Note: Descriptions are shown in the official language in which they were submitted.
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STABLE PACKAGING ~r~ T.TNF~ PRODUCING PSEUDC~
R~!; 1 KO~J 1KU ~
Rela~ Applications
This application is a Continuation-in-Part of U.S.S.N.
08/555,15S, entitled ~Stable Packaging Cell Line Producing
Pseudotyped Retroviruses For Gene Transfer", by Daniel S.
Ory, Michel Sadelain and Richard C. Mulligan, filed
November 8, 1995 and of U.S.S.N. 08/651,050, entitled "A
Method For Generation Of Retroviral cDNA Expression
Libraries With A Vesicular Stomatitis Virus-G (VSV-G) Host
Range ~or Expression Cloning By Complementation", by Daniel
S. Ory and Jean E. Schaffer, filed May 21, 1996. The
teachings of each are incorporated herein by reference in
their entirety.
Fundinq Statement
Work described herein was supported by the National
Institutes of Health grant, K11 HL02910. The U.S.
Government has certain rights in the invention.
Backqround
Recombinant retroviruses are useful for in vivo and in
vitro gene expression and for production of proteins of
interest in eukaryotic host cells. Generally, recombinant
retroviruses are produced by introducing a suitable
proviral DNA vector into ~ ~lian cells that produce the
necessary viral proteins for encapsidation of the desired
recombinant RNA and generation of infectious recombinant
virions. Since, for most gene transfer applications, the
generation of pure stocks of recombinant virus free of
replication-competent helper virus is desirable, there has
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been considerable interest in developing cell lines that
produce the necessary viral gene products for the
generation of recombinant retrovirus, yet do not themselves
either yield detectable helper virus or transfer viral
genes (Coffin, J., In:RNA Tumor Viruses, Weiss, R. et al.
(ed.). Cold Spring Harbor Laboratory, Vol. 2, pp.36-73
(1985); Mann, R. et al ., Cell, 33:153-159 (1983); Watanabe,
S., et al., Mol. Cell Biol., 3:2241-2249 (1983); Cone,
R.D., et al., PNAS, USA, 81:6349-6353 (1984); Miller, A.D.,
et al., Mol. Cell Biol., 6:2895-2902 (1986); Bosselman,
R.A., et al., Mol. Cell Biol., 7:1797-1806 (1986). One
approach to doing so is to use mutated proviral genome to
develop retroviral packaging cell lines. However,
production of helper virus and/or transfer of packaging
function (i.e., viral genes) may still occur.
Thus, an improved retroviral packaging cell line is
needed which limits the potential for generation of helper
virus.
Summary of the Invention
The present invention relates to a stable packaging
cell line which produces helper-free pseudotyped
retroviruses and is of mammalian origin, preferably of non-
murine origin, such as stable packaging human cell lines.
These are referred to herein respectively, as stable
pseudotyped retrovirus packaging cell lines and stable
pseudotyped retrovirus packaging human cell lines. The
packaging cell line comprises one or more non-retroviral
expression constructs, such as an expression construct with
the human cytomegalovirus (CMV) immediate early promoter or
derivatives of this promoter (e.g., pMD), which direct
expression of: a) the retroviral gag gene and the
retroviral pol gene, referred to as the retroviral gagpol
genes, and b) a non-retroviral gene which is under the
control of an inducible operator system and whose gene
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product pseudotypes retroviral core virions. The gagpol
gene products package the desired recombinant RNA into core
virions, which are pseudotyped by the non-retroviral gene
product, resulting in production of a stable, pseudotyped
retrovirus packaging cell line capable of generating
helper-free recombinant retrovirus.
In one ~-hoAi -nt, the present invention relates to a
stable, pseudotyped retrovirus packaging cell line capable
of generating helper-free recombinant pseudotyped
retrovirus with a pantropic host range. These cell lines
generate helper-free recombinant pseudotyped retrovirus.
The packaging cells comprise one or more non-retroviral
expression constructs which direct expression of retroviral
gagpol genes and a gene for the Vesicular Stomatitis Virus
G (VSV-G) glycoprotein. The VSV-G glycoprotein, which is
under the control of an inducible operator system (e.g.,
tet operator), provides an envelope protein that
pseudotypes the retroviral core virion generated by the
gagpol proteins. The result is a stable, pseudotyped
retrovirus packaging cell line (e.g., H29 gagpol) which
generates helper-free recombinant pseudotyped retrovirus
with a pantropic host range. In another embodiment,
altered (e.g., mutated) retroviral gagpol genes are used to
produce a stable, pseudotyped retrovirus packaging cell
line (e.g., H29 new gagpol cell line).
The present invention further relates to a method of
making a stable, pseudotyped retrovirus packaging cell line
which generates helper-~ree recombinant pseudotyped
retrovirus. In the method of the present invention,
mammalian cells are co-transfected with one or more non-
retroviral expression constructs which direct the
expression of: a) retroviral gagpol genes and b) a non-
retroviral gene which is under the control of an inducible
operator system and provides a pseudotyped envelope for
retroviral core virions. The gagpol proteins package the
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desired recombinant RNA into core virions, which are
pseudotyped by the non-retroviral gene product, resulting
in production of a stable, pseudotyped retrovirus packaging
cell line which generates helper-free recombinant
pseudotyped retrovirus.
In one embodiment, the present invention relates to a
method of making a stable, pseudotyped retrovirus packaging
cell line which generates helper-free recombinant
pseudotyped retrovirus with a pantropic host range. In
this embodiment, mammalian cells are co-transfected with
one or more non-retroviral expression constructs which
direct the expression of retroviral gagpol genes (e.g.,
wild type or altered) and a VSV-G gene. The VSV-G gene,
which is under the control of an inducible operator system,
provides a pseudotyped envelope protein for the retroviral
core virions, which are produced by the gagpol proteins.
This results in production of a stable, pseudotyped
retrovirus packaging cell line which generates helper-free
recombinant pseudotyped retrovirus with a pantropic host
range.
In another embodiment, the present invention relates
to a method of making a stable, pseudotyped retrovirus
packaging cell line which generates helper-free recombinant
pseudotyped retrovirus with a pantropic host range. In the
method, mammalian host cells are co-transfected with a
first non-retroviral construct which expresses the gene for
tet transactivator fusion protein (tTA) (Gossen, M. and
Bujard, M., Proc. Natl . Acad. Sci., 89:5547-5551 (1992))
and a second non-retroviral construct which expresses a
gene for the VSV-G glycoprotein under the control of tet
operator ( ;n;~l human CMV immediate early promoter
incorporating tet binding sequences). The transfected
cells are screened for tetracycline-inducible VSV-G
expression; VSV-G protein is not detected in the presence
of tetracycline and is detected in the absence of
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tetracycline. Such cells are transfected with a third non-
retroviral construct which expresses the retroviral gagpol
genes, and screened for production of retroviruses.
Transfected cells which produce retroviruses are stable,
pseudotyped retrovirus packaging cells which generate
helper-free recombinant pseudotyped retrovirus with a
pantropic host range.
The present invention further relates to the
particular packaging cell lines described herein (H29
gagpol or 293GPG cell line, H29 new gagpol cell line) and
the particular cells and constructs (e.g., packaging
elements) used to produce the stable, pseudotyped
retrovirus packaging cell line described herein (H29 cells
and pMD, pMDtet, pMDtet.G, PMD.gagpol, pMD.new gagpol
constructs).
Another aspect of the invention is a retroviral vector
for producing a cDNA library for expression in ~m~l ian
cells. The retroviral vector comprises two retroviral long
terminal repeats (LTRs) (e.g., a 5' retroviral LTR and a 3'
retroviral LTR), a cloning site for insertion of cDNA and a
cytomegalovirus (e.g., human) promoter. In one embodiment,
the two LTRs are Moloney murine leukemia virus (MMLV). In
a specific embodiment, the 3' MMLV LTR is unmodified and
the 5' is a modified or chimeric MMLV LTR in which the U3
region of the 5' MMLV LTR is replaced with the
cytomegalovirus (e.g., human) promoter or the
cytomegalovirus enhancer-promoter.
The present invention also relates to a cDNA library
for expression in mammalian cells. The library comprises
retroviral vectors which comprise two retroviral LTRs, cDNA
and a cytomegalovirus promoter. The cDNA is positioned at
a unique cloning site within the retroviral vector,
preferably between the two LTRs, and is operably linked to
the cytomegalovirus promoter.
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The present invention also relates to a method of
expression cloning in - -lian cells. The method
comprises the steps of introducing into mammalian cells a
cDNA expression library comprising retroviral vectors of
5 the present invention and maintA; n ing the mammalian cells
containing the expression library under conditions
appropriate for expression of the cDNA expression library,
whereby the cDNAs in the expression library are expressed
in the ~ ~lian cells. In one embodiment, the cDNA
lO expression library is introduced into mammalian cells by
infection with pseudotyped retroviruses produced in a
stable ~mm~lian (e.g., human, murine) packaging cell line.
The stable mammalian packaging cell line can be selected to
produce pseudotyped retroviruses with pantropic, ecotropic
15 or amphotropic host range, preferably pantropic host range.
In a particular embodiment, the present invention relates
to a method of expression cloning in mammalian cells. The
method comprises the steps of introducing a cDNA expression
library comprising retroviral vectors which comprise two
20 retroviral LTRs, cDNA and a cytomegalovirus promoter into a
packaging cell line which produces pseudotyped
retroviruses; maintaining the packaging cell line
containing the expression library under conditions
appropriate for generation of pseudotyped retroviral
25 particles containing the cDNA expression library; infecting
~ lian cells with the pseudotyped retroviral particles,
under conditions appropriate for infection of the m~mm~lian
cells; and maintaining the resulting mammalian cells under
conditions appropriate for expression of the cDNA in the
30 ~ ~lian cells. In a particular embodiment, the packaging
cell line is a stable human embryonic kidney cell line and,
specifically, a human 2s3-derived cell line.
The present invention also relates to a method of cDNA
expression cloning in m~Tnm~lian cells, wherein VSV-G
3S pseudotyped retrovirus particles which contain RNA produced
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by transcription of cDNA in a cDNA library are produced in
a packaging cell line. The cDNA library comprises vectors
which, in turn, comprise two retroviral LTRs, cDNA and a
cytomegalovirus promoter; the cDNA is positioned between
the LTRs and operably linked to the promoter. Mammalian
cells are then infected with the VSV-G pseudotyped
retroviral particles produced under conditions appropriate
for transcription of RNA contained in the VSV-G pseudotyped
retrovirus particles and production of protein encoded by
cDNA in the cDNA library (by translation of the RNA in the
retrovirus particles) in the mammalian cells. Mammalian
cells which contain the RNA contained in the VSV-G
pseudotyped retrovirus particles or protein encoded by the
cDNA in the cDNA library are detected, using known methods.
For example, RNA can be detected using in situ
hybridization. Alternatively, immunodetection can be used,
where the cDNA encodes protein which is expressed at the
cell surface and the expressed proteins can be detected
using antibodies which bind the protein expressed by the
cDNA of interest (see e.g., U.S. Patent No. 5,506,126). In
addition, epitope tags can be used to detect the protein
expressed. In addition, functional assays can be used to
detect the function of a protein expressed by the cDNA of
interest (e.g., a protein which confers an adhesive
phenotype on a cell).
The present invention also relates to a method of
identifying a gene defect responsible for a mutant
phenotype using cDNA expression cloning by complementation
in mammalian cells. In this method, VSV-G pseudotyped
retrovirus particles which contain RNA produced by
transcription of cDNA in a cDNA library are produced in a
packaging cell line. The cDNA library comprises two
retrovirus LTRs, cDNA and a cytomegalovirus promoter; the
cDNA is positioned between the LTRs and operably linked to
the promoter. Mammalian cells which display a mutant
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phenotype are infected with the VSV-G pseudotyped
retroviral particles under conditions appropriate for
transcription of RNA contained in the VSV-G pseudotyped
retrovirus particles and production of protein encoded by
cDNA in the cDNA library (by translation of the RNA in the
retrovirus particles). Mammalian cells with the mutant
phenotype which display the wild type phenotype upon
expression of the cDNA are identified. The cDNA which
confers the wild type phenotype in the ~ lian cells is
then identified, thereby determining the gene defect
responsible for the mutant phenotype.
Development of the stable, pseudotyped retrovirus
packaging cell lines described herein limits the formation
of helper virus. As a result, stable pseudotyped
retrovirus packaging cell lines are particularly valuable
reagents for in vivo gene transfer studies aimed at cell
lineage analysis and the development of human gene
replacement therapies.
Use of the retroviral vectors of the present invention
enables retroviral cDNA expression cloning in any mammalian
cell type, obviating the need for specialized cells for
efficient expression cloning. In any mutant mammalian cell
type for which there is a phenotype distinct from the wild-
type parental cell type (e.g., primary human cells derived
from patients, primary or established cell lines derived
from mutant animal strains, primary or established cell
lines derived from knockout mice, mutant cell lines
generated in cell culture) the genetic difference between
the mutant and wild type cell (the genetic alteration(s) or
defect(s)) can be rapidly identified by expression cloning
by complementation using this invention.
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Brief Descxiption of the Fiqures
Figure 1 is a schematic representation of the pMD
construct.
Figure 2 is a schematic representation of the pMDtet
construct.
Figure 3 is a schematic representation of the pMD.G
construct.
Figure 4 is a schematic representation of the pMDtet.G
construct.
Figure 5 is a schematic representation of the
pMD.gagpol construct.
Figure 6 is a schematic representation of the pMD.new
gagpol construct.
Figure 7 is a schematic representation of the plasmid,
pBC.tTA
Figure 8 is a schematic representation of the plasmid,
MFG.SnlsLacZ.
Figure 9A is a schematic representation of the ~U3
retroviral construct, ~U3nlsLacZ
Figure sB is a schematic representation of the ~U3
retroviral construct, ~U3Bam.
Detailed Descri~tion of the Invention
The present invention relates to a stable retrovirus
packaging cell line of mammalian origin, preferably of non-
murine origin, such as stable packaging human cell lines,
which produce pseudotyped retroviruses. These are referred
to herein, respectively, as stable pseudotyped retrovirus
packaging cell lines and stable pseudotyped retrovirus
packaging human cell lines for producing pseudotyped
retroviruses for retroviral gene transfer. The packaging
cell lines of the present invention comprise one or more
non-retroviral constructs for expression of retroviral
gagpol proteins, which produce a retroviral core virion,
and a protein which provides a pseudotyped envelope for the
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retroviral core virion. The protein which provides a
pseudotyped envelope for retroviral core proteins is under
control of an inducible operator system. That is,
production of retrovirus by the packaging cell line
described herein is controlled by the inducible expression
of the protein which provides a pseudotyped envelope for a
retroviral core virion. Packaging cell lines of the
present invention l~- ~in viable when uninduced (e.g., in
the presence of tetracycline when a tet operator is used)
and express retroviral gagpol proteins which are non-
enveloped; the uninduced packaging cell lines are capable
of generating (producing) recombinant pseudotyped
retroviral particles when induced (e.g., in the absence of
tetracycline when a tet operator is used). Thus, the
pseudotyped retrovirus packaging cell line of the present
invention is stable. Once induced, the packaging cell
lines generate (produce) recombinant pseudotyped retroviral
particles.
In addition, the packaging cell line of the present
invention limits the potential for generation of helper
virus. Use of non-retroviral constructs and a non-
retroviral protein which produces a pseudotyped envelope
for the retroviral core virion contributes to the limited
generation of helper virus. Potential for helper virus
formation can be further limited by using non-murine cells
(e.g., human cells). Murine cell lines (e.g., NIH 3T3
cells) are typically used to generate retrovirus packaging
cell lines. However, the presence of endogenous murine
retrovirus in the genome of murine cell lines, such as
NIH 3T3 cells (Danos, et al., Proc. Natl. Acad. sci.,
85:6460-6466 (1988), could facilitate recombination events
between the host cell genome, the retroviral expression
constructs and the retroviral vectors, thereby contributing
to production of helper virus.
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In addition, the retroviral gagpol genes can be
altered (e.g.,mutated), further limiting the potential for
the production of helper virus. An example of mutated
gagpol sequences (i.e., new 5' gagpol; new 3' gagpol) is
described in Example 1.
As described in Example 2, packaging cell lines of the
present invention can be derived from human 293 cells which
incorporate a novel non-retroviral, human CMV immediate
early promoter expression construct (pMD) to express the
gagpol gene and pseudotyped envelope which limits the
potential for generation of helper virus. In addition,
silent mutagenesis of gagpol coding sequences minimizes
homology with retroviral vector sequences, further limiting
the potential for generation of helper virus. As further
described in herein, the packaging cell lines of the
present invention express the Vesicular Stomatitis Virus G
(VSV-G) glycoprotein which efficiently pseudotypes the
retroviral core virions. The VSV-G glycoprotein has a
broad host range. Therefore, VSV-G pseudotyped
retroviruses demonstrate a broad host range (pantropic) and
are able to efficiently infect cells that are resistant to
infection by ecotropic and amphotropic retroviruses (Yee,
J.-K., et al., Proc. Natl. Acad. sci . , 91:9564-9568
(1994)). High levels of expression of VSV-G are cytotoxic
and therefore, VSV-G expression in the new packaging cell
line is controlled by an inducible operator system, such as
the inducible tet operator system, allowing for tight
regulation of gene expression (i.e., generation of
retroviral particles) by the concentration of tetracycline
in the culture medium.
Finally, as demonstrated herein, VSV-G pseudotyped
retroviral particles can be concentrated more than 100-fold
by ultracentrifugation (Burns, J.C., et al., Proc. Natl.
Acad. Sci., 90:8033-8037 (1993)). The stable VSV-G
pseudotyped retrovirus packaging cell lines permit
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generation of large scale viral preparations (e.g. from 10
to 50 liters supernatant) to yield retroviral stocks in the
range of 107 to 10ll retroviral particles per ml.
The H29 cells that express the inducible VSV-G protein
have been observed in cell culture for better than 20
passages. The H29 cells at passage 20 remain viable and
continue to express in an inducible manner detectable VSV-G
protein (e.g., by cell fusion studies, Western blotting) at
levels equivalent to cells at an early passage.
The expression construct for use in the present
invention is a non-retroviral vector which directs
expression of retroviral gagpol genes used to produce a
retroviral core virion, and a protein which provides a
pseudotyped envelope for the retroviral core virion. As
described in Example 1, a suitable expression construct for
use in the present invention is a human cytomegalovirus
(CMV) ;~;ate early promoter construct. Other examples
of constructs which can be used to practice the invention
include constructs that use SV40, RSV and rat ~-actin
promoters.
One or more of the non-retroviral expression
constructs can be used to express the gagpol genes and the
protein which provides a pseudotyped envelope, using skills
known in the art. For example, the proteins can be
expressed using one non-retroviral expression construct.
In addition, two non-retroviral expression constructs can
be used wherein one construct expresses the gagpol genes
and the other construct expresses the genes (VSV-G, tTA)
which provide a pseudotyped envelope under control of an
inducible operator. Alternatively, as described in Example
1, three non-retroviral constructs can be used: the first
non-retroviral construct codes for the inducible tet
transactivator protein (tTA) which controls expression of
the gene that expresses a pseudotyped envelope, the second
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non-retroviral construct expresses the genes which provides
the pseudotyped envelope and the third non-retroviral
construct expresses the gagpol genes. Further, the gag and
pol sequences can be expressed separately, requiring a
fourth non-retroviral construct (e.g., in which the third
retroviral construct expresses the gag gene and the fourth
retroviral expression construct expresses the pol gene).
As referred to herein, a "pseudotype envelope" is an
envelope protein other than the one that naturally occurs
with the retroviral core virion, which encapsidates the
retroviral core virion (resulting in a phenotypically mixed
virus). A suitable protein which provides a pseudotyped
envelope is the Vesicular Stomatitis Virus G (VSV-G)
glycoprotein, as described in Example 1. Any suitable
serotype (e.g., Indiana, New Jersey, Chandipura, Piry) and
strain (e.g., VSV Indiana, San Juan) of VSV-G can be used
in the present invention. The protein chosen to pseudotype
the core virion determines the host range of the packaging
cell line. VSV-G interacts with a specific phospholipid on
the surface of mammalian cells (Schlegel, R., et al., Cell,
32:639-646 (1983); Supertzi, F., et al., ~. Gen Virol.,
68:387-399 (1987)). Thus, the packaging cell line which
utilizes VSV-G to provide a pseudotyped envelope for the
retroviral core virion has a broad host range (pantropic).
Other suitable proteins which can be used to provide a
pseudotyped envelope for a retroviral core virion include
type C murine retroviral envelope proteins; HTLV-1 envelope
protein, Gibbon ape leukemia virus envelope protein, and
derivatives of a suitable protein which provide a
pseudotyped envelope (e.g., proteins which include
insertions, deletions or mutations to prepare targeted
envelope sequences such as ecotropic envelope with the EPO
- ligand, synthetic and/or other hybrid envelopes;
derivatives of the VSV-G glycoprotein). In addition,
3S derivatives of murine retroviral envelope proteins can be
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used. For example, derivatives of the VSV-G protein can be
obtained in which the portion of the VSV--G protein which is
responsible for binding to the cell surface is replaced by
a specific ligand and the portion of the VSV-G protein
5 responsible for membrane fusion is retained. The portion
of the VSV-G protein responsible for binding to the cell
surface is determined for example, by performing point
mutation and deletion sequence analysis of the VSV-G
sequence. The ability of each mutated VSV--G protein to
lO bind to the cell surface is determined using an appropriate
binding assay. Retroviral particles incorporating such
derivatives of VSV-G protein would now be able to be
targeted to specific cell populations.
As discussed above, an inducible operator is used for
15 controlled expression of the gene which provides a
pseudotyped envelope. For example, high levels of VSV--G
expression are cytotoxic (Yee, J.--K., et al., Proc. Natl.
Acad. Sci., 91:9564-9568 (1994) ) . Thus, an inducible
tetracycline (i.e., tet) operator system is used to allow
20 for tight regulation of VSV-G expression by the
concentration of tetracycline in the culture medium of the
packaging cell line. That is, with the tet operator
system, in the presence of tetracycline, the tetracycline
is bound to the tet transactivator fusion protein (tTA),
25 preventing binding of tTA to the tet operator sequences and
allowing expression of the gene under control of the tet
operator sequences (Gossen, M. and Bujard, M., Proc. Natl.
Acad. Sci., 89:5547-5551 (1992) ) . In the absence of
tetracycline, the tTA binds to the tet operator sequences
30 preventing expression of the gene under control of the tet
operator. Examples of other inducible operator systems
which can be used for controlled expression of the protein
which provides a pseudotyped envelope are l) inducible
eukaryotic promoters responsive to metal ions (e.g., the
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metallothionein promoter), glucocorticoid hormones and
2) the lac repressor/operator/inducer system of E. coli.
The nucleotide sequences which are encoded by the non-
retroviral constructs can be obtained from a variety of
suitable sources for use in the present invention. For
example, nucleotide sequences expressing the operator
system, the pseudotyped envelope and the gagpol sequences
can be purified from natural sources, produced by chemical
synthesis or produced by recombinant DNA t~rhniques. For
example, as described in Example 1, the gagpol sequence can
be obtained using the pCripen~ construct.
The cells used to prepare the packaging cells are
- ~lian cells, preferably non-murine cells. In a
particular embodiment, the cells used to produce the
packaging cell line are human cells (e.g., 293 cells,
Graham, F., et al., J. Gen. Virol., 36:59-72 (1977); tsa
201 cells, Heinzel, S., et al ., J. Virol ., 62:3738 (1988)).
The packaging cell lines of the present invention can
be used to produce recombinant pseudotyped retroviruses to
enable gene transfer, in vitro and in vivo, for purposes of
expressing all or a portion of a desired gene in eukaryotic
cells. For example, using known techniques, the packaging
cell lines described herein can be used to produce
recombinant pseudotyped retroviruses which are used to
introduce a gene which encodes a particular mRNA, protein
or polypeptide (e.g., therapeutic proteins or polypeptides,
such as insulin, human growth hormone, erythropoietin, gene
replacement for cystic fibrosis (CFTR), familial
hypercholesterolemia (LDL receptor), ADA Deficiency (ADA),
Gaucher's Disease (glucocerebrosidase), antisense therapy
by expression of inhibitory mRNA sequences) into eukaryotic
cells in order to produce the mRNA or protein in quantities
which are useful in administration for therapeutic purposes
or in a diagnostic context (Yee, J. -K., et al . , Proc. Natl .
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Acad. sci., 91:9564-9568 (1994); Dranoff, G., et al., Proc.
Natl. Acad. sci., 90:3539-3543 (1993); Miller, A.D., et
al ., Meth. in Enz., 217:581-599 (1993)). That is,
pseudotyped recombinant virus can be harvested from the
packaging cell lines and used as viral stock to infect
recipient cells in culture or in vivo using known methods.
In the case of secreted proteins or proteins expressed in
hematopoietic cells, sensitive assays such as ELISA or
Western blotting can be used to assess gene transfer
efficiency. Alternatively, high titer viral stocks
produced by packaging cell lines provide superior gene
transfer efficiency in transducing cells (e.g.,
hematopoietic cells) and reduce contamination as compared
with current co-cultivation techni~ues.
The packaging cell lines of the present invention can
also be used to produce pseudotyped retroviruses containing
DNA of interest for introducing DNA or genes of interest
into mammalian cells, such as human cells, which will
subsequently be at' i n; ~tered into localized areas of the
body (e.g., ex vivo infection o~ autologous white blood
cells for delivery of protein into localized ares the body,
see e.g., U.S. Patent No. 5,399,346).
In addition, the packaging elements used to generate
the stable, pseudotyped retrovirus packaging cell can be
used in a variety of ways. For example, the H29 cell line,
which demonstrates inducible VSV-G expression, can be used
to generate retroviral libraries for expression cloning.
The potential for production of high titer viral stocks
will improve the representation of rare cDNAs in a given
library. The packaging cell lines of the present invention
can also serve as the basis for further generation of
pseudotyped packaging cell lines.
The packaging elements can be used as expression
constructs for purposes of efficient constitutive (e.g.,
pMD) and inducible (pMDtet) gene expression. The pMDtet.G
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-17-
construct can be used for other applications for inducible
expression of VSV-G. The pMD gagpol and pMD new gagpol can
be used to develop new generations of retrovirus packaging
cell lines. The pMD, pMDtet, pMDtet.G and pMD.gagpol
constructs can be used for efficient expression of a
heterologous gene.
Thus, as described herein, a stable cell line
expressing VSV-G in an inducible fashion has been
generated. In addition, a new CMV expression vector (pMD)
and its derivatives (pMD.G, pMDtet.G, pMD.gagpol, pMD.new
gagpol) which use genomic human ~-globin sequences for high
levels of expression has been developed. As further
described herein, a stable 293-based packaging cell line
that uses CMV expression constructs, as compared with
mutated proviral constructs, which limit the potential for
helper virus has been developed and use of a mutated gagpol
expression construct in a stable cell line to limit the
potential for helper virus has been demonstrated.
The present invention also relates to a retroviral
vector for producing a cDNA expression library, for
expression in A ~lian cells, comprising two retroviral
LTRs, a cloning site for insertion of cDNA, and a
cytomegalovirus promoter. In one embodiment, the two LTRs
are Moloney murine leukemia virus (MMLV). In a specific
embodiment, the 3' MMLV LTR is unmodified and the 5' is a
modified or ~-hi ~Aric MMLV LTR in which the U3 region of the
5' MMLV LTR is replaced with the cytomegalovirus promoter
or the cytomegalovirus enhancer-promoter.
The retroviral LTRs can be derived from any suitable
retroviral vector, preferably a retroviral vector which
results in high titer or expression of retroviral proteins
The two LTRs can be derived from the same retroviral vector
or different retroviral vectors. For example, as described
in Example 3, both retroviral LTRs can be derived from the
Moloney murine leukemia virus (MMLV). Other suitable
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retroviral LTRs include, for example, those derived from
murine sarcoma virus (MSV), murine papillary sarcoma virus
(MPSV), and Friend virus.
The cloning site of the retroviral vector can be a
variety of cloning sites. For example, as described in
Example 3, the cloning site can be a BamHI cloning site.
Other suitable cloning sites for use in the retroviral
vectors of the present invention include, for example, any
unique or infrequent restriction site within the gagpol or
env genome, or within the U3 region.
The cytomegalovirus promoter can be obtained from any
suitable source. For example, as described in Example 3,
the complete cytomegalovirus enhancer-promoter is derived
from the human cytomegalovirus (HCMV). Part or all of
previously described CMV promoter could be used in the
present invention. Other suitable sources for obtaining a
cytomegalovirus promoter include commercial sources, such
as Clontech, Invitrogen and Stratagene.
The retroviral vectors of the present invention can be
used for expression cloning in mammalian cells, wherein a
cDNA expression library comprising the retroviral vectors
described herein are introduced into mammalian cells under
conditions appropriate for expression of the cDNA
expression library. In one embodiment, the present
invention relates to a cDNA expression library for
expression in - ~ian cells, wherein the library
comprises two retroviral LTRs, a cytomegalovirus promoter
and cDNA, wherein the cDNA is positioned between the
retroviral LTRs and is operably linked to the
cytomegalovirus promoter.
The cDNA for use in the present invention is any cDNA
which is of interest for expression in mammalian cells.
The cDNA can be from any type of cells, such as blood
cells, cells from tissue samples, or cultured cells.
Generally the cDNA will be from the same type of cell in
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which the cDNA is being expressed in those cases where
expression cloning by complementation is being carried out.
The cDNA library for use in the present invention can
be obtained using routine methods (e.g., Seed and Aruffo,
Proc. Natl. Acad. Sci, USA, 84:3365-3369 (1987)). For
example, mRNA can be prepared from any cell and the cDNA
synthesized using standard t~hniques (Sambrook, J., et
al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press (1989)) and commercially available
cloning kits (e.g., Pharmacia, Invitrogen, Stratagene).
The retroviral vectors of the present invention can be
introduced into mammalian cell using any technique which
results in expression of the cDNA expression library in the
cell (e.g., electroporation, calcium phosphate
precipitation, cationic lipids, liposomes). In one
embodiment, the cDNA expression library is introduced into
a packaging cell line to produce retroviral particles,
containing RNA transcribed from the cDNA expression
library, which are used to infect mammalian cells resulting
in expression of the cDNA expression library in the
infected mammalian cell. In this embodiment, a packaging
cell line (e.g., the 293GPG packaging cell line described
herein) can be used to produce pseudotyped retroviral
particles useful for expression cloning in mammalian cells,
as described in Example 4. Other suitable packaging cell
lines for use in the present invention include other human
cell line derived (e.g., embryonic cell line derived)
packaging cell lines and murine cell line derived packaging
cell lines, such as Psi-2 cells (Mann, R., et al., Cell,
33:153-159 (1983); FLY (Cossett, F.L., et al., Virol.,
193:385-395 (1993)), BOSC 23 cells (Pear, W.S., et al.,
Proc. Natl. Acad. sci, USA, 90:8392-8396 (1993), PA317
cells (Miller, A.D. and C. Buttimore, Molec. and Cell.
Biol., 6:2895-2902 (1986)), Kat cell line, (Finer, M.H., et
al., Blood, 83 :43-50 (1994)) GP+E-86 cells and GP+EM12
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cells (Markowitz, D., et al ., J. Virol ., 62: 1120-1124
(1988), and Psi Crip and Psi Cre cells (U.S. Patent
No. 5,449,614; Danos, 0. and Mulligan, R.C., PNAS, USA,
85:6460-6464 (1988)). Also see Yang, Y., et al ., Human
Gene Ther., 6:1203-1213 (1995). The packaging cell lines
for use in the present invention can produce retroviral
particles having a pantropic amphotropic or ecotropic host
range. Therefore, in this - h5~; ?nt, the cDNA expression
library of the present invention can be expressed in any
cell within the host range of the retroviral particle
produced by the packaging cell line. Further, in this
embodiment, the promoter of the retroviral vector of the
present invention can be any promoter which produces
sufficient levels of transcription of the retroviral vector
in the particular packaging cell line (e.g., SV40 promoter,
RSV promoter, ~-actin promoter).
The retroviral vectors of the present invention (e.g.,
the ~U3 retroviral vectors) can be used for transient
transfection of packaging cell lines which produce
pseudotyped retroviruses (e.g., the 293 GPG cells described
herein, which are also referred to herein as H29 gagpol
cells) for production of high titer pseudotyped (e.g., VSV-
G) retrovirus. The retroviral vectors of the present
invention permit construction of cDNA expression libraries
(in the retroviral vectors) for transfection of retroviral
packaging cell lines, in which pseudotyped retrovirus
particles containing the cDNA expression libraries are
produced. Each pseudotyped retroviral particle generally
contains multiple mRNA molecules.
As described in Example 4, high titer VSV-G
pseudotyped virus using the ~U3 retroviral vectors have
been produced using the 293GPG cells which can be used for
expression cloning by complementation in any cell line
having a VSV-G host range. The retroviral vector,
~U3nlsLZ, has been used to transfect 293GPG cells which
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produce retroviruses at titers up to 3 x 106 infectious
units (i.u.)/ml. In addition, as described in Example 5,
the retroviral cDNA cloning vector, ~U3BAM, has been used
to transfect 293GPG cells with levels of expression and
viral titers ~_ -rable to the ~U3nlsLZ vector. Assessment
of the effect of 5' untranslated sequences on cDNA
expression and viral titer showed that the viral ~U3Bam
vector can acc~ odate up to 165 base pairs of 5' upstream
non-coding DNA sequences with only a modest reduction in
expression or viral titer (compared with a MFG-derived
based retroviral vector which has been optimized for high
cDNA expression and generation of high viral titers). The
VSV-G retroviral pseudotypes produced by 293GPG cells
described herein have broad host range and will permit
infection of any mammalian type (Yee, J.-K, et al., PNAS,
91:9564-9568).
Thus, the present invention provides retroviral
vectors which can be used with a variety of mammalian
packaging cell lines to produce pseudotyped retroviral
particles which can, in turn, be used to infect a variety
of ~ -lian cells containing the expression product
(protein, polypeptide) encoded by the cDNA. In a specific
example, the retroviral vectors are used to transiently
transfect 293GPG cells to produce high titer virus with a
VSV-G host range. The ~U3BAM retroviral vector will allow
cloning of any cDNA library into the vector. Transfection
of the retroviral cDNA library into 293GPG cells produces
retrovirus at titers >1o6 i.u./ml that are capable of
infecting any mammalian cell type. Infection of host cells
with this retroviral vector results in stable integration
- of the proviral genome, facilitating long-term high level
expression of the cDNA in the retroviral construct.
The novel methodology described herein makes use of
retroviral vectors (e.g., ~U3Bam retroviral vectors) and a
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packaging cell line (e.g., the 293GPG cells), and provides
for retroviral cDNA expression cloning in any mammalian
cell type, obviating the need for specialized cells (e.g.
Cos7 cells, oocytes) for efficient expression cloning. In
any mutant ~ ~lian cell type for which there is a
phenotype distinct from the wild-type parental cell type
(e.g., primary human cells derived from patients, primary
or established cell lines derived from mutant animal
strains, primary or established cell lines derived from
knockout mice, mutant cell lines generated in cell culture)
the gene defect(s) can be rapidly identified by expression
cloning by complementation using this invention.
An extension of this methodology is the use of the
retroviral vectors of the present invention to produce
pseudotyped retrovirus in the packaging cells, for
expression of candidate cDNA clones in cells that are
derived from patients with genetic defects and established
phenotypes. This will permit screening to determine the
basis for genetic defects (e.g., altered expression of a
gene involved in metabolism) in patients by complementation
analysis. For example, numerous patients have been
characterized biochemically and genetically to have single
gene defects in fatty acid metabolism. However, the mutant
genes have not been established by conventional methods. A
panel of retroviral constructs which encode candidate cDNAs
for various enzymes in fatty acid metabolism can be tested,
for example, for complementation in primary fibroblasts
from these patients.
The retroviral vectors of the present invention are
retroviral-derived vectors (e.g., Moloney murine leukemia
virus-derived vectors) in which the retroviral enhancer-
promoter (e.g., HCMV) has been precisely replaced with the
cytomegalovirus enhancer-promoter in order to facilitate
high levels of expression in the packaging cell lines
(e.g., 293GPG).
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Further, the retroviral vectors of the present
invention can be used to generate retroviral cDNA
expression libraries to allow expression cloning in any
- ~lian cell type. In addition, identification of mutant
genes responsible for human genetic defects by expression
cloning by complementation can be accomplished using
retroviral vectors of the present invention. Expression
cloning of mutant genes from cultured cell lines that have
been mutagenized in culture and have a known phenotype or
from primary or established cell lines derived from animals
with mutant phenotypes can be performed. For example, the
expression cDNA library of the present invention is
introduced into a cell having a mutated phenotype and
identification of the gene(s) which complements the defect
is determined. Thus, an important advantage provided by
the present invention is that expression cloning by
complementation as described herein can be used to identify
gene(s) responsible for a phenotype caused by a mutation
and obtain proof of the function of the responsible
gene(s). Further, the example of the present invention
will allow identification in knockout mice of gene products
which complement the introduced mutation, or of gene
products which function within the pathway(s) affected by
the mutation (i.e., identification of downstream effectors
by suppressor analysis) can be determined with the
retroviral vectors of the present invention. Retroviral
cDNA libraries constructed in the retroviral vectors for
commercial distribution are also provided.
The invention is further illustrated by the following
examples, which are not intended to be limiting in any way.
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ExemPlification
E~nle 1 ~xpression Vector Constructions
pMD (see Figure 1) was constructed with the 3.1 kb
EcoRI-BamHI fragment from pBC12.AB that includes the pXF3
backbone and human CMV ; -~iate early promoter regions and
a 1.34 kb BamHI-Xbal fragment derived from pUCMd~s(R)S
(Sadelain, M., et al ., Proc. Natl . Acad . sci . USA, 92: 6728-
6732 (1995)) that includes the genomic human ~-globin
sequences from the BamHI site in exon 2 through 690 bp in
the 3' untranslated region. The plasmid pUCMd~s(R)S,
however, differs from the genomic sequence in that there is
a 374 bp deletion in the second intron between the first
and third RsaI sites. pBC12.AB is a derivative of
pBC12/CMV/IL-2 lB. Cullen, Cell 46:973 (1986)) in which the
IL-2 sequences (bp 756-1439) have been replaced with a
polylinker. The 3.1 kb EcoRI-BamHI and 1.34 kb BamHI-Xbal
fragments were ligated after the EcoRI and Xbal overhangs
were blunt-ended by treatment with the Klenow fragments.
pMD.G (see Figure 3) was constructed with a 1.6 kb
EcoRI fragment containing the VSV G gene that was derived
from pSVGL (Rose and Bergman, Cell 34:513 (1983)) and was
cloned into the EcoRI site in pMD which is within exon 3 of
the genomic human ~-globin sequence.
pMDtet (see Figure 2) was generated with a 0.47 kb
Xhol-BamHI fragment from pUHC 13-3 (Gossen and Bujard,
Proc. Natl . Acad. sci. 89:5547-5551 (1992)), which contains
the tet operator and ~in;~l CMV promoter sequences, the
1.34 BamHI-Xbal fragment from pUCMd~s(R)S and a 3.06 kb
Xbal-Xhol fragment from pSL301 (Invitrogen).
To construct pMDtet.G (see Figure 4), the 1.6 kb EcoRI
fragment containing the VSV G gene (pSVGL) was cloned into
the EcoRI site in pMD.tetG which is within exon 3 of the
genomic human ~-globin sequence.
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To construct pMD.gagpol (see Figure 5), PCR was
performed with pCRIPenv- (Danos and Mulligan, Proc. Natl.
Acad. Sci., 85:6460-6466 (1988)) and the following pairs of
primers: 5'-CGGAATTCATGGGCCAGACTGTTACC-3' (SEQ ID No:1) and
5'-AGCAACTGGCGATAGTGG-3' (SEQ ID No:2), 5'-
CGGAATTCTTAGGGGGCCTCGCGG-3~ (SEQ ID No:3) and 5'-
ACTACATGCTGAACCGGG-3' (SEQ ID No:4). The PCR products were
digested with EcoRI and Xhol and with EcoRI and HindIII,
respectively, to generate 0.94 kb EcoRI-Xhol and 0.94 kb
HindIII-EcoRI fragments. These fragments were ligated with
the 3.3 kb Xhol-HindIII fragment from pCRIPenv- and pUC19
which had been linearized with EcoRI and phosphatase
treated to produce pUCl9.gagpol. The 5.2 kb EcoRI fragment
from pUCl9.gagpol was cloned into the EcoRI site in pMD,
which is within exon 3 of the genomic human ~-globin
sequence, to yield pMD.gagpol.
To construct pMD.new gagpol (see Figure 6), PCR was
performed with pBCIL2.gagpol (Chung and Mulligan,
unpublished results), which encodes a mutated gagpol
sequence and the following pairs of primers: 5'-
CGGAATTCATGGGTCAGACTGTTACTAC-3' (SEQ ID No: 5) and 5'-
AGCAACTGGCGATAGTGG-3' (SEQ ID No: 2), 5'-
CGGAATTCTTAGGGAGCTTCTCTTGTTAG-3' (SEQ ID No: 6) and 5'-
ACTACATGCTGAACCGGG-3' (SEQ ID No: 4). The mutated gagpol
sequences are as follows:
New 5'gagpol:
5'-
ATGGGTCAGACTGTTACTACCCCTCTAAGTTTAACTTTGGGCCATTGGAAAGATGTAGAGAGGATCGCCCACAACCAGAGTGTAGACGTTAAGAAAAGACGTTGGGTCA~lllllGTT
CTGCAGAGTGGCCTACCTTCAACGTAGGCTGGCCAAGAGATGGTACTTTTAACAGAGAC
CTTATTACCCAGGTCAAGATCAAA~~ lAGTCCAGGCCCTCACGGACATCCAGATCA
GGTCCCTTACATTGTCACCTGGGAAGCTCTTGCCTTTGACCCTCCCCCTTGGGTGAAGC
CTTTTGTCCACCCTAAGCCCCCACCTCCCTTGCCTCCAAGTGCTCCTTCCCTCCCTCTT
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GAACCCCCTCGCAGTACTCCACCTCGATCCAGTCTCTATCCTGCCCTA-3' (SEQ ID
No: 7)
New 3' gagpol:
5'-
ATTCTATACGGAGCCCCTCCCCCTTTAGTTAACTTTCCAGACCCTGATATGACTAGAGTAACAAACTCTCCTAGTCTTCAGGCACACCTCCAAGCCCTGTACCTAGTCCAACATGAAG
TGTGGAGACCCTTAGCAGCTGCATACCAGGAACAGCTTGACAGGCCTGTAGTCCCCCAC
CCGTACAGAGTGGGAGACACTGTATGGGTCCGACGCCACCAAACAAAAAACTTAGAGCC
TCGATGGAAGGGCCCCTACACTGTACTACTCACAACCCCTACAGCCCTGAAGGTTGACG
GGATAGCTGCCTGGATTCACGCTGCACACGTGAAAGCAGCTGACCCTGGAGGGGGTCCC
TCTAGCAGATTAACCTGGCGCGTACAAAGATCCCAGAATCCTCTGAAAATCAGGCTAAC
AAGAGAAGCTCCCTAA-3' (SEQ ID No: 8)
The PCR products were digested with EcoRI and XhoI and
with EcoRI and HindIII, respectively, to generate 0.94 kb
EcoRI-Xhol and 0.94 kb HindIII-EcoRI fragments. These
fragments were ligated with the 3.3 kb Xhol-HindIII
fragment from pCRIPenv- and pUC19 which had been linearized
with EcoRI and phosphatase treated to produce pUCl9.new
gagpol. The 5. 2 kb EcoRI fragment from pUCl9.new gagpol
was cloned into the EcoRI site in pMD, which is within exon
3 of the genomic human ~-globin sequence, to yield pMD.new
gagpol.
A novel CMV expression vector (pMD) was constructed
for expression of the wild-type gagpol (pMD.gagpol) and for
the mutagenized gagpol (pMD.new gagpol). For pMD.gagpol,
reverse transcriptase assays have been performed which
demonstrate production of retroviral particles under
conditions of both transient and stable expression. For
pMD.new gagpol, reverse transcriptase assays have been
performed which demonstrate production of retroviral
particles under conditions of transient expression.
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~rle 2 Construction of Packaqinq Cell Lines
H29 cell line.
Human 293 cells (gift, B. Panning; Graham, F., et al . ,
J. Gen . Virol ., 36: 59-72 (1977)) were grown in DMEM with 5%
inactivated fetal bovine serum, supplemented with 2 mM L-
glutamine, penicillin and streptomycin (293 growth media)
and incubated at 37 C with 5% C02. The 293 cells were co-
transfected by the calcium phosphate precipitation method
(Pear, et al ., PNAS, 90: 8392-8396 (1994)) with 5 ~g pBC.tTA
(see Figure 7; T. Chung and R. Mulligan, unpublished
results), 5 ~g pMDtet.G and 1 ~g pJ6~puro (gift, J.
Morgenstern). During transfection the 293 growth media was
supplemented with 1.0 ~g/ml tetracycline (Sigma). The
transfected cells were plated into selection 48 hours post-
transfection in 293 growth media supplemented with l.0~g/ml tetracycline and 2 ~g/ml puromycin (Sigma). 72
independent clones were selected for clonal expansion and
screened for tetracycline-inducible VSV-G expression. To
screen the clones, each clone was plated in parallel into
two 35 mm tissue culture dish (Corning) at 30% confluence.
The following day one plate was washed twice with 2 ml 293
growth media without tetracycline and the media changed to
standard 293 media supplemented with 2 ~g/ml puromycin. At
48 hours the cells were harvested for total cellular
protein and the paired samples run on a 7.5% SDS-
polyacrylamide gel under reducing conditions. The gels
were transferred onto nitrocellulose (Schleicher & Schuell,
0.45 mm) with a semi-dry electroblotter (Owl Scientific).
Western blotting was performed using standard procedures.
For the primary antibody a murine monoclonal anti-VSV-G IgG
(Sigma) was used at a dilution of 1:800. For the secondary
antibody an HRP-coupled donkey anti-mouse IgG F(ab)2
fragment (Pharmingen) was used at a dilution of 1:10,000.
Chemiluminescent detection was performed with the Dupont
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NEN Renaissance kit. Positive cell lines (e.g. H29) were
selected on the basis of no detectable VSV-G expression in
the presence of tetracycline in the growth media and the
detection of inducible VSV-G expression in the absence of
tetracycline in the growth media.
H29 gaqPol cell line (293 GPG cell line).
H29 cells were grown in 293 growth media supplemented
with 1.0 ~g/ml tetracycline and 2 ~g/ml puromycin (H29
media) and co-transfected by the calcium phosphate
precipitation method with 10 ~g pMD.gagpol linearized with
Scal and 2 ~g pSV2neo. During transfection the H29 media
was supplemented with 1.0 ~g/ml tetracycline. The
transfected H29 cells were plated into selection 48 hours
post-transfection in H29 media supplemented with 1.0 ~g/ml
tetracycline and 0.3 mg/ml G418 (Gibco). Sixty-nine
independent clones were selected for clonal expansion and
were screened for reverse transcriptase activity (Goff, et
al ., ~. Virology, 38:239-248 (1981)).
Of the sixty-nine independent clones selected for
clonal expansion, 10 clones had reverse transcriptase
activity that exceeded the positive control (i.e., ~ Cre
Cells). These 10 clones will be further characterized.
H29 new qaqpol cell line
H29 cells are grown in 293 growth media supplemented
with 1.0 ~g/ml tetracycline and 2 ~g/ml puromycin (H29
media) and co-transfected by the calcium phosphate
precipitation method with 10 ~g pMD.new gagpol linearized
with Scal and 2 ~g pSV2neo. During transfection the H29
media was supplemented with 1.0 ~g/ml tetracycline. The
transfected H29 cells are plated into selection 48 hours
post-transfection in H29 media supplemented with 1.0 ~g/ml
tetracycline and 0.3 mg/ml G418 (Gibco). Independent
clones are selected for clonal expansion and screened for
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reverse transcriptase activity (Goff, et al., ~. Virology
(1981)).
Discussion
Thus, the derivative of pMD, pMDtet.G, provides for
tetracycline inducible expression of VSV.G. Inducible
expression of VSV-G has been demonstrated in a transient
assay and in a stable cell line, H29. H29 is derived from
293 cells (Graham, F., et al., ~. Gen. Virol., 36:59-72
(1977)) and was selected after co-transfection with pBC.tTA
(the Tet transactivator), pMDtet.G and pJGnpuro. The H29
cells show inducible VSV-G expression by western blotting
that is only 5-fold less than transient VSV-G expression in
parental 293 cells. The H29 cells passaged in culture for
20 passages continue to demonstrate inducible VSV-G
expression.
Exam~le 3 Construction of the AU3 retroviral cloninq
vectors
The AU3nlsLacZ retroviral vector was constructed by
precise replacement of the U3 region in the 5' LTR of
MFG.SnlsLacZ (Berns, et al., Numan Gene Therapy, 6:347-368
(1995); see Figure 8) with the HCMV enhancer-promoter (nt
-671 to -2) (Boshart, M., Cell, 41:521-530 (1985)). In
~U3nlsLacZ the entire 5' genomic flanking region and all
but 65 bp from the 3' genomic flanking region from
MFG.SnlsLacZ is eliminated.
The pMD plasmid was constructed as described in
Example 1. For the construction of the ~U3nlsLacZ, a 701
bp fragment encoding the HCMV promoter was generated by PCR
with the pMD plasmid as the template with the pair of
primers, 5'-GGGCCCAAGCTTCCCATTGCATACGTTGTATC-3' (SEQ ID
N0: 9) and 5'-GGACTGGCGCCGGTTCACTAAACGAGCTC-3' (SEQ ID
N0: 10), creating a 5' Hind III site and a 3' Ras I site.
The PCR product was digested with Hind III and Kas I to
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yield a 677 bp fragment. The 91 bp ~as I-Sty I was
isolated from the 3' LTR of MFG (Riviere, I., et al., PNAS,
92:6733-6737 (1995)). The 253 bp Sty I-Eag I and the 4994
bp Eag I-Sca I fragments were isolated from MFG.SNlsLacZ
(Berns, et al., Human Gene Therapy, 6:342-368 (1995)), and
the backbone for ~U3nlsLacZ is a 2.65 kb ~ind III-Sma I
fragment from pUC18.
For the construction of ~U3Bam, a 561 bp fragment was
generated by PCR with ~U3nlsLacZ as the template with the
pair of primers, 5'-GTGACCTGGGAAGCCTTGGC-3' (SEQ ID N0: 11)
and 5'CGGGATCCAGTCTAGAGGATGGTCCACC-3' SEQ ID N0: 12),
creating a 5' Ras I site and a 3' Bam N I site. The PCR
product was digested with Ras I and Bam H I to yield a 389
bp fragment. The 389 bp Kas I-Bam ~ I fragment was ligated
with 4466 bp Bam H I-Eag I and 695 bp Eag I-Kas I fragments
that were derived from ~U3nlsLacZ.
Figure 9A displays the structure of the ~U3nlsLacZ
vector and Figure 9B displays the structure of the ~U3Bam
vector.
~0 Exam~le 4 Production of VSV-G ~seudotyped retrovirus bY
transient transfection of 293GPG cells
The plasmid pBC.tTA was constructed from pBC12/CMV/IL-
2 (Cullen, B.R., Cell, 46:973-982 (1986)) by replacement of
the IL-2 sequences (bp 756-1439) with the tet
transactivator gene from pUHD10-1 (Gossen, M., et al.,
Proc. Natl. Acad. Sci., 89:5547-551 (1992)). Figure 7
displays the structure of the pBC.tTA plasmid.
The pMD, pMD.G, pMDtet, pMDtet.G, and pMD.gagpol
constructs and the 293GPG cell line were constructed as
described in Example 1.
Transient transfections with 293GPG cells were
performed on 60 mm dishes where 4-5 x 106 cells were plated
the night prior in 4 ml 293 GPG media. 4 ug of ~U3nlsLacZ
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was diluted into 300 ul opti-MEM (Gibco BRL) and incubated
at room temperature for 30 minutes with 25 ul Lipofectamine
~Gibco BRL) diluted into 300 ul opti-MEM. 2.4 ml opti-MEM
was added to the DNA-Lipofectamine mixture and layered on
top of the 293GPG cells, which had been rinsed 30 minutes
prior to transfection and had media replaced with 2 ml
opti-MEM. 2 ml 293 media was added at 7-8 hours post-
transfection and the media was changed at 24 hours. The
supernatant was harvested at 72 hours and viral titers
determined as described below.
Assay for ~-Galactosidase ActivitY and Determination of
Viral Titers
To stain cells for ~-galactosidase activity, cells
were washed with phosphate buffered saline supplemented
with 1 mM magnesium (PBS+) and fixed with 1% glutaraldehyde
in PBS+ for 10 minutes at 37~C (Lim, K., et al.,
Biotechniques, 7:576-579 (1989). The fixative was
aspirated and the cells incubated with 3.3 mM potassium
ferricyanide (Sigma), 3.3 mM potassium ferrocyanide (Sigma)
and 0.2% X-gal (Molecular Probes) in PBS+ for 2 hours at
37~C. Quantitative ~-galatosdase activity was determined
using a commercially available luminescent assay
(Clontech). To determine viral titers, NIH 3T3 cells were
plated at 1 x 105 cells per well in 6-well culture dishes
16 hours prior to infection and incubated for 24 hours with
serial dilutions of viral supernatants containing 8 ug/ml
polybrene (Sigma). Viral titer was determined as the
average number of cells with blue nuclei (~-galactosidase-
producing cells) per twenty 1 mm2 fields (2--3x 104 cells)
multiplied by a factor to account for plate size, dilution
of viral stock and division of target cells in tissue
culture wells.
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Production of VSV-G pseudoty~ed retrovirus by transient
transfections of 293GPG cells
The 293GPG clone was used to produce VSV-G pseudotyped
retrovirus by transient transfection, taking advantage of
the high transfectability of 293 cells. For transient
transfection of the 293GPG cells, the ~U3nlsLacZ retroviral
construct was generated by substitution of the U3 region of
the 5' LTR of MFG.nlsLacZ with the HCMV enhancer-promoter
and deletion of the genomic 5' and 3' flanking regions of
MFG.SnlsLacZ. When compared to MFG.SnlsLacZ, aU3nlsLacZ
enabled a 20-fold increase in expression in transient
transfections. The 293GPG clone was transiently
transfected by lipofectamine with an average efficiency of
40% with the ~U3nlsLacZ construct. A 48 hour virus
supernatant was collected between 24 and 72 hours post-
transfection and removal of tetracycline from the growth
medium. Viral titers in the range of 1-3 x 106 i.u./ml
were achieved transiently.
ExamPle 5 Effect of 5' Untranslated Sequences on cDNA
ex~ression and viral titer
There are several differences between previously used
vectors and the ~U3 retroviral cloning vectors. First, the
AU3 vectors are specifically modified for high transient
expression in 293-derived cell lines (e.g. 293GPG cells) by
precise replacement of the U3 region in the 5' LTR by the
complete human CMV enhancer-promoter (Boshart et al., Cell,
41:521-530 (1985)). Second, the ~U3 vectors are derived
from MFG which is an established high titer and high
expression vector. Finally, unlike the vectors used for
previous retroviral expression cloning, the effect of 5'
untranslated sequences on cDNA expression and cDNA viral
titer was examined. This is important because in the
course of construction of cDNA libraries (Seed, B. and
CA 02237000 1998-0~-07
W O 97/17457 PCT~US96/17807
Aruffo, S., PNAS, 84: 3365-3369 (1987)) the cDNA inserts
often include up to 200 base pairs of 5' untranslated
sequences. Therefore, the retroviral cloning vector must
be able to accommodate these additional sequences so that
bias will not be introduced into the library.
The effect of 5' untranslated sequences on cDNA
expression and viral titer was ~ ;ned by insertion of the
lacZ gene with 0-165 base pairs of 5' untranslated
sequences into the ~U3Bam vector. The following is the
data normalized to 0 base pairs of untranslated sequence
(i.e., ~U3LacZ which does not contain the modified Bam H I
cloning site):
# of 5' expression (%) viral titer (%)
untranslated bp
0 100 100
43 51 34
106 36 31
165 19 27
These results demonstrate that the ~U3Bam vector can
accommodate up to 165 bp of 5~ untranslated sequences
within the cDNA insert with only a modest reduction in
expression or viral titer as compared with a non-cloning
MFG-based retroviral vector. This data supports the
capability of the AU3Bam vector to promote efficient
packaging of the cDNA inserts and efficient transfer of the
cDNA to the target calls.
E~uivalents
~ 10 Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
described specifically herein. Such equivalents are
CA 02237000 1998-05-07
W O 97/17457 PCTAUS96/17807
intended to be encompassed in the scope of the following
claims.
CA 02237000 1998-0~-07
W O 97/17457 PCT~US96/17807
-35-
~u~.._~ LISTING
- ( 1 ) G~NT~'R~T. INFORMATION:
(i) APPLICANT:
~A'I NAME: ~ ~AD IN~ I~1VL~ FOR BIOMEDICAL ~T.'.~T.'AT~
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GI TELEPHONE:
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(i) APPLICANT/ lNV~I~ lOR:
A'l NAMB: SADELAIN, MICHEL
IBI STREET: 401 Ea~t 89th Street
,C, CITY: New York
,DJ STATE/PROVINCE: New York
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(i) APPLICANT/lNv~ OR:
,'A'I NAMB: MULLIGAN, RICHARD C.
,B'I STRBET: Two Sandy Pond Road
'C, CITY: Lincoln
DI STATE/PROVINCE: Ma~qachuqetts
~EJ COUhl~Y: United State~
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IG TELEPHONE:
~I~ TELEFAX:
(i) APPLICANT/lNV~;h10R:
(A) NAMB: SCHAFFER, JEAN B.
(B) STRBET: 8 Greenbriar Street
(C) CITY: St. Louiq
CA 02237000 1998-0~-07
WO 97/l7457 PCTAUS96/17807
~D'l STATE/PROVINCE: M; ~80~
EI couhL~r: United State5
~F~ POSTAL CODE/ZIP: 63124
~GI TELEPHONE:
~I~ TELEFAX:
(ii) TITLE OF lNV~l~ ~ lON: STA8LE PACXAGING CELL LINE PRODUCING
PSEUDOTYPED RETROVIRUSES
(iii) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
~A) ADDRESSEE: Hamilton, 8rook, Smith ~ Reynold~, P.C.
,BI STREET: Two Militia Drive
,C, CITY: Lexington
Dl STATE: Ma~Rachu~etts
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(v) COMPUTER p~n~RT.T FORM:
~A~ MEDIUM TYPE: Floppy di8k
~BJ COMPUTER: IBM PC compati~le
~C, OPERATING SYSTEM: PC-DOS/MS-DOS
,DJ SOFTWARE: PatentIn Release #1.0, vercion #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WUMS96-01
(B) FILING DATE: May 21, 1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WHI95-07
(B) FILING DATE: N~v. ~er 8, 1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME: Granahan, Patricia
(B) REGISTRATION NUMBER: 32,227
(C) X~r~-~CE/DOCKET NUMBER: WHI95-07M PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 861-6240
(B) TELEFAX: (617) 861-9540
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 26 baQe pairs
~B TYPE: nucleic acid
,'CI STRANDEDNESS: single
~D, TOPOLOGY: linear
CA 02237000 1998-0~-07
WO 97/17457 PCT~US96/17807
-37-
(ii) ~OLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /de~c ~ "oligonucleotide"
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:1:
CGGAATTCAT GGGCCAGACT GTTACC 26
(2) INFORMATION FOR SEQ ID NO:2:
( i ) ~yU~N~: CHARACTERISTICS:
'A'I LENGTH: 18 ba~e pair~
BI TYPE: nucleic acid
C, STRANDEDNESS: ~ingle
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /de~c = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AGCAACTGGC GATAGTGG 18
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
~A'l LENGTH: 24 ba~e pair~
~BJ TYPE: nucleic acid
~CJ STRANDEDNESS: single
~D,, TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CGGAATTCTT AGGGGGCCTC GCGG 24
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A'l LENGTH: 18 ba~e pairs
IBI TYPE: nucleic acid
,C STRANDEDN~SS: ~ingle
~Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID No:4:
ACTACATGCT GAACCGGG 18
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WO 97/17457 PCT~US96/17807
-38-
(2) INFORMATION FOR SEQ ID NO:5:
(i) s~Q~ CHARACTERISTICS:
~A' LENGTH: 28 ba~e pair~
~BJ TYPE: nucleic acid
'C, STRANDEDNESS: single
~D,l TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:5:
CGGAATTCAT GGGTCAGACT GTTACTAC 28
(2) INFORMATION FOR SEQ ID No:6:
( i ) S~U~N~ CHARACTERISTICS:
'A'l LENGTH: 29 ba~e pairs
BI TYPE: nucleic acid
~,C, STRANDEDNESS: ~ingle
~D, TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTIO~: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGGAATTCTT AGGGAGCTTC ~l L~7 ~ lAG 29
(2) INFORMATION FOR SEQ ID NO:7:
(i) S~U~:N~ CHARACTERISTICS:
,'A'I LENGTH: 402 ba~e pair~
~Bl TYPE: nucleic acid
~C, STRANDEDNESS: single
D; TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) S~U~N~ DESCRIPTION: SEQ ID NO:7:
ATGGGTCAGA CTGTTACTAC CC~l~lAAGT TTAACTTTGG GCCATTGGAA AGATGTAGAG 60
AGGATCGCCc A~Ac~r-A~7 TGTAGACGTT AA~.AAAA~AC GTTGGGTCAC 'Ll111'~71 l~'L 120
GCAGAGTGGC CTACCTTCAA CGTAGGCTGG CrAA~-~-ATG GTACTTTTAA cA~-A~-ACCTT 180
ATTACC~AGG TCAAGATCAA A~l~ AGT CCAGGCCCTC ACGGACATCC AGATCAGGTC 240
CCTTACATTG TCACCTGGGA AGCTCTTGCC TTTGACCCTC CCC~lLGGGT GAAGCCTTTT 300
GTCCACCCTA AGCCCC~ACC ~lCC~lLGCCT CCAAGTGCTC ~llCC~LCCC TCTTGAACCC 360
CA 02237000 1998-0~-07
W O 97/17457PCTtUS96tl7807
-39-
CCTCGCAGTA CTCCACCTCG ATCCAGTCTC TATCCTGCCC TA 402
(2) lNr ~KMATION FOR SEQ ID NO:8:
U~N~ CHARACTERISTICS:
(A', LENGTH: 429 ba~e Pair~
,B TYPE: nUC1eiC aCid
~C~ STRANDEDNESS: ~ing1e
~DJ TOPOLOGY: 1inear
(ii) MOLECULE TYPE: DNA (9~-- ;C)
(Xi) ~U~N~: DESCRIPTION: SEQ ID NO:8:
ATTCTATACG GAGCCC~1CC CC~L.1AGTT AA~1~C~AG ACCCTGATAT GACTAGAGTA 60
AC~ArTCTC CTA~LC1--~A GGCACACCTC CAAGCCCTGT ACCTAGTCCA ACATGAAGTG 120
TGrArArCCT TAGCAGCTGC A~ACr~eGAA CAGCTTGACA GGCCTGTAGT CCCCrACCCG 180
TAe~rArTGG GAGACACTGT ATGGGTCCGA CGCr~Cr~A~ CAAAAAACTT AGAGCCTCGA 240
TGGAAGGGCC CCTACACTGT ACTACTCACA ACCCCTACAG CCCTGAAGGT TGACGGGATA 300
GCTGCCTGGA TTCACGCTGC ACACGTGAAA GCAGCTGACC CTGGAGGGGG 1CC~ ~AGC 360
AGATTAACCT GGCGCGTACA AAGATCCCAG AA~C~1~A A~ATCAGGCT ~CA~r~r~ 420
GCTCCCTAA 429
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
'A', LENGTH: 32 ba~e Pair~
,B, TYPE: nUC1eiC acid
~C~ STRANDEDNESS: ~ing1e
~,D~ TOPOLOGY: 1inear
(ii) MOLECULE TYPE: DNA (genOmiC)
(Xi) ~:QU~:N~ DESCRIPTION: SEQ ID NO:9:
GGGCCCAAGC TTCCCATTGC ATAC~ LA TC 32
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A~, LENGTH: 29 baSe Pair~
~B, TYPE: nUC1eiC ac id
~C STRANDEDNESS: qing1e
,D, TOPOLOGY: 1inear
(ii) MOLECULE TYPE: DNA (genOmiC)
CA 02237000 1998-05-07
W O 97/17457 PCTrUS96/17807
-40-
(xi) ~yu~:N~ DESCRIPTION: SEQ ID NO:lû:
GGACTGGCGC CGG~ACTA AACGAGCTC 29
t2) INFORMATION FOR SEQ ID NO:ll:
(i) s~Q~:N~ CHARACTERISTICS:
Aj LENGTH: 20 base p~irn
BI TYPE: nucleic acid
~C, STRANDEDNESS: single
,DJ TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (gen~ ic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
GTGACCTGGG AAGCCTTGGC 20
~2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
,A' LENGTH: 28 ba~e pairs
BI TYPE: nucleic acid
~C, STRANDEDNESS: single
fD TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CGGGATCCAG TCTAGAGGAT GGTCCACC 28
