U.S. patent application number 10/205179 was filed with the patent office on 2003-01-02 for packaging cells.
Invention is credited to Barber, Jack R., Chang, Stephen M.W., Jolly, Douglas J., Respess, James G..
Application Number | 20030003567 10/205179 |
Document ID | / |
Family ID | 27538819 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003567 |
Kind Code |
A1 |
Barber, Jack R. ; et
al. |
January 2, 2003 |
Packaging cells
Abstract
The invention described herein allows the production of
recombinant retroviruses (retroviral vector particles) from
producer cells which are safer and of higher titer than normal. In
addition, methods are provided for making helper cells which, when
a recombinant retrovirus genome is introduced to make a producer
line, produce particles that are targeted toward particular cell
types. Methods are also provided for making recombinant retrovirus
systems adapted to infect a particular cell type, such as a tumor,
by binding the retrovirus or recombinant retrovirus in the
particular cell type. Methods are also provided for producing
recombinant retroviruses which integrate in a specific small number
of places in the host genome, and for producing recombinant
retroviruses from transgenic animals.
Inventors: |
Barber, Jack R.; (San Diego,
CA) ; Jolly, Douglas J.; (La Jolla, CA) ;
Respess, James G.; (San Diego, CA) ; Chang, Stephen
M.W.; (San Diego, CA) |
Correspondence
Address: |
CHIRON CORPORATION
Intellectual Property
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
27538819 |
Appl. No.: |
10/205179 |
Filed: |
July 24, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10205179 |
Jul 24, 2002 |
|
|
|
09389525 |
Sep 2, 1999 |
|
|
|
09389525 |
Sep 2, 1999 |
|
|
|
07586603 |
Sep 21, 1990 |
|
|
|
07586603 |
Sep 21, 1990 |
|
|
|
07565606 |
Aug 10, 1990 |
|
|
|
07565606 |
Aug 10, 1990 |
|
|
|
07395932 |
Aug 18, 1989 |
|
|
|
07395932 |
Aug 18, 1989 |
|
|
|
07170515 |
Mar 21, 1988 |
|
|
|
Current U.S.
Class: |
435/235.1 ;
435/366; 435/5 |
Current CPC
Class: |
C12N 2740/13023
20130101; A01K 2217/05 20130101; A61K 39/00 20130101; C12N
2740/13052 20130101; C12N 2740/13043 20130101; A61K 2039/57
20130101; A61K 38/217 20130101; C12N 2740/13022 20130101; A61K
38/2013 20130101; C12N 15/86 20130101; C07K 2319/00 20130101; C12N
15/8509 20130101; A61K 35/13 20130101; C07K 2319/32 20130101; A01K
67/033 20130101; C12N 2740/15022 20130101; C07K 14/70514 20130101;
A01K 2267/01 20130101; C12N 2740/16222 20130101; A61K 48/00
20130101; C12N 7/00 20130101; C07K 14/005 20130101; A61K 2039/53
20130101; C12N 9/1211 20130101; A61K 2039/5256 20130101 |
Class at
Publication: |
435/235.1 ;
435/366; 435/5 |
International
Class: |
C12Q 001/70; C12N
007/00; C12N 005/08 |
Claims
1. A method of selecting packaging cells which produce high levels
of a primary agent selected from a packaging protein and a gene
product of interest, comprising: (a) providing in packaging cells a
genome comprising a primary gene which expresses a primary agent
therein, and a selectable gene which expresses a selectable protein
therein at lower levels than the primary agent, the expression
levels of the primary gene and selectable gene being proportional;
(b) exposing the packaging cells to a selecting agent which enables
identification of those cells which express the selectable protein
at a critical level; and (c) detecting those packaging cells which
express high levels of the primary agent.
2. A method of producing a recombinant retrovirus, comprising:
generating gag, pol, and env proteins from a cell line infected by
a recombinant virus which is capable of producing the proteins; and
contacting the proteins with viral vector RNA, tRNA, liposomes, and
a cell extract to complement missing functions for particle
assembly, so as to produce recombinant retroviruses carrying the
viral vector RNA.
3. A method of producing a recombinant retrovirus, comprising: (a)
generating recombinant viral vectors which separately or in
combination, code for gag/pol, env and a retroviral vector genome;
(b) producing high titre stocks of the vectors; and (c)
co-infecting primary or other cells to generate recombinant
retroviral vectors.
4. A method of producing a recombinant retrovirus, comprising
growing a producer cell having a genome comprising: (a) a gene of
interest along with a packaging signal of a first retroviral
phenotype; (b) gag and pol genes of the first retroviral phenotype,
absent a packaging signal; (c) a hybrid env gene absent a packaging
signal, the product of said hybrid env gene comprising a
cytoplasmic segment of the first retroviral phenotype, and a
binding segment exogenous to the first retroviral phenotype.
5. A method of producing a recombinant retrovirus, comprising
growing a producer cell having a genome comprising: (a) a gene of
interest along with a packaging signal of a first retroviral
phenotype; (b) gag and pol genes of the first retroviral phenotype,
absent a packaging signal; and (c) a gene coding for a ligand which
is expressed on the surface of the producer cell and which is
subsequently exhibited on the surface of the vector particle.
6. The method of claim 5 wherein the ligand is CD4.
7. A hybrid env gene useful for preparing a retrovirus which can
selectively carry a gene of interest to a target cell, the env gene
coding for: (a) a cytoplasmic segment of a first retroviral
phenotype; and (b) a binding segment exogenous to the first
retroviral phenotype, the binding segment being capable of
selectively binding to the target cell.
8. A method of producing a recombinant retrovirus which is capable
of integrating its genome into a preselected site on a target
cell's genome, comprising: packaging a vector in a capsid and
envelope, and including in the viral particle a modified form of
integrase which is capable of integrating the retroviral genome
into the preselected site.
9. A method of producing recombinant retroviruses, comprising:
mating a transgenic animal or insect containing a gag/pol-env viral
construct, with a transgenic animal or insect containing a vector
construct containing a promoter; isolating the progeny of said
transgenic animals or insects; isolating selected cells from the
progeny; growing said cells in an appropriate medium; and isolating
recombinant retroviruses from the cells.
10. A method of producing a transgenic packaging animal or insect,
comprising: mating a transgenic animal or insect containing a
vector construct coding for some, but not all viral proteins
necessary for packaging, with a transgenic animal or insect
containing a vector construct coding for the remainder of said
necessary viral proteins; and isolating the progeny of said
transgenic animals or insects.
11. The method of claim 10, further comprising the step of mating
said progeny with a transgenic animal or insect containing a vector
construct, to produce primary cells capable of producing high titre
recombinant retrovirus.
12. The method of claim 10, further comprising the step of
infecting cells explanted from said progeny with a recombinant
retrovirus containing a vector construct to produce primary cells
capable of producing high titre recombinant retrovirus.
13. A non-mouse packaging cell line that produces at least a
ten-fold increase in viral packaging protein, as compared to a
standard mouse amphotropic packaging cell line.
14. The cell line of claim 13 wherein the viral packaging protein
is the gag/pol protein.
15. The cell line of claim 13 wherein the cell line is an
amphotropic packaging cell line.
16. The packaging cell line of any one of claims 13-15 wherein the
packaging cell line, upon introduction of a vector construct,
produces at least a ten-fold increase in vector titre as compared
to a standard mouse amphotropic packaging cell line.
17. The packaging cell line of any one of claims 13-15 wherein the
packaging cell line, upon introduction of a vector construct,
produces vector particles capable of infecting human cells.
18. A xenotropic packaging cell line which, upon introduction of a
vector construct, is capable of producing vector particles
substantially uncontaminated by replication competent virus.
19. The packaging cell line of claim 18 wherein the cell line
produces at least equal vector titre as compared to a standard
mouse amphotropic packaging cell line when HT1080 cells are
infected.
20. A polytropic packaging cell line.
21. The packaging cell line of claim 20 which, upon introduction of
a vector construct, is capable of producing vector particles
substantially uncontaminated by replication competent virus.
22. The packaging cell line of claim 20 wherein the packaging cell
line, upon introduction of a vector construct, produces at least a
ten-fold increase in vector titre as compared to a standard mouse
amphotropic packaging cell line when 293 cells are infected.
23. A polytropic packaging cell line wherein the packaging cell
line, upon introduction of a vector construct, produces vector
particles capable of infecting cells of kidney lineage, but not
cells of fibroblast, epithelial, T-cell or monocyte lineage.
24. A non-mouse packaging cell line carrying on separate operons
the genes for gag/pol and env, said operons lacking retroviral LTR
sequences and which, upon introduction of an N2 type vector
construct, produces substantially no helper virus after at least
twenty days passage in culture.
25. The cell line of claim 24 wherein the cell line is an
amphotropic packaging cell line.
26. The cell line of claim 24 wherein the cell line is a polytropic
packaging cell line.
27. The cell line of claim 24 wherein the cell line is a xenotropic
packaging cell line.
28. A method of producing a recombinant retrovirus, comprising: (a)
introducing packaging genes from a retroviral vector system into a
cell line, said cell line having substantially no endogenous
proviruses which produce transcripts packageable by the retroviral
vector system; and (b) selecting for cells that produce at least a
tenfold increase in viral packaging protein as compared to a
standard mouse amphotropic packaging cell line, and that, upon
introduction of a vector construct, produce at least a ten-fold
increase in vector titre as compared to a standard mouse
amphotropic packaging cell line.
29. A method of producing a recombinant retrovirus, comprising: (a)
introducing packaging genes from a retroviral vector system capable
of infecting human cells into a cell line, said cell line having
substantially no endogenous proviruses which produce transcripts
packageable by the retroviral vector system; and (b) selecting for
cells that, upon introduction of a vector construct, produce vector
titres at least equivalent to those of a standard mouse amphotropic
packaging cell line, and which produce vector particles capable of
infecting human cells.
30. A cell line selected from the group consisting of CA, 2A, DA,
DA2, DX, HX, and HP.
31. A method of producing a vector capable of infecting a selected
cell type, comprising: (a) continuously passaging a virus in cells
of the selected cell type until the virus has genetically mutated
and a predominant fast growing strain has evolved; (b) isolating
the mutated and fast growing strain; (c) identifying and isolating
the components of the mutated strain responsible for the
preferential growth of the mutated virus; (d) inserting the
identified and isolated components as substitutes for counterpart
components in a producer cell based upon the virus prior to its
continuous passage; and (e) culturing the producer cell to produce
the vector.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
07/586,603, which is a continuation-in-part of U.S. Ser. No.
07/565,606, filed Aug. 10, 1990, which is a continuation-in-part of
U.S. Ser. No. 07/395,932, filed Aug. 18, 1989, which is a
continuation-in-part of U.S. Ser. No. 07/170,515, filed Mar. 21,
1988, which application is now abandoned.
TECHNICAL FIELD
[0002] The present invention relates generally to retroviruses, and
more specifically, to recombinant retroviruses which are capable of
delivering vector constructs to susceptible target cells. These
vector constructs are typically designed to express desired
proteins in target cells, including proteins which can have a
therapeutic effect in a number of ways, and hence, constitute a
"drug" transport system for allowing transport of proteins (or RNA)
into cells. The specificity of proteins (and RNA) for enzymatic
reaction, for binding of cellular components, for immunological
action, or for other biological effects, allows for correspondingly
specific actions on target cells if the protein or RNA molecule can
be transported into the cells. Such actions include the repair of
genetic defects, production of antisense RNA to block cellular
process, the enzymatic potentiation of prodrugs, and stimulation of
the cellular immune system, as well as many other therapies based
on the intracellular production of proteins.
BACKGROUND OF THE INVENTION
[0003] Retroviruses are RNA viruses which can replicate and
integrate into a host cell's genome through a DNA intermediate.
This DNA intermediate, or provirus, may be stably integrated into
the host's cellular DNA. Due to their efficiency at integrating
into host cells, retroviruses are considered to be one of the most
promising vectors for use in human gene therapy. These vectors have
a number of properties that lead them to be considered as one of
the most promising techniques for genetic therapy of disease. These
include: (1) efficient entry of genetic material (the vector
genome) into cells; (2) an active efficient process of entry into
the target cell nucleus; (3) relatively high levels of gene
expression; (4) minimal pathological effects on target cells; and
(5) the potential to target to particular cellular subtypes through
control of the vector-target cell binding and the tissue-specific
control of gene expression. For example, a foreign gene of interest
may be incorporated into the retrovirus in place of the normal
retroviral RNA. When the retrovirus injects its RNA into a cell,
the foreign gene is also introduced into the cell, and may then be
integrated into the host's cellular DNA as if it were the
retrovirus itself. Expression of this foreign gene within the host
results in expression of the foreign protein by the host cell.
[0004] Most retroviruses which have been developed for gene therapy
are murine retroviruses. Briefly, these retroviruses exist in two
forms, as proviruses integrated into a host's cellular DNA, or as
free virions. The virion form of the virus contains the structural
and enzymatic proteins of the retrovirus (including reverse
transcriptase), two RNA copies of the viral genome, and portions of
the cell's plasma membrane in which is embedded the viral envelope
glycoprotein. The genome is organized into four main regions: the
Long Terminal Repeat (LTR), and the qag, pol. and env genes may be
found at both ends of the proviral genome, is a composite of the 5'
and 3' ends of the RNA genome, and contains cis-acting elements
necessary for the initiation and termination of transcription. The
three genes gag, pol, and env are located between the terminal
LTRs. The gag and pol genes encode, respectively, internal viral
structures and enzymatic proteins. The env gene encodes the
envelope glycoprotein which confers infectivity and host range
specificity of the virus.
[0005] An important consideration in using retroviruses for gene
therapy is the availability of "safe" retroviruses. Packaging cell
lines have been developed to meet this concern. Briefly, this
methodology employs the use of two components, a retroviral vector
and a packaging cell. The retroviral vector contains long terminal
repeats (LTRs), the foreign DNA to be transferred and a packaging
sequence (i). This retroviral vector will not reproduce by itself
because the genes which encode structural and envelope proteins are
not included within the vector. The packaging cell contains genes
encoding the gag, pol, and env proteins, but does not contain the
packaging signal ".psi." Thus, a packaging cell can only form empty
virion particles by itself. Within this general method, the
retroviral vector is introduced into the packaging cell, thereby
creating a "producer cell." This producer cell manufactures virion
particles containing only the retroviral vector's (foreign) DNA,
and therefore has previously been considered to be a safe
retrovirus for therapeutic use.
[0006] There are several shortcomings in the current use of this
approach. One issue involves the generation of "live virus" (i.e.,
competent replicating retrovirus) by the producer cell line.
Preparations of human therapeutics which are contaminated with
retroviruses are not currently considered suitable for use in human
therapy. For example, extreme measures are taken to exclude
retroviral contamination of for imaging and therapy. Live virus can
in conventional producer cells when: (1) The vector genome and the
helper genomes recombine with each other; (2) The vector genome or
helper genome recombines with homologous cryptic endogenous
retroviral elements in the producer cell; or (3) Cryptic endogenous
retroviral elements reactivate (e.g., xenotropic retroviruses in
mouse cells).
[0007] Another issue is the propensity of mouse based producer
lines to package endogenous retroviral-vector-like elements (which
can contain onc gene sequences) at efficiencies close to that with
which they package the desired vector. Such elements, because of
their vector-like structure, are transmitted to the target
treatment cell at frequencies that parallel its transfer of the
desired vector sequence.
[0008] A third issue is the ability to make sufficient vector
particles at a suitable concentration to: (1) treat a large number
of cells (e.g., 10.sup.8-10.sup.10); and (2) manufacture vector
particles at a commercially viable cost. Finally, the only producer
lines currently used for transfer of genes to human cells are
amphotropic producer lines, known for the eponymous murine
retroviral envelope gene, which has receptors in most human
cells.
[0009] In order to construct safer packaging cell lines,
researchers have generated additional deletions in the 3' LTR and
portions of the 5' LTR (see, Miller and Buttimore, Mol. Cell.
Biol., 6:2895-2902, 1986). When such cells are used, two
recombination events are necessary to form the wild-type genome.
Nevertheless, results from several laboratories have indicated that
even when several mutations are present, wild-type virus may still
be generated (see, Bosselman et al., Mol. Cell. Biol. 7:1797-1806,
1987; Danos and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6460-6464,
1988).
[0010] Many of the helper cell lines that have been described to
date have been limited to a host cell range of murine, avian, rat
and dog cell lines have been generated using amphotropic retroviral
vector systems, which can infect human cells as well as a broad
range of other mammalian cells (see, Sorge et al., Mol. Cell. Biol.
4:1720-1737, 1984), amphotropic packaging lines developed thus far
have retained portions of one or more of the viral LTRs, and, thus,
even when multiple mutations are present, have remained capable of
generating a replication-competent genome. Amphotropic vector
systems with multiple mutations and reduced propensities toward
generating infectious virus generally exhibit unsatisfactorily low
titres of retroviral particles.
[0011] One of the more recent approaches to constructing safer
packaging cell lines involves the use of complementary portions of
helper virus, divided among two separate plasmids, one containing
gag and pol, and the other containing env (see, Markowitz et al.,
J. Virol. 62:1120-1124; and Markowitz et al., Virology 167:
600-606, 1988. One benefit of this double-plasmid system is that
three recombination events are required to generate a replication
competent genome. Nonetheless, these double-plasmid vectors have
also suffered from the drawback of including portions of the
retroviral LTRs, and therefore remain capable of producing
infectious virus. Cell lines containing both 3' and 5' LTR
deletions have been constructed, but have thus far not proven
useful since they produce relatively low titers (Daugherty et al.,
J. Virol. 63:3209-3212, 1989).
[0012] The present invention overcomes difficulties of prior
packaging cell lines, and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for producing
recombinant retroviruses in which the retroviral genome is packaged
in a capsid and envelope, preferably through the use of a packaging
cell. The packaging cells are provided with viral protein-coding
sequences, preferably in the form of two plasmids integrated into
the genome of the cell, which produce all proteins necessary for
production of viable retroviral particles, a DNA viral construct
which codes for an RNA which will carry the desired gene, along
with a packaging signal which will direct packaging of the RNA into
the retroviral particles.
[0014] The present invention additionally provides a number of
techniques for producing recombinant retroviruses which can
facilitate:
[0015] i) the production of higher titres from packaging cells;
[0016] ii) the production of higher titres of helper free
recombinant retrovirus from packaging cell lines that are
non-murine (to avoid production of recombinant or endogenously
activated retroviruses, and to avoid packaging of defective murine
retroviral sequences) and which will infect human cells;
[0017] iii) the production of helper free recombinant retroviruses
with higher titres using alternative non-hybrid envelopes such as
xenotropic or polytropic envelope proteins (to allow infection of
cells poorly infectable with amphotropic recombinant retroviruses
or to allow specificity of cell type infection).
[0018] iv) packaging of vector constructs by means not involving
the use of packaging cells;
[0019] v) the production of recombinant retroviruses which can be
targeted for preselected cell lines;
[0020] vi) the construction of retroviral vectors with
tissue-specific (e.g., tumor) promoters; and
[0021] vii) the integration of the proviral construct into a
preselected site or sites in a cell's genome.
[0022] One technique for producing higher titres from packaging
cells takes advantage of the discovery that of the many factors
which can limit titre from a packaging cell, one of the most
limiting is the level of expression of the packaging proteins,
namely, the gag, pol, and env proteins, as well as the level of
expression of the retroviral vector RNA from the proviral vector.
This technique allows the selection of packaging cells which have
higher levels of expression (i.e., produce higher concentrations)
of the foregoing packaging proteins and vector construct RNA. More
specifically, this technique allows selection of packaging cells
which produce high levels of what is referred to herein as a
"primary agent," which is either a packaging protein (e.g., gag,
pol, or env proteins) or a gene of interest to be carried into the
genome of target cells (typically as a vector construct). This is
accomplished by providing in packaging cells a genome carrying a
gene (the "primary gene") which expresses the primary agent in the
packaging cells, along with a selectable gene, preferably
downstream from the primary gene. The selectable gene expresses a
selectable protein in the packaging cells, preferably one which
conveys resistance to an otherwise cytotoxic drug. The cells are
then exposed to a selecting agent, preferably the cytotoxic drug,
which enables identification of those cells which express the
selectable protein at a critical level (i.e., in the case of a
cytotoxic drug, by killing those cells which do not produce a level
of resistance protein required for survival).
[0023] Preferably, in the technique briefly described above, the
expression of both the selectable and primary genes is controlled
by the same promoter. In this regard, it may be preferable to
utilize a non-MLV retroviral 5' LTR. In order to maximize titre of
a recombinant retro-virus from packaging cells, this technique is
first used to select packaging cells expressing high levels of all
the required packaging proteins, and then is used to select which
of these cells, following transfection with the desired proviral
construct, produce the highest titres of the recombinant
retrovirus.
[0024] Techniques are also provided to select cells that produce
higher titres of helper free recombinant retroviruses in non-murine
cells. These cell lines produce recombinant retroviruses capable of
efficiently infecting human cells. These techniques involve
screening potential parent cells for their ability to produce
recombinant retroviruses in the presence of a replicating virus.
Subsequently, uninfected cultures of candidate cell lines chosen by
the above procedure are infected with a vector expressing a
retroviral gag/pol, and clones which synthesize high levels of
gag/pol are identified. A clone of this type is then reinfected
with a vector expressing env, and clones expressing high level of
env (and gag/pol) are identified. Within the context of the present
invention, "high levels" means discernibly greater than that seen
in the standard mouse packaging line, PA317 on a Western blot
analysis. Many non-mouse cell lines such as human or dog have never
been known to spontaneously generate competent retrovirus, do not
carry possible recombination partners for recombinant murine
retroviral packaging or gene sequences; and do not carry genes
which make RNA which may be packaged by the MLV system. Techniques
are provided to generate cell lines which produce high titres of
recombinant retroviruses using alternative envelopes such as
xenotropic or polytropic by techniques similar to those described
above. Such retroviruses may be used in infecting amphotropic
resistant cells (xenotropic envelope) or infecting only a subset of
cells (polytropic).
[0025] A technique suitable for producing recombinant retroviruses
which can be targeted for preselected cell lines utilizes
recombinant retroviruses having one or more of the following: an
env gene comprised of a cytoplasmic segment of a first retroviral
phenotype, and an extracellular binding segment exogenous to the
first retroviral phenotype (the binding segment being from a second
viral phenotype or from another protein with desired binding
properties which is selected to be expressed as a peptide which
will bind to the desired target); another viral envelope protein;
another ligand molecule in place of the normal envelope protein; or
another ligand molecule along with an envelope protein that does
not lead to infection of the target cell type. Preferably, in the
technique briefly described above, an env gene comprised of a
cytoplasmic segment of a retroviral phenotype is combined with an
exogenous gene encoding a protein having a receptor-binding domain
to improve the ability of the recombinant retrovirus to bind
specifically to a targeted cell type, e.g., a tumor cell. In this
regard, it may be preferable to utilize a receptor-binding domain
which binds to receptors expressed at high levels on the surface of
the target cell (e.g., growth factor receptors in tumor cells) or
alternatively, a receptor-binding domain binding to receptors
expressed at a relatively higher level in one tissue cell type
(e.g., epithelial cells, ductal epithelial cells, etc., in breast
cancer). One potential advantage to targeting with hybrid envelopes
with specificity for growth factor or activation receptors (like
EGF or CD3 receptors is that binding of the vector itself may then
lead to cell cycling, which is necessary for viral integration and
expression. Within this technique, it may be possible to improve
and genetically alter recombinant retroviruses with specificity for
a given tumor by repeated passage of a replicating recombinant
retrovirus in tumor cells; or by linking the vector construct to a
drug resistance gene and selecting for drug resistance.
[0026] Techniques for integrating a retroviral genome at a specific
site in the DNA of a target cell involve the use of homologous
recombination, or alternatively, the use of a modified integrase
enzyme which will recognize a specific site on the target cell
specific insertion allows genes to be inserted at sites on the
target cells' DNA, which will minimize the chances of insertional
mutagenesis, minimize interference from other sequences on the DNA,
and allow insertion of sequences at specific target sites so as to
reduce or eliminate the expression of an undesirable gene (such as
a viral gene) in the DNA of the target cell.
[0027] It will be appreciated that any of the above-described
techniques may be used independently of the others in particular
situations, or can be used in conjunction with one or more of the
remainder of the techniques.
[0028] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A depicts four plasmids designed to express retroviral
proteins in mammalian cells. pSVgp and pRSVenv are cotransfected
with a selectable marker, while pSVgp-DHFR and pRSVenv-phleo are
the equivalent plasmids with the selectable marker placed
downstream of the viral protein-coding regions.
[0030] FIG. 1B depicts vectors which lead- to expression of: 1. MLV
core proteins (pSCV10); 2-4 MLV amphotropic env (pCMvenv AmDra,
pCMVenv AnNhe, pMLPenv AmSph); 5. MLV xenotropic env (PCMV xeno).
6. MLV MCFenv (pCMV MCF); 7. MLV amphotropic env as a retroviral
vector (pLARNL).
[0031] FIG. 1C depicts the results of the screening procedure for
assessing the intrinsic ability of cell lines to make retroviral
vectors in the presence of helper virus (Example 2B).
[0032] FIG. 1D depicts the results of selecting clones of cells
into which pSCV10 had been introduced and examining these clones
for gag production as compared to PA317 by Western blots.
[0033] FIG. 1E depicts the results of Western blot experiments to
compare levels of amphotropic env in cell lysates from DA, CA, 2A
and PA317.
[0034] FIG. 1F depicts the results of Western blot experiments to
compare levels of xenotropic env in cell lysates from transient
transfections of CF gag/pol and permanently expressing lines XF7,
X6, X10 and PA317 (ampho env).
[0035] FIG. 1G depicts the results of Western blot 10 experiments
to compare levels of MCF (polytropic) env in HT1080 derived clones,
PA317 (amphotropic env) and HX (xenotropic env).
[0036] FIG. 2 depicts three sites of fusion of HIV env and MOMLV
env after site-directed mutagenesis. The joint at the extracellular
margin of the transmembrane region is designated as A, while B and
C indicate locations of joints at the middle of the transmembrane
region and cytoplasmic margin, respectively. The numbering is
according to nucleotide numbers (RNA Tumor Viruses, Vol. II, Cold
Spring Harbor, 1985). ST, SR, SE are the starts of tat, rev and env
while TT, TR, and TE are the corresponding termination sites.
[0037] FIG. 3 depicts the substitution of U3 in a 5' LTR by a
heterologous promoter/enhancer in which can be fused to either the
Sac I, Bssh II or other site in the region.
[0038] FIG. 4 illustrates a representative method for crossing
transgenic mice expressing viral protein or vector RNA. 30
DETAILED DESCRIPTION OF THE INVENTION
[0039] In one aspect, the present invention is based, in part, upon
the discovery of the major causes of low recombinant virus titres
from packaging cell lines (PCL), and of techniques to correct those
causes. Basically, at least five factors may be postulated as
causes for low recombinant virus titres:
[0040] 1. the limited availability of viral packaging proteins;
[0041] 2. the limited availability of retroviral vector RNA
genomes;
[0042] 3. the limited availability of cell membrane for budding of
the recombinant retroviruses;
[0043] 4. the limited intrinsic packaging efficiency of the
retroviral vector genome; and
[0044] 5. the density of the receptor specific for the envelope of
a given retrovirus.
[0045] 6. The limited availability of host cell constituents (such
as RNA or myristoylation, phosphorylation, glycosylation or
proteolytic functions).
[0046] As noted above, the limited availability of viral packaging
proteins is the initial limiting factor in recombinant retrovirus
production from packaging cells. When the level of packaging
protein in the packaging cells is increased, titre increases to
about 10.sup.5 infectious units/milliliter, following which
increasing packaging protein level has no further effect on titres.
However, titres can be further augmented by also increasing the
level of retroviral vector genome available for packaging. Thus, as
described herein, it is advantageous to select producer cells that
manufacture the maximum levels of packaging proteins and retroviral
vector genomes. It has been discovered that the methods of
identifying, and thus selecting, packaging cells and producer
cells, described earlier under the section entitled "Background of
the Invention," tend to lead to selection of many producer cells
which produce low titres for the reasons described below.
[0047] The present invention takes advantage of the previously
disadvantageous fact that the protein expression level of a gene
downstream from the 5' LTR or other promoter, and spaced therefrom
by an intervening gene, is substantially less than if the
intervening gene were absent. In the present invention, the
selectable gene is placed downstream from a gene of the packaging
genome or the gene of interest carried by the vector construct, but
is still transcribed under the control of the viral 5' LTR or other
promoter without any splice donor or splice acceptor sites. This
accomplishes two things. First, since the packaging genes or genes
of interest are now upstream with no intervening gene between
themselves and the promoter, their corresponding proteins
(packaging protein or protein of interest) will be expressed at a
higher level (five- to twentyfold) than the selectable protein.
Second, the selectable protein will be expressed on average at a
lower level, with the distribution of level of expression shifting
toward lower levels. However, the selection level for resistance to
phleomycin remains the same, so that only the top-end expressing
cells survive. The levels of the packaging protein or of the
protein of interest will still be proportional, only in this case,
a higher level of selectable protein corresponds to a much higher
level of packaging protein or protein of interest.
[0048] Preferably, the foregoing procedure is performed using a
plasmid carrying one of the proviral gag/pol or env packaging
genes, along with a first selectable gene. These cells are then
screened for the cells producing the highest levels of protein by
reaction with an antibody against gag/pol (or possibly env), a
second enzyme or labelled antibody, and then sorted on a
fluorescence-activated cell sorter (FACS) or detected on a western
blot. Alternatively, other tests for protein level may be used.
Subsequently, the procedure and screening are repeated using those
selected cells, and the other of the gag/pol or env packaging
genes. In this step, a second selectable gene (different from the
first) would be required downstream from the packaging gene and the
cells producing the largest amount of the selected. This cell line
is a packaging cell line (PCL) that may be used with any available
vector. The procedure and screening are then repeated using the
surviving cells, with a plasmid carrying the proviral vector
construct bearing the gene of interest and a third selectable gene,
different from the first or second selectable gene. As a result of
this procedure, cells producing high titres of the desired
recombinant retrovirus will be selected, and these can be cultured
as required to supply recombinant retrovirus. In addition, gag and
pol can be independently introduced and selected.
[0049] Example 1 describes the construction of gag/pol and env
plasmids designed to use these procedures.
EXAMPLE 1
Plasmids Designed to Make High Levels of Packaging Proteins (FIG.
1)
[0050] 1. The 2.7 kb Xba I fragment from pPAM (Miller et al., Mol.
Cell. Biol. 5:431, 1985), which contains the amphotrophic env
segment, was cloned in pUC18 at the Xba I site, then removed with
Hind III and Sma I. This fragment was cloned into the vector pRSV
neo (Gorman et al., Mol. Cell. Biol. 2:1044, 1982; Southern et al.,
J. Mol. Appl. Genet. 1:327, 1982) cut with Hind III and Pvu II, to
give pRSV env. A 0.7 kb Bam HI to BstE II fragment from the plasmid
pUT507 (Mulsant et al., Somat. Cell. Mol. Genet. 14:243, 1988) with
the BstE II end filled in carries the phleo resistance coding
sequence. The 4.2 kb Bam HI to Xho I fragment, the contiguous 1.6
kb Xho I to Xba I (Xba I filled in) from RSVenv, and the phleo
fragment were ligated to give pRSVenv-phleo.
[0051] 2. A fragment from the Pst I site at nucleotide 563 of MLV
(RNA Tumor Viruses, Vol. II, Cold Spring Harbor, 1985) to the Sca I
site at 5870 was derived from pMLV-K (Miller et al., 1985, op.
cit.) and cloned in the Pst I to Bam HI (Bam HI filled-in) fragment
from p4aA8 (Jolly et al., Proc. Natl. Acad. Sci. USA 80:477, 1983)
that has the SV40 promoter, the pBR322 ampicillin resistance and
origin of replication and the SV40 poly A site. This gives pSVgp.
pSVgpDHFR was made using the following fragments: the 3.6 kb Hind
III to Sal I fragment from pSVgp containing the SV40 promoter plus
MLV gag and some pol sequences; the 2.1 kb Sal I to Sca I fragment
from PMLV-K with the rest of the pol gene, the 3.2 kb Xba I (Xba I
filled-in) to Pst I fragment from pF400 with the DHFR gene plus
poly A site, pBR322 origin and half the ampicillin resistance gene;
the 0.7 kb Pst I to Hind III fragment from pBR322 with the other
half of the ampicillin resistance gene. This gives pSVgp-DHFR. All
these constructs are shown in FIG. 1. These plasmids can be
transfected into 3T3 cells or other cells, and high levels of gag,
pol or env obtained.
[0052] An additional method for accomplishing selection is to use a
gene selection in one round and its antisense in a subsequent
round. For example, gag/pol may be introduced into an
HPRT-deficient cell with the HPRT gene and selected for the
presence of this gene using that media which requires HPRT for the
salvage of purines. In the next round, the antisense to HPRT could
be delivered downstream to env and the cell selected in 6
thioguanine for the HPRT-deficient phenotype. Large amounts of
antisense HPRT would be required in order to inactivate the HPRT
gene transcripts, assuming no reversion occurred. A further method
of accomplishing selection is described below. Co-transfection of a
10.times. stoichiometric excess of the expression vector over the
separate selectable marker ensures high copy number of expression
vector in drug resistant cell clones. In most of the examples noted
herein, the gag/pol and envelope expression vectors were introduced
independently (i.e., separate transfections) so that the two
structural genes would not recombine or concatamerize (as
transfected integrated DNA tends to do), assuring that the genes
are unlinked in the genome. The steady-state level of intracellular
MLV gag/pol and env was measured by protein immunoblotting. The
relative ease, sensitivity, and reproducibility of immunoblotting
allowed rapid, quantitative analysis of a large number of cell
clones necessary to identify over-expressors of the MLV structural
proteins (gag/pol in particular) (see Example 2).
[0053] In addition to the gag/pol expressing constructs which begin
at nucleotide 563 of MOMLV, several others can be constructed which
contain upstream lead sequences. It has been observed by Prats et
al. (RNA Tumor Viruses Meeting, Cold Spring Harbor, N.Y., 1988)
that a glycosylated form of the gag protein initiates at nucleotide
357 and a translation enhancer maps in the region between
nucleotides 200-270. Therefore, gag/pol expressing constructs may
be made beginning at the Bal I site (nucleotide 212) or Eag I site
(nucleotide 346) to include these upstream elements and enhance
vector production. A preferred method of accomplishing this is to
include degenerate mutations to inactivate the packaging signal
found here, without affecting the coding potential of the nucleic
acid.
[0054] Envelope Substitutions
[0055] The ability to express gag/pol and env function separately
allows for manipulation of these functions independently. A cell
line that expresses ample amounts of gag/pol can be used, for
example, to address questions of titre with regard to env. One
factor resulting in low titres is the density of appropriate
receptor molecules on the target cell or tissue. A second factor is
the affinity of the receptor for the viral envelope protein. Given
that env expression is from a separate unit, a variety of envelope
genes (requiring different receptor proteins), such as xenotropic,
polytropic, or amphotrophic envs from a variety of sources, can be
tested for highest titres on a specific target tissue.
[0056] Envelope proteins from one retrovirus can often substitute,
to varying degrees, for that of another retrovirus. For instance,
the envelope of murine virus 4070A, HTLV I, GALV, and BLV can each
substitute for that of MOMLV, albeit with a lower efficiency (Cone
and Mulligan, Proc. Natl. Acad. Sci. USA 81:6349-53, 1984; Wilson
et al., J. Virol. 63, 2374-78, 1989; Ban et al., J. Gen. Virol.
70:1987-93, 1989). To increase the number of cell types that could
be infected with MLV-based vectors, PCLs were generated which
individually express either amphotropic, xenotropic, or polytropic
envelopes. Vector produced from any of these PCLs can be used to
infect any cell which contains the corresponding distinct receptor
(Rein and Schultz, Virology 136:144-52, 1984). Some cell types may,
for instance, lack the amphotropic receptor and thus be resistant
to infection with amphotropic vector, but express the xenotropic
receptor and therefore be infectable by xenotropic vector. One
report suggests that xenotropic vector, in the presence of
replication-complement xenotropic virus, may more effectively
infect human hematopoietic progenitor cells (Eglitis et al.,
Biochem. Biophys. Res. Comm. 151:201-206, 1988). Xenotropic vector,
in the presence of replication-competent xenotropic virus, also
infects cells from other species which are not easily infectable by
amphotropic virus such as bovine, porcine, and equine (Delouis et
al., Biochem. Biophys Res. Comm. 169:80-14, 1990). The xenotropic
PCLs will therefore be useful for veterinary purposes in these
species. Another example would be utilization of the spleen
focus-forming virus (SFFV) envelope gene which may allow targeting
to cells containing the erythropoietin receptor (J. P. Li et al.,
Nature 343:762-764, 1990).
[0057] As a specific example, all of the amphotropic PCLs described
herein (canine and human fibroblasts) were infectable by xenotropic
vector but were resistant to infection by amphotropic vector,
presumably due to the phenomenon of "viral interference" (cf. A.
Rein, Virology 120:251-57, 1982). The xenotropic PCL therefore
allows the facile infection of these amphotropic PCLs, which in
turn produces 10-100.times.-higher titre than PCLs whose vector has
been introduced by other means (Miller et al., Somat. Cell Mol.
Genet. 12:175-83, 1986). In principle, a PCL expressing any viral
envelope which can function with the MLV vector and packaging
system and whose corresponding cellular receptor is found in a
given PCL, is useful for allowing vector infection of that PCL.
[0058] Vector produced from the polytropic PCL described herein has
a more restricted host range on human cells than vector produced
from either amphotropic or xenotropic PCLs (see data below). The
polytropic PCL may therefore be particularly useful for targeting
vector to a specific human cell type. The reduced homology between
both xenotropic and polytropic envelopes with the MOMLV gag/pol and
with the vector makes these PCLs even less likely to generate
replication-competent retrovirus by homologous recombination than
amphotropic PCLs. Examples of the use of these methods are set
forth below (see Example 2).
[0059] Furthermore, envelopes from nonmurine retrovirus sources can
be used for pseudotyping a vector. The exact rules for pseudotyping
(i.e., which envelope proteins will interact with the nascent
vector particle at the cytoplasmic side of the cell membrane to
give a viable viral particle (Tato, Virology 88:71, 1978) and which
will not (Vana, Nature 336:36, 1988), are not well characterized.
However, since a piece of cell membrane buds off to form the viral
envelope, molecules normally in the membrane are carried along on
the viral envelope. Thus, a number of different potential ligands
can be put on the surface of viral vectors by manipulating the cell
line making gag and pol in which the vectors are produced or
choosing various types of cell lines with particular surface
markers. One type of surface marker that can be expressed in helper
cells and that can give a useful vector-cell interaction is the
receptor for another potentially pathogenic virus. The pathogenic
virus displays on the infected cell surface its virally specific
protein (e.g., env) that normally interacts with the cell surface
marker or receptor to give viral infection. This reverses the
specificity of the infection of the vector with respect to the
potentially pathogenic virus by using the same viral
protein-receptor interaction, but with the receptors on the vector
and the viral protein on the cell.
[0060] It may be desirable to include a gene which encodes for an
irrelevant envelope protein which does not lead to infection of
target cells by the vector so produced, but does facilitate the
formation of infectious viral particles. For example, one could use
human Sup T1 cells as a helper line. This human T-cell line
expresses CD4 molecules at high levels on its surface. Conversion
of this into a helper line can be achieved by expressing gag/pol
with appropriate expression vectors and also, if necessary, the
Moloney ecotropic env gene product as an irrelevant (for human
cells) envelope protein (the Moloney ecotropic env only leads to
infection of mouse cells). Vectors produced from such a helper line
would have CD4 molecules on their surfaces and therefore be capable
of infecting only cells which express HIV env, such as HIV-infected
cells.
[0061] In addition, hybrid envelopes (as described below) can be
used in this system as well, to tailor the tropism (and effectively
increase titres) of a retroviral vector. A cell line that expresses
ample amounts of a given envelope gene can be employed to address
questions of titre with regard to gag and pol.
[0062] Furthermore, it is also possible to add ligand molecules
exogenously to the viral particles which either incorporate
themselves in the lipid envelope or can be linked chemically to the
lipid or protein constituents.
[0063] Cell Lines
[0064] The most common packaging cell lines used for MOMLV vector
systems (psi2, PA12, PA317) are derived from murine cell lines.
There are several reasons why a murine cell line is not the most
suitable for production of human therapeutic vectors:
[0065] 1. They are known to contain endogenous retroviruses, some
of which are closely related in sequence and viral type to the MLV
vector system used here.
[0066] 2. They contain nonretroviral or defective retroviral
sequences that are known to package efficiently.
[0067] 3. There may be deleterious effects caused by the presence
of murine cell membrane components.
[0068] Several non-murine cell lines are potential parents for
packaging lines. These include Vero cells which are used in Europe
to prepare polio vaccine, WI38 which are used in the U.S. in
vaccine production, CHO cells which are used in the U.S. for TPA
preparation, D17 or other dog cells that may have no endogenous
viruses, and those described in Example 2.
[0069] The most important safety concern for the production of
retroviral vectors is the inherent propensity of retroviral PCLs to
generate replication-competent retrovirus after introduction of a
vector (Munchau et al., Virology 176:262-65, 1990). This can occur
in at least two ways: 1) homologous recombination can occur between
the therapeutic proviral DNA and the DNA encoding the MOMLV
structural genes ("gag/pol" and "env") present in the PCL
(discussed below under "Generation of PCLs"); and 2) generation of
replication-competent virus by homologous recombination of the
proviral DNA with the very large number of defective endogenous
proviruses found in murine cells (Steffen and Weinberg, Cell
15:1003-10, 1978); Canaani and Aaronson, Proc. Natl. Acad. Sci.,
USA 76:1677-81, 1979; Stoye and Coffin, J. Virol. 61:2659-69 1987).
In addition, even murine cell lines lacking vector can produce
virus spontaneously or after induction, (e.g., xenotropic virus
which can replicate in human cells, Aaronson and Dunn, J. Virol.
13:181-85, 1974; Stephenson and Aaronson, Proc. Natl. Acad. Sci.,
USA 71:4925-29, 1974; Aaronson and Stephenson, Biochem. Biophys.
Acta 458:323-54, 1976). Another safety concern with the utilization
of murine cells for the production of murine retroviral vectors is
the observation that some of the many endogenous proviral genes
(retrovirus-like genes) in the murine genome are expressed,
recognized by the retroviral structural gene products of murine
PCLs, and delivered and expressed in target cells with an
efficiency at least comparable to that of the desired vector
(Scolnick et al., J. Virol. 29:964-72, 1979; Scadden et al., J.
Virol. 64:424-27, 1990). These observations strongly suggest that
murine cell lines are an unsafe choice for the production of murine
retroviral vectors for human therapeutics. To circumvent the
inherent safety problems associated with murine cells, PCLs have
been generated exclusively from non-murine cell lines (e.g., canine
and human cell lines) which are known to lack genomic sequences
homologous to that of MOMLV by hybridization analysis (data not
shown) (Martin et al., Proc. Natl. Acad. Sci., USA 78:4892-96,
1981). Those skilled in the art will recognize that the packaging
cells described herein will have a low, but inherent capability of
packaging random RNA molecules. Such RNA molecules will not be
permanently transmitted to the pseudo-infected target cell.
[0070] In addition to issues of safety, the choice of host cell
line for the PCL is of importance because many of the physical
(such as stability) and biological properties (such as titre) of
retroviral particles are dictated by the properties of the host
cell. For instance, the host cell must efficiently express
(transcribe) the vector RNA genome, prime the vector for first
strand synthesis with a cellular tRNA, tolerate and covalently
modify the MLV structural proteins (proteolysis, glycosylation,
myristylation, and phosphorylation), and the maturing virion buds
from the cell membrane, carrying many of the membrane components
with it. For example, it has been found that vector made from the
mouse packaging line PA317 is retained by a 0.3 micron filter,
while that made from the CA line described herein will pass
through.
EXAMPLE 2
Packaging Cell Selection
[0071] A. MLV Structural Gene Expression Vectors
[0072] To decrease the possibility of replication-competent virus
being generated by genetic interactions between the MLV proviral
vector DNA and the structural genes of the PCL, separate expression
vectors, each lacking the viral LTR, were generated to express the
gag/pol and env genes independently. To further decrease the
possibility of homologous recombination with MLV vectors and the
resultant generation of replication-competent virus, minimal
sequences other than the protein coding sequences were used. In
order to express high levels of the MLV structural proteins in the
host cells, strong transcriptional promoters (CMV early and Ad5
major late promoters) were utilized. An example of the construction
of a MoMLV gag/pol expression vector (pSCV10, see FIG. 1B.1)
follows:
[0073] 1. The 0.7 Kb HinCII/XmaIII fragment encompassing the human
cytomegalovirus (CMV) early transcriptional promoter (Boshart et
al., Cell 41:521-30, 1985) was isolated.
[0074] 2. A 5.3 Kb PstI(partial)/ScaI fragment from the MoMLV
proviral plasmid, MLV-K (Miller et al., Mol. Cell Biol. 5:531,
1985) encompassing the entire gag/pol coding region was
isolated.
[0075] 3. A 0.35 Kb DraI fragment from SV40 DNA (residues
2717-2363) encompassing the SV40 late transcriptional termination
signal was isolated.
[0076] 4. Using linkers and other standard recombinant DNA
techniques, the CMV promoter-MoMLV gag/pol-SV40 termination signal
was ligated into the bluescript vector SK.sup.+.
[0077] An example of the construction of an MLV amphotropic
envelope expression vector (pCMVenvAmDra, see FIG. 1B.2)
follows.
[0078] 1. A 2.7 Kb XbaI/NheI fragment containing the coding
sequence of amphotropic envelope from the 4070A proviral clone
(Chattopadhyay et al., J. Virol. 39:777-91, 1981) was isolated.
[0079] 2. Using linkers and other standard DNA techniques, the CMV
early promoter and SV40 late termination signal described for the
gag/pol expression above (pSCV10) were ligated in the order CMV
promoter-envelope-termination signal.
[0080] A second example of the construction of an MLV amphotropic
envelope expression vector (PCMVenvAmNhe, see FIG. 1B.3)
follows.
[0081] 1. A 2.7 Kb XbaI/NheI fragment containing the coding
sequence of amphotropic envelope from the 4070A proviral clone
described above was isolated.
[0082] 2. Using linkers and other standard recombinant DNA
techniques, the CMV early promoter described for the gag/pol
expression above (pSCV10) was ligated in the plasmid pUC18 in the
order CMV promoter-envelope (no added transcriptional termination
signal).
[0083] A third example of the construction of an MLV amphotropic
envelope expression vector (pMLPenvAmSph, see FIG. 1B.4)
follows.
[0084] 1. A 0.9 Kb EcoRI/HindIII fragment containing the Adenovirus
5 left end, major late transcriptional promoter, and tripartite
leader sequence was isolated.
[0085] 2. A 0.85 Kb EcoRI/BamHI fragment containing the SV40 small
t intron and transcriptional termination signal from clone pJD204
(De Wit et al., Mol. Cell. Biol. 7:725-37, 1987) was isolated.
[0086] 3. A 3 Kb SphI/SmaI fragment containing the coding sequence
of amphotropic envelope from the 4070A proviral clone described
above was isolated.
[0087] 4. Using linkers and other standard recombinant DNA
techniques, the MLP, amphotropic envelope and the SV40 termination
signal were ligated in plasmid pBR322 in the order
MLP-envelope-SV40.
[0088] An example of the construction of an MLV xenotropic envelope
expression vector (pCMMVxeno, see FIG. 1B.5) follows.
[0089] 1. A 2.2 Kb NaeI/NheI fragment containing the coding region
of the xenotropic envelope obtained from clone NZB9-1 (O'Neill et
al., J. Virol. 53:100-106, 1985) was isolated.
[0090] 2. Using linkers and other standard recombinant DNA
techniques, the CMV early promoter and SV40 late termination signal
described for the gag/pol expression above (pSCV10) were ligated in
the order CMV promoter-envelope-termination signal.
[0091] An example of the construction of an MLV polytropic envelope
expression vector (pCMVMCF, see FIG. 1B.6) follows.
[0092] 1. A 2 Kb BamHI/NheI fragment containing the coding region
of the polytropic envelope obtained from clone MCF-247W (Holland et
al., J. Virol. 53:152-57, 1985) was isolated.
[0093] 2. Using linkers and other standard recombinant DNA
techniques, the CMV early promoter and SV40 late termination signal
described for the gag/pol expression above (pSCV10) were ligated in
the order CMV promoter-envelope-termination signal.
[0094] An example of the construction of an MLV ampho env Neo.sup.+
retroviral vector (pLARNL, FIG. 1B.7) follows.
[0095] 1. The vector PLRNL vector (Emi et al., J. Virol.
65:1202-1207, 1991) was digested with BamHI.
[0096] 2. A 2.7 Kb XbaI fragment containing the envelope protein
coding region of retrovirus 4070A (Chattopadhyay et al., J. Virol.
39:777-91, 1981) was isolated.
[0097] 3. Fragments from procedures 1 and 2 above were ligated.
[0098] B. Host Cell Selection
[0099] Host cell lines were screened for their ability to
efficiently (high titre) rescue a drug resistance retroviral vector
(A alpha N2) using replication competent retrovirus to produce the
gag/pol and env structural genes ("MA" virus). Titre was measured
from confluent monolayers 16 h after a medium change by adding
filtered supernatants (0.45 um filters) to 5.times.10.sup.4 NIH 3T3
TK.sup.- cells on a 6 cm tissue culture plate in the presence of 4
.mu.g/ml polybrene followed by selection in G418.
[0100] Data from the screening process is shown in FIG. 2. Among
the non-murine cell lines which demonstrate the ability to package
MoMLV-based vector with high titre are the cell lines CF2, D17,
293, and HT1080. These cell lines were used herein as examples,
although any other cells may be tested by such means.
[0101] C. Generation of Packaging Cell
[0102] (i) gag/pol Intermediate
[0103] As examples of the generation of gag/pol intermediates for
PCL production, D17, 293, and HT1080 were co-transfected with 1 ug
of the methotrexate resistance vector, pFR400 (Graham and van der
Eb, Virology 52:456-67, 1973), and 10 ug of the MOMLV gag/pol
expression vector, pSCV10 (above) by calcium phosphate
co-precipitation (D17 and HT1080, see Graham and van der Eb,
Virology 52:456-67, 1973), or lipofection (293, see Felgner et al.,
Proc. Natl. Acad. Sci., USA 84:7413-17, 1987). After selection for
transfected cells in the presence of the drugs dipyrimidol and
methotrexate, individual drug resistant cell colonies were expanded
and analyzed for MOMLV gag/pol expression by extracellular reverse
transcriptase (RT) activity (modified from Goff et al., J. Virol.
38:239-48, 1981) and intracellular p30.sup.gag by western blot
using anti p30 antibodies (goat antiserum #77S000087 from the
National Cancer Institute). This method identified individual cell
clones in each cell type which expressed 10-50.times. higher levels
of both proteins compared with that of a standard mouse amphotropic
PCL, PA317 (FIG. 1D and Table 1).
1TABLE 1 PROPERTIES OF MoMLV GAG/POL-EXPRESSING CELLS LARNL RT
p30.sup.gag TITRE CELL NAME ACTIVITY (CPM) EXPRESSION (CFU/ML) 3T3
800 - N.D. PA317 1350 +/- 1.2 .times. 10.sup.3 D17 800 - N.D. D17
4-15 5000 + + + + + 1.2 .times. 10.sup.4 D17 9-20 2000 + + + 6.0
.times. 10.sup.3 D17 9-9 2200 + + 1.0 .times. 10.sup.3 D17 9-16
6100 + + + + + 1.5 .times. 10.sup.4 D17 8-7 4000 - N.D. HT1080 900
- N.D. HTSCV21 16400 + + + + + 8.2 .times. 10.sup.3 HTSCV25 7900 +
+ + 2.8 .times. 10.sup.3 HTSCV42 11600 + + 8.0 .times. 10.sup.2
HTSCV26 4000 - <10 293 600 - N.D. 293 2-3 6500 + + + + + 7
.times. 10.sup.4 293 5-2 7600 + + + + + N.D.
[0104] The biological activity of these proteins was tested by
introducing a retroviral vector, LARNL (see FIG. 1B) which
expresses both the amphotropic envelope and a Neo.sup.+ marker
which confers resistance to the drug, G418. In every case,
co-expression of gag/pol in the cell line and env from the vector
allowed efficient packaging of the vector as determined by
cell-free transfer of G418 resistance to 3T3 cells (titre). Titre
was measured from confluent monolayers 16 h after a medium change
by adding filtered supernatants (0.45 um filters) to
5.times.10.sup.4 NIH3T3 TK.sup.- cells on a 6 cm tissue culture
plate in the presence of 4 ug/ml polybrene followed by selection in
G418. Significantly, the vector titres from the cell lines
correlated with the levels of p30.sup.gag (Table 1). Since the
level of env should be the same in each clone and is comparable to
the level found in PA317 (data not shown), this indicates that
titre was limited by the lower levels of gag/pol in these cells
(including PA317). The titre correlated more closely with the
levels of p30.sup.gag than with the levels of RT.
[0105] (ii) Conversion of gag/pol Lines Into Amphotropic Packaging
Lines
[0106] As examples of the generation of amphotropic PCLs, the
gag/pol over-expressors for 293 (termed 2-3) and D17 (termed 4-15)
were co-transfected by the same techniques described above except
that 1 ug of the phleomycin resistance vector, pUT507 (Mulsant et
al., Somat. Cell Mol. Genet. 14:243-52, 1988), and 10 ug of the
amphotropic envelope expression vectors, pMLPenvAmSph (for 2-3) or
pCMVenvAmNhe (for 4-15) were used. After selection for transfected
cells in the presence of phleomycin, individual drug resistant cell
colonies were expanded and analyzed for intracellular gp80.sup.env
expression by western blot using anti gp7o (goat antiserum
#79S000771 from N.C.I.). Several clones were identified which
expressed relatively high levels of both gag/pol and ampho env
(PCLs, see FIG. 1 for representative data).
[0107] In another example of the generation of an ampho PCL, CF2
cells were electroporated (cf. Chu et al., Nucl. Acids Res.
15:1311-26, 1987) with 2 ug of the phleomycin resistance marker,
pUT507, 10 ug of pSCV10 (above), and 10 ug of pCMVenvAmNhe (above).
After selection for transfected cells in the presence of
phleomycin, individual drug resistant cell colonies were expanded
and analyzed for intracellular expression of MLV p30.sup.gag and
gp80.sup.env proteins by western blot using specific antisera. A
clone was identified which expressed relatively high levels of both
gag/pol and ampho env (FIG. 1E).
[0108] (iii) Performance of Amphotropic Packaging Cell Lines
[0109] A number of these ampho PCLs were tested for their capacity
to package retroviral vectors by measuring titre after the
introduction of retroviral vectors (Table 2). The measurements were
performed using uncloned PCLs, so that the average performance of
the lines was calculated.
2TABLE 2 VECTOR TITRE AND HELPER VIRUS GENERATION IN AMPHOTROPIC
PCLs VECTOR TITRE.sup.a (+/- HELPER VIRUS.sup.b) CELL TYPE b-Gal
KT-1 N2 PA317 3.5 .times. 10.sup.2 (N.D.) 1.0 .times. 10.sup.4
(N.D.) 3.0 .times. 10.sup.5 (+).sup.c CA 5.0 .times. 10.sup.4
(N.D.) 3.0 .times. 10.sup.5 (-).sup.d 2.0 .times. 10.sup.6
(-).sup.d 2A 4.0 .times. 10.sup.4 (N.D.) 2.0 .times. 10.sup.5
(-).sup.e N.D. DA N.D. N.D. 2.0 .times. 10.sup.5 (-).sup.d DA2 N.D.
3.9 .times. 10.sup.5 (-).sup.d N.D. .sup.acfu/ml .sup.bas judged by
marker rescue assay with MA virus as positive control .sup.cafter
20 days in culture .sup.dafter 60 days in culture .sup.eafter 90
days in culture
[0110] Highest titres are obtained when retroviral vectors were
introduced into PCLs by infection (Miller et al., Somat. Cell Mol.
Genet. 12:175-83, 1986). However, although amphotropic MLV vectors
are known to infect these host cell types, the PCLs are blocked for
infection by ampho vector since they express ampho env ("viral
interference"). To overcome this problem, vectors containing other
viral envelopes (such as xenotropic env or VSV G protein, which
bind to cell receptors other than the ampho receptor) were
generated in the following manner. Ten ug of the vector DNA of
interest was co-transfected with 10 ug of DNA which expresses
either xeno env (pCXvxeno, above) or a VSV G protein expression
vector, MLP G, onto a cell line which expresses high levels of
MOMLV gag/pol such as 2-3 cell (see above). The resultant vector
containing xenotropic env or VSV G protein, respectively, was
produced transiently in the co-transfected cells and after 2 days
cell free supernatants were added to the potential PCLs, and
vector-infected cells were identified by selection in G418. Both
types of vector efficiently infected the ampho-blocked cells and
after G418 selection cell free supernatants were collected from the
confluent monolayers and titred on NIH 3T3 TK.sup.- cells as
described above. The cell clones with the highest titre were chosen
as PCLs and referred to as DA (D17 ampho), 2A (293 ampho), and CA
(CF2 ampho), respectively. In no case was helper virus detected in
the currently described PCLs, even when a retroviral vector (N2)
which has a high probability of generating helper virus (Armentano
et al., J. Virol. 62:1647-50, 1987) was introduced into the PCLs
and the cells passaged for as long as 2 months (3 months for vector
KT-3). On the other hand, the same vector introduced into the PA317
cell line generated helper virus within 3 weeks of continual
passaging.
[0111] (iv) Conversion of gag/pol Lines into Xenotropic Packaging
Cell Lines
[0112] As examples of the generation of xenotropic PCLS, the
gag/pol over-expressors for D17 (4-15) and HT1080 (SCV21) were
co-transfected by the same techniques described above except that 1
ug of either the phleomycin resistance vector, pUT507 (for SCV21),
or the hygromycin B resistance marker, pY3 (for 4-15, see
Blochlinger and Diggelmann, Mol. Cell Biol. 4:2929-31, 1984), and
10 ug of the xenotropic envelope expression vector, pCMVxeno
(above) was used. After selection for transfected cells in the
presence of phleomycin or hygromycin, respectively, individual drug
resistant cell colonies were expanded and analyzed for
intracellular expression of MLV p30.sup.gag and gp75.sup.env
proteins by western blot using specific antisera. Clones were
identified which expressed relatively high levels of both gag/pol
and xeno env (FIG. 1F).
[0113] (v) Performance of Xenotropic Packaging Cell Lines
[0114] A number of these potential xeno PCLs were tested for their
capacity to package retroviral vectors by measuring titre after the
introduction of retroviral vectors (Table 3).
3TABLE 3 VECTOR TITRE ON XENOTROPIC PCLs KT-1 TITRE (CFU/ML) CELL
CLONE ON HT1080 CELLS HT1080 SCV21 XF1 1.0 .times. 10.sup.5 XF7 1.0
.times. 10.sup.5 XF12 (HX) 4.5 .times. 10.sup.5 D17 4-15 X6 9.0
.times. 10.sup.4 X10 (DX) 1.3 .times. 10.sup.5 X23 8.0 .times.
10.sup.4
[0115] As described above, vector containing VSV G protein was
produced transiently in 2-3 cells. After 2 days, cell free
supernatants were added to the xeno PCLs and after G418 selection
cell free supernatants were collected from the confluent monolayers
and titred as described above except that HT1080 cells, which are
infectable by xeno vector, was used instead of NIH 3T3 TK.sup.-
cells which are resistant to xeno vector. The cell clones with the
highest titre were chosen as PCLs and referred to as DX (D17 xeno)
and HX (HT1080 xeno), respectively.
[0116] The propensity of the PCLs described above to generate
helper virus was tested even more stringently by co-cultivating
ampho and xeno PCLs containing the vector, N2. Since ampho vector
can infect the xeno PCLs and vice versa, this allows continuous
cross-infection events, each of which increases the probability of
generating helper virus. As an example, 2A cells containing N2 were
co-cultivated with HX cells containing N2. After 23 days, the
cultures were still free of ampho and xeno viruses as judged by a
vector rescue assay on 293 or Mus dunni cells, both of which can
detect ampho and xeno viruses (Table 4).
4TABLE 4 HIGH STRINGENCY ANALYSIS FOR PCL TENDENCY TO GENERATE
HELPER VIRUS TEST MATERIAL HELPER VIRUS ASSAY AMPHOTROPIC VIRUS +
XENOTROPIC VIRUS + PA317 + N2 (21d) + 2A + HX + N2 (23d) -
[0117] (vi) Conversion of gag/pol Lines into Polytropic Packaging
Cell Lines
[0118] As an example of the generation of a polytropic PCL, the
gag/pol over-expressor for HT1080 (SCV21) was co-transfected by the
same techniques described above, except that 1 ug of the phleomycin
resistance vector, pUT507, and 10 ug of the polytropic envelope
expression vector, pCMVMCF (above) was used. After selection for
transfected cells in the presence of phleomycin, individual drug
resistant cell colonies were expanded and analyzed for
intracellular expression of MLV gp.sub.70env protein by western
blot using specific antiserum. Clones were identified which
expressed relatively high levels of both gag/pol (not shown) and
polytropic env (FIG. 1G).
[0119] (vii) Performance of Polytropic Packaging Cell Lines
[0120] One of these potential poly PCLs (clone 3) was tested for
the capacity to package retroviral vectors by measuring titre after
the introduction of retroviral vectors (Table 5).
5TABLE 5 HOST-RANGE OF POLYTROPIC VECTOR FROM HP CELLS CELL LINE
SPECIES .beta.-Gal TITRE 3T3 MURINE 1.0 .times. 10.sup.4 PA317
MURINE 1.0 .times. 10.sup.4 208F/C5 RAT 4.0 .times. 10.sup.4
Mv-1-Lu MINK 5.0 .times. 10.sup.3 FRhL MACAQUE <10 HT1080 HUMAN
<10 HeLa HUMAN <10 WI 38 HUMAN <10 DETROIT 551 HUMAN
<10 SUP TI HUMAN <10 CEM HUMAN <10 U937 HUMAN <10 293
HUMAN 2.0 .times. 10.sup.4 AAT HUMAN <10 Vandenberg HUMAN
<10
[0121] This cell clone was chosen as PCL and referred to as HP
(HT1080 poly). As described above, vector containing VSV G protein
was produced transiently in 2-3 cells and after 26 days, cell free
supernatants were added to the polytropic PCL (HP). After G418
selection, cell free supernatants were collected from the confluent
monolayers and titred as described above on a variety of cell
lines. The infection of human cells was very restricted, with all
cell lines tested being negative with the exception of 293
cells.
[0122] Although the factors that lead to efficient infection of
specific cell types by retroviral vectors are not completely
understood, it is clear that because of their relatively high
mutation rate, retroviruses may be adapted for markedly improved
growth in cell types in which initial growth is poor, simply by
continual reinfection and growth of the virus in that cell type
(the adapter cell). This can also be achieved using viral vectors
that encode some viral functions (e.g., env), and which are passed
continuously in cells of a particular type which have been
engineered to have the functions necessary to complement those of
the vector to give out infectious vector particles (e.g., gag/pol).
For example, one can adapt the murine amphotropic virus 4070A to
human T-cells or monocytes by continuous growth and reinfection of
either primary cell cultures or permanent cell lines such as Sup T1
(T-cells) or U937 (monocytes). Once maximal growth has been
achieved, as measured by reverse transcriptase levels or other
assays of virus production, the virus is cloned out by any of a
number of standard methods, the clone is checked for activity
(i.e., the ability to give the same maximal growth characteristic
on transfection into the adapter cell type) and this genome used to
make defective helper genomes and/or vectors which in turn, in an
appropriately manufactured helper or producer line, will lead to
production of viral vector particles which infect and express in
the adapter cell type with high efficiency (10.sup.7-10.sup.9
infectious units/ml).
[0123] VII. Alternative Viral Vector Packaging Techniques
[0124] Two additional alternative systems can be used to produce
recombinant retroviruses carrying the vector construct. Each of
these systems takes advantage of the fact that the insect virus,
baculovirus, and the mammalian viruses, vaccinia and adenovirus,
have been adapted recently to make large amounts of any given
protein for which the gene has been cloned. For example, see Smith
et al. (Mol. Cell. Biol. 3:12, 1983); Piccini et al. (Meth.
Enzymology, 153:545, 1987); and Mansour et al. (Proc. Natl. Acad.
Sci. USA 82:1359, 1985).
[0125] These viral vectors can be used to produce proteins in
tissue culture cells by insertion of appropriate genes into the
viral vector and, hence, could be adapted to make retroviral vector
particles.
[0126] Adenovirus vectors are derived from nuclear replicating
viruses and can be defective. Genes can be inserted into vectors
and used to express proteins in mammalian cells either by in vitro
construction (Ballay et al., EMBO J. 4:3861, 1985) or by
recombination in cells (Thummel et al., J. Mol. Appl. Genetics
1:435, 1982).
[0127] One preferred method is to construct plasmids using the
adenovirus Major Late Promoter (MLP) driving: (1) gag/pol, (2) env,
(3) a modified viral vector construct. A modified viral vector
construct is possible because the U3 region of the 5' LTR, which
contains the viral vector promoter, can be replaced by other
promoter sequences (see, for example, Hartman, Nucl. Acids Res.
16:9345, 1988). This portion will be replaced after one round of
reverse transcriptase by the U3 from the 3' LTR.
[0128] These plasmids can then be used to make adenovirus genomes
in vitro (Ballay et al., op. cit.), and these transfected in 293
cells (a human cell line making adenovirus E1A protein), for which
the adenoviral vectors are defective, to yield pure stocks of
gag/pol, env and retroviral vector carried separately in defective
adenovirus vectors. Since the titres of such vectors are typically
10.sup.7-10.sup.11/ml, these stocks can be used to infect tissue
culture cells simultaneously at high multiplicity. The cells will
then be programmed to produce retroviral proteins and retroviral
vector genomes at high levels. Since the adenovirus vectors are
defective, no large amounts of direct cell lysis will occur and
retroviral vectors can be harvested from the cell supernatants.
[0129] Other viral vectors such as those derived from unrelated
retroviral vectors (e.g., RSV, MMTV or HIV) can be used in the same
manner to generate vectors from primary cells. In one embodiment,
these adenoviral vectors are used in conjunction with primary
cells, giving rise to retroviral vector preparations from primary
cells.
[0130] In some cases, gene products from other viruses may be used
to improve the properties of retroviral packaging systems. For
instance, HIV rev protein might be included to prevent splicing of
HIV env or HIV gag/pol MLV vectors or HIV sor might increase the
infectivity of T cells by free virus as it does with HIV (See
Fischer et al., Science 237:888-893, 1987).
[0131] In an alternative system (which is more truly
extracellular), the following components are used:
[0132] 1. gag/pol and env proteins made in the baculovirus system
in a similar manner as described in Smith et al. (supra) (or in
other protein production systems, such as yeast or E. coli);
[0133] 2. viral vector RNA made in the known T7 or SP6 or other in
vitro RNA-generating system (see, for example, Flamant and Sorge,
J. Virol. 62:1827, 1988);
[0134] 3. tRNA made as in (2) or purified from yeast or mammalian
tissue culture cells;
[0135] 4. liposomes (with embedded env protein); and
[0136] 5. cell extract or purified necessary components (when
identified) (typically from mouse cells) to provide env processing,
and any or other necessary cell-derived functions.
[0137] Within this procedure (1), (2) and (3) are mixed, and then
env protein, cell extract and pre-liposome mix (lipid in a suitable
solvent) added. It may, however, be necessary to earlier embed the
env protein in the liposomes prior to adding the resulting
liposome-embedded env to the mixture of (1), (2), and (3). The mix
is treated (e.g., by sonication, temperature manipulation, or
rotary dialysis) to allow encapsidation of the nascent viral
particles with lipid plus embedded env protein in a manner similar
to that for liposome encapsidation of pharmaceuticals, as described
in Gould-Fogerite et al., Anal. Biochem. 148:15, 1985). This
procedure allows the production of high titres of replication
incompetent recombinant retroviruses without contamination with
pathogenic retroviruses or replication-competent retroviruses.
[0138] VIII. Cell Line-Specific Retroviruses--"Hybrid Envelope"
[0139] The host cell range specificity of a retrovirus is
determined in part by the env gene products. For example, Coffin,
J. (RNA Tumor Viruses 2:25-27, Cold Spring Harbor, 1985) notes that
the extracellular component of the proteins from murine leukemia
virus (MLV) and Rous Sarcoma virus (RSV) are responsible for
specific receptor binding. The cytoplasmic domain of envelope
proteins, on the other hand, are understood to play a role in
virion formation. While pseudotyping (i.e., the encapsidation of
viral RNA from one species by viral proteins of another species)
does occur at a low frequency, the envelope protein has some
specificity for virion formation of a given retrovirus. The present
invention recognizes that by creating a hybrid env gene product
(i.e., specifically, an env protein having cytoplasmic regions and
exogenous binding regions which are not in the same protein
molecule in nature) the host range specificity may be changed
independently from the cytoplasmic function. Thus, recombinant
retroviruses can be produced which will specifically bind to
preselected target cells.
[0140] In order to make a hybrid protein in which the receptor
binding component and the cytoplasmic component are from different
retroviruses, a preferred location for recombination is within the
membrane-spanning region of the cytoplasmic component. Example 10
describes the construction of a hybrid env gene which expresses a
protein with the CD4 binding portion of the HIV envelope protein
coupled to the cytoplasmic domain of the MLV envelope protein.
EXAMPLE 3
Hybrid HIV-MLV Envelopes
[0141] A hybrid envelope gene is prepared using in vitro
mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985)
to introduce a new restriction site at an appropriate point of
junction. Alternatively, if the two envelope sequences are on the
same plasmid, they can be joined directly at any desired point
using in vitro mutagenesis. The end result in either case is a
hybrid gene containing the 5' end of the HIV gp 160 and the 3' end
of MLV pl5E. The hybrid protein expressed by the resulting
recombinant gene is illustrated in FIG. 2 and contains the HIV
gp120 (CD4 receptor binding protein), the extracellular portion of
HIV gp 41 (the gp 120 binding and fusigenic regions), and the
cytoplasmic portion of MLV piSE, with the joint occurring at any of
several points within the host membrane. A hybrid with a fusion
joint at the cytoplasmic surface (joint C in FIG. 2) causes
syncytia when expressed in Sup T1 cells. The number of apparent
syncytia are approximately one-fifth that of the nonhybrid HIV
envelope gene in the same expression vector. Syncytia with the
hybrid occurs only when the rev protein is co-expressed in trans. A
hybrid with a fusion joint at the extracellular surface (joint A in
FIG. 2) gives no syncytia while hybrid B (in the middle of
transmembrane regions) gives approximately five-fold less syncytium
on Sup T1 cells than hybrid C.
[0142] While Example 3 illustrates one hybrid protein produced from
two different retroviruses, the possibilities are not limited to
retroviruses or other viruses. For example, the receptor binding
portion of human interleukin-2 may be combined with the envelope
protein of MLV to target vectors to cells with IL-2 receptors. In
this case, a recombination would preferably be located in the gp 70
portion of the MLV env gene, leaving an intact p15E protein.
Furthermore, the foregoing technique may be used to create a
recombinant retrovirus with an envelope protein which recognizes
antibody Fc segments. Monoclonal antibodies which recognize only
preselected target cells only could then be bound to such a
recombinant retrovirus exhibiting such envelope proteins so that
the retrovirus would bind to and infect only those preselected
target cells. Alternatively, a hybrid envelope with the binding
domain of avidin would be useful for targeting cells' "images" in a
patient or animal with biotinylated antibodies or other ligands.
The patient would first be flooded with antibodies, and then
antibody binding nonspecifically allowed to clear from the
patient's system, before administering the vector. The high
affinity (10.sup.-15) of the avidin binding site for biotin would
then allow accurate and efficient targeting to the original tissue
identified by the monoclonal "image."
[0143] The approach may also be used to achieve tumor-specific
targeting and killing by taking advantage of three levels of
retroviral vector specificity; namely, cell entry, gene expression,
and choice of protein expressed. Retroviral vectors enter cells and
exert their effects at intracellular sites. In this respect their
action is quite unique. Using this property, and the three levels
of natural retroviral specificity (above), retroviral vectors may
be engineered to target and kill tumor cells.
[0144] The overall goal of targeting of retrovirus to tumor cells
may be accomplished by two major experimental routes; namely, a)
selection in tissue culture (or in animals) for retrovituses that
grow preferentially in tumor cells; or b) construction of
retroviral vectors with tissue (tumor)-specific promoters with
improvements being made by in vitro passage, and negative and
positive drug-sensitivity selection.
[0145] Vectors suitable for selectively infecting selected cell
types, such as a tumor cell, may generally be prepared by (a)
continuously passaging a virus in cells of the selected cell type
until the virus has genetically mutated and a predominant fast
growing strain has evolved; (b) isolating the mutated and fast
growing strain; (c) identifying and isolating the components of the
mutated strain responsible for the preferential growth of the
mutated virus; (d) inserting the identified and isolated components
as substitutes for counterpart components in a producer cell based
upon the virus (prior to continuous passage); and (e) culturing the
producer cell to produce the vector.
[0146] At least four selective protocols may be utilized to select
for retrovirus which grow preferentially in tumor cells; namely, 1)
"Env Selection by Passage In Vitro," wherein selection of
retrovirus with improved replicative growth ability is accomplished
by repeated passage in tumor cells; 2) "Selection with a Drug
Resistance Gene," wherein genetic selection for tumor "specific"
retroviruses is based on viral constructs containing a linked drug
resistance gene; 3) "Hybrid-Env," wherein selection (by protocol #1
or #2, above) of retrovirus with tumor-"specificity" is initiated
from a construct containing a hybrid envelope gene which is a
fusion of a tumor receptor gene (i.e., an anti-tumor antibody
H-chain V-region gene fused with env; or, a growth receptor fused
with env); in this case selection begins at a favorable starting
point, e.g., an env which has some specificity for tumor cells; or
4) "Selection by Passage In Vitro and Counter Selection by
Co-cultivation with Normal Cells," wherein growth in tumor cells is
selected-for by repeated passage in mixtures of drug-resistant
tumor cells and drug-sensitive normal cells.
[0147] With respect to retroviral vector constructs carrying tissue
(tumor)-specific promoters, biochemical markers with different
levels of tissue-specificity are well known, and genetic control
through tissue-specific promoters is understood in some systems.
There are a number of genes whose transcriptional promoter elements
are relatively active in rapidly growing cells (i.e., transferring
receptor, thymidine kinase, etc.) and others whose
promoter/enhancer elements are tissue specific (i.e., HBV enhancer
for liver, PSA promoter for prostate). Retroviral vectors and
tissue-specific promoters (present either as an internal promoter
or within the LTR) which can drive the expression of selectable
markers and cell cycle genes (i.e., drug sensitivity, Eco gpt; or
HSVTK in TK-cells). Expression of these genes can be selected for
in media containing mycophenolic acid or HAT, respectively. In this
manner, tumor cells containing integrated provirus which actively
expresses the drug resistance gene will survive. Selection in this
system may involve selection for both tissue-specific promoters and
viral LTRs. Alternatively, specific expression in tumor cells, and
not in normal cells, can be counter-selected by periodically
passaging virus onto normal cells, and selecting against virus that
express Eco gpt or HSVtk (drug sensitivity) in those cells (by
thioxanthine or acyclovir). Infected cells containing integrated
provirus which express Eco gpt or tk phenotype will die and thus
virus in that cell type will be selected against.
[0148] IX. Site-Specific Integration
[0149] Targeting a retroviral vector to a predetermined locus on a
chromosome increases the benefits of gene-delivery systems. A
measure of safety is gained by direct integration to a "safe" spot
on a chromosome, i.e., one that is proven to have no deleterious
effects from the insertion of a vector. Another potential benefit
is the ability to direct a gene to an "open" region of a
chromosome, where its expression would be maximized. Two techniques
for integrating retroviruses at specific sites are described
below.
[0150] (ii) Integrase Modification
[0151] Another technique for integrating a vector construct into
specific, preselected sites of a target cell's genome involves
integrase modification.
[0152] The retrovirus pol gene product is generally processed into
four parts: (i) a protease which processes the viral gag and pol
products; (ii) the reverse transcriptase; and (iii) RNase H, which
degrades RNA of an RNA/DNA duplex; and (iv) the endonuclease or
"integrase."
[0153] The general integrase structure has been analyzed by Johnson
et al. (Proc. Natl. Acad. Sci. USA 83:7648-7652, 1986). It has been
proposed that this protein has a zinc binding finger with which it
interacts with the host DNA before integrating the retroviral
sequences.
[0154] In other proteins, such "fingers" allow the protein to bind
to DNA at particular sequences. One illustrative example is the
steroid receptors. In this case, one can make the estrogen
receptor, responding to estrogens, have the effect of a
glucocorticoid receptor, responding to glucocorticoids, simply by
substituting the glucocorticoid receptor "finger" (i.e., DNA
binding segment) in place of the estrogen receptor finger segment
in the estrogen receptor gene. In this example, the position in the
genome to which the proteins are targeted has been changed. Such
directing sequences can also be substituted into the integrase gene
in place of the present zinc finger. For instance, the segment
coding for the DNA binding region of the human estrogen receptor
gene may be substituted in place of the DNA binding region of the
integrase in a packaging genome. Initially, specific integration
would be tested by means of an in vitro integration system (Brown
et al., Cell 29:347-356, 1987). To confirm that the specificity
would be seen in vivo, this packaging genome is used to make
infectious vector particles, and infection of and integration into
estrogen-sensitive and estrogen-nonsensitive cells compared in
culture.
[0155] Through use of this technique, incoming viral vectors may be
directed to integrate into preselected sites on the target cell's
genome, dictated by the genome-binding properties of site-specific
DNA-binding protein-segments spliced into the integrase genome. It
will be understood by those skilled in the art that the integration
site must, in fact, be receptive to the fingers of the modified
integrase. For example, most cells are sensitive to glucocorticoids
and hence their chromatin has sites for glucocorticoid receptors.
Thus, for most cells, a modified integrase having a glucocorticoid
receptor finger would be suitable to integrate the proviral vector
construct at those glucocorticoid receptor-binding sites.
[0156] X. Production of Recombinant Retroviral Vectors in
Transgenic Animals
[0157] Two problems previously described with helper line
generation of retroviral vectors are: (a) difficulty in generating
large quantities of vectors; and (b) the current need to use
permanent instead of primary cells to make vectors. These problems
can be overcome with producer or packaging lines that are generated
in transgenic animals. These animals would carry the packaging
genomes and retroviral vector genomes. Current technology does not
allow the generation of packaging cell lines and desired
vector-producing lines in primary cells due to their limited life
span. The current technology is such that extensive
characterization is necessary, which eliminates the use of primary
cells because of senescence. However, individual Lines of
transgenic animals can be generated by the methods provided herein
which produce the packaging functions, such as gag, pol or env.
These lines of animals are then characterized for expression in
either the whole animal or targeted tissue through the selective
use of housekeeping or tissue-specific promoters to transcribe the
packaging functions. The vector to be delivered is also inserted
into a line of transgenic animals with a tissue-specific or
housekeeping promoter. As discussed above, the vector can be driven
off such a promoter substituting for the U3 region of the 5' LTR
(FIG. 3). This transgene could be inducible or ubiquitous in its
expression. This vector, however, is not packaged. These lines of
animals are then mated to the gag/pol/env animal and subsequent
progeny produce packaged vector. The progeny, which are essentially
identical, are characterized and offer an unlimited source of
primary producing cells. Alternatively, primary cells making
gag/pol and env and derived from transgenic animals can be infected
or transfected in bulk with retrovirus vectors to make a primary
cell producer line. Many different transgenic animals or insects
could produce these vectors, such as mice, rats, chickens, swine,
rabbits, cows, sheep, fish and flies. The vector and packaging
genomes would be tailored for species infection specificity and
tissue-specific expression through the use of tissue-specific
promoters and different envelope proteins. An example of such a
procedure is illustrated in FIG. 4.
[0158] Although the following examples of transgenic production of
primary packaging lines are described only for mice, these
procedures can be extended to other species by those skilled in the
art. These transgenic animals may be produced by microinjection or
gene transfer techniques. Given the homology to MLV sequences in
mice genome, the final preferred animals would not be mice.
EXAMPLE 4
Production of Gag/Pol Proteins Using Housekeeping Promoters for
Ubiquitous Expression in Transgenic Animals
[0159] An example of a well-characterized housekeeping promoter is
the HPRT promoter. HPRT is a purine salvage enzyme which expresses
in all tissues. (See Patel et al., Mol. Cell Biol. 6:393-403, 1986
and Melton et al., Proc. Natl. Acad. Sci. 81:2147-2151, 1984). This
promoter is inserted in front of various gag/pol fragments (e.g.,
Bal I/Sca I; Aat II/Sca I; Pst I/Sca I of MOMLV; see RNA Tumor
Viruses 2, Cold Spring Harbor Laboratory, 1985) that are cloned in
Bluescript plasmids (Strategene, Inc.) using recombinant DNA
techniques (see Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, 1982). The resulting plasmids are
purified (Maniatis et al., op. cit.) and the relevant genetic
information isolated using Geneclean (Bio 101) or electroelution
(see Hogan et al. (eds.), Manipulating the Mouse Embryo: A
Laboratory Manual, Cold Spring Harbor, 1986).
[0160] These fully characterized DNAs are microinjected in the
pronucleus of fertilized mouse ova at a concentration of 2 ug/ml.
Live-born mice are screened by tail blot analyses (see Hogan et
al., op. cit.). Transgenic-positive animals are characterized for
expression levels of gag/pol proteins by immunoprecipitation of
radiolabeled primary cells, such as fibroblast (see Harlow et al.
(eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor, 1988).
Animals then bred to homozygosity for establishment of animal lines
that produce characterized levels of gag/pol.
EXAMPLE 5
Production of env Proteins/Hybrid Envelope Proteins Using
Housekeeping Promoters for Ubiquitous Expression in Transgenic
Animals
[0161] This example utilizes the HPRT promoter for expression of
either envelope or hybrid envelope proteins. The envelope proteins
can be from any retrovirus that is capable of complementing the
relevant gag/pol, in this case that of MLV. Examples are ecotropic
MLV, amphotrophic MLV, xenotropic MLV, polytropic MLV, or hybrid
envelopes. As above, the envelope gene is cloned behind the HPRT
promoter using recombinant DNA techniques (see Maniatis et al., op.
cit.). The resulting "minigene" is isolated (see Hogan et al., op.
cit.), and expression of envelope protein is determined (Harlow et
al., op. cit.). The transgenic envelope animals are bred to
homozygosity to establish a well-characterized envelope animal.
EXAMPLE 6
Production of gag/pol-env Animals Using Housekeeping Promoters for
Ubiquitous Expression in Transgenic Animals
[0162] This uses the well-characterized gag/pol animals, as well as
the animals for the establishment of a permanent gag/pol/envelope
animal line. This involves breeding to homozygosity and the
establishment of a well-characterized line. These lines are then
used to establish primary mouse embryo lines that can be used for
packaging vectors in tissue culture. Furthermore, animals
containing the retroviral vector are bred into this line.
EXAMPLE 7
Production of Tissue-Specific Expression of gag/pol-env or Hybrid
Envelope in Transgenic Animals
[0163] This example illustrates high level expression of the
gag/pol, envelope, or hybrid envelope in specific tissues, such as
T-cells. This involves the use of CD2 sequences (see Lang et al.,
EMBO J. 7:1675-1682, 1988) that give position and copy number
independence. The 1.5 kb Bam HI/Hind III fragment from the CD2 gene
is inserted in front of gag/pol, envelope, or hybrid envelope
fragments using recombinant DNA techniques. These genes are
inserted into fertilized mouse ova by microinjection. Transgenic
animals are characterized as before. Expression in T-cells is
established, and animals are bred to homozygosity to establish
well-characterized lines of transgenic animals. Gag/pol animals are
mated to envelope animals to establish gag/pol-env animals
expressing only in T-cells. The T-cells of these animals are then a
source for T-cells capable of packaging retroviral vectors. Again,
vector animals can be bred into these gag/pol-env animals to
establish T-cells expressing the vector.
[0164] This technique allows the use of other tissue-specific
promoters, such as milk-specific (whey), pancreatic (insulin or
elastase), or neuronal (myelin basic protein) promoters. Through
the use of promoters, such as milk-specific promoters, recombinant
retroviruses may be isolated directly from the biological fluid of
the progeny.
EXAMPLE 8
Production of Either Housekeeping or Tissue-Specific Retroviral
Vectors in Transgenic Animals
[0165] The insertion of retroviruses or retroviral vectors into the
germ line of transgenic animals results in little or no expression.
This effect, described by Jaenisch (see Jahner et al., Nature
298:623-628, 1982), is attributed to methylation of 5' retroviral
LTR sequences. This technique would overcome the methylation effect
by substituting either a housekeeping or tissue-specific promoter
to express the retroviral vector/retrovirus. The U3 region of the
5' LTR, which contains the enhancer elements, is replaced with
regulatory sequences from housekeeping or tissue-specific promoters
(see FIG. 20). The 3' LTR is fully retained, as it contains
sequences necessary for polyadenylation of the viral RNA and
integration. As the result of unique properties of retroviral
replication, the U3 region of the 5' LTR of the integrated provirus
is generated by the U3 region of the 3' LTR of the infecting virus.
Hence, the 3' is necessary, while the 5' U3 is dispensable.
Substitution of the 5' LTR U3 sequences with promoters and
insertion into the germ line of transgenic animals results in lines
of animals capable of producing retroviral vector transcripts.
These animals would then be mated to gag/pol-env animals to
generate retroviral-producing animals (see FIG. 4).
[0166] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *