natl. Acad. Sci.,
USA, 91:6196-6200 (June 1994); and Engelhardt et al, Hum. Gene
Ther., 5:1217-1229 (Oct. 1994), both incorporated by reference
herein.
[0102] These recombinant adenoviruses were isolated following
transfection [Graham, Virol., 52:456-467 (1974)], and were
subjected to two rounds of plaque purification. Lysates were
purified by cesium chloride density centrifugation as previously
described [Englehardt et al, Proc. Natl. Acad. Sci. USA,
88:11192-11196 (1991)]. Cesium chloride was removed by passing the
virus over a BioRad DG10 column using phosphate-buffered
saline.
EXAMPLE 8
LacZ Gene Transfer into Mouse
A. Transfer into Mouse Muscle
[0103] Five to six-week old male C57B/6 mice were anesthetized.
Anterior tibialis muscles were exposed and directly injected with
either recombinant adenovirus H5.000CBLacZ, H5.010CBLacZ or
H5.001CBLacZ as follows: 25 .mu.L of purified viral suspension at a
stock concentration of 5.times.10.sup.11 virus particles/mL was
injected by inserting the tip of the 33 gauge needle of a 100 .mu.L
Hamilton syringe into the belly of the muscle.
[0104] Animals were sacrificed on day 4, 14, 28 and 60 post
injection. The muscles were dissected and frozen in liquid nitrogen
cooled isopentane. Six .mu.M sections were cut in a cryostat, fixed
and stained for .beta.-galactosidase activity for 6 hours at
37.degree. C.
[0105] While the blue stained recombinant virus was found for each
virus in the day 4 and day 14 (most abundant) stains, by day 28,
the H5.001CBLacZ clearly demonstrated more virus on day 28. By day
60, the only virus which stained positive was the H5.001CBLacZ.
B. Transfer into Mouse Lung and Circulation
[0106] Recombinant adenovirus H5.000CBLacZ (control), and
H5.001CBLacZ (1.times.10.sup.11 viral particles) were administered
to six week old C57BL/6 female mice by tail vein injection and
trachea installation. The animals were sacrificed and their liver
and lung tissues were harvested at days 4, 9, 21, 28 and 35
post-administration. The transgene and viral late gene expression
were compared.
[0107] At therapeutic doses of virus, there was diminished
expression of late viral proteins at all time points in comparison
with transgene.
C. Dose Responses in Liver
[0108] Dose responses of E4-deleted and E4 intact recombinant
adenoviruses in the liver of C57BL/6 mice were studied by tail vein
administration of 1.5.times.10.sup.11, 5.times.10.sup.10,
1.7.times.10.sup.10, 5.6.times.10.sup.9, and 1.9.times.10.sup.9
viral particles and comparing the transgene and viral late gene
expression at day 4, 21, 28, 35, and 42 post administration.
[0109] At therapeutic doses of virus, there was diminished
expression of late viral proteins at all time points in comparison
with transgene.
EXAMPLE 9
Other Gene Transfers
A. Human OTC Gene Transfer
[0110] The human OTC gene [A. L. Horwich et al, Science,
224:1068-174 (1984)] or the human CFTR gene [Riordan et al,
Science, 245:1066-1073 (1989)] was used to replace the LacZ as the
transgene in the recombinant E1/E4 deleted adenoviruses described
above, using the techniques analogous for the construction of the
above-described LacZ vectors.
[0111] The resulting human OTC-containing recombinant viruses were
administered at an MOI of 10 to 30 to human hepatocytes. The E1/E4
deleted recombinant adenovirus demonstrated less replication and
less late gene expression than when the E1/E4 deleted recombinant
adenoviruses are administered to muscle, as described in the
example above. However, the results of this gene transfer are
better than comparable transfers with recombinant adenoviruses
containing only a deletion in the E1 gene or a deletion in the E1
gene and a point mutation in the E2a gene.
[0112] Similar results are demonstrated when the transgene is CFTR
and the method of administration is intratracheal into lungs.
[0113] All references recited above are incorporated herein by
reference. Numerous modifications and variations of the present
invention are included in the above-identified specification and
are expected to be obvious to one of skill in the art. Such
modifications and alterations to the compositions and processes of
the present invention, such as selections of different transgenes
and plasmids for the construction of the packaging cell lines and
recombinant adenoviruses, or selection or dosage of the viruses or
immune modulators, are believed to be within the scope of the
claims appended hereto.
Sequence CWU 1
1
* * * * *