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Description  |
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FIELD OF THE INVENTION
This invention relates to genetically engineered cell lines and cell
transplantation therapy. In particular, it relates to oncogene-transformed
cell lines useful for transplantation.
BACKGROUND OF THE INVENTION
Insulin is synthesized, processed and secreted by pancreatic .beta.cells,
the major endocrine cell type in the islets of Langerhans that are
distributed throughout the pancreas. Pancreatic .beta.cells secrete
insulin in response to an increase in extracellular glucose concentration.
The two major forms of diabetes, insulin-dependent diabetes mellitus (IDDM)
and non-insulin-dependent diabetes mellitus (NIDDM) are both characterized
by an inability to deliver insulin in an amount and with the precise
timing that is needed for control of glucose homeostasis. The inadequate
insulin delivery is caused by: .beta.-cell destruction by autoimmune
mechanisms in IDDM, and .beta.-cell dysfunction closely coupled to insulin
resistance in NIDDM. Despite these differences in etiology, a common
therapeutic goal for the two disorders is to restore the capacity for
glucose-mediated insulin release to its normal level.
Treatment of IDDM requires insulin replacement, either by conventional
administration of the hormone or by transplantation of insulin-secreting
tissue. Since the latter strategy has thus far relied largely on the use
of scarce human pancreas as the insulin source, it has not been feasible
for general application. Some investigators have proposed the use of
xenografts, e.g., porcine, as a means of overcoming the problem of tissue
availability. However, the immune barrier to xenografts is formidable,
even using techniques such as encapsulation to help them evade the host
immune response.
A number of investigators have developed pancreatic .beta.-cell lines using
transgenic mice expressing dominant oncogenes, particularly SV40
T-antigen, under control of the insulin promoter {Newgard, C. B.,
Diabetes, 43:341-350 (1994) and Hanahan, D., Nature, 315:33-40 (1985)}.
Mice expressing T-antigen under the control of the rat insulin gene
promoter develop .beta.-cell tumors at 12-20 weeks after birth.
Unfortunately, most {see Knaack, et al., Diabetes, 43:1413-1417, (1994)}
.beta.-cell lines derived from these animals do not retain normal
glucose-responsive insulin production {Tal, M., et al., Mol. Cell Biol.,
12:422-32 (1992)}.
In the absence of spontaneously arising cell lines with the desired
properties, cell lines can be created by transfer of dominant oncogenes
into primary cells {Chou, J. Y., Mol. Endocrinol., 3:1511-14 (1989)}. Such
cell lines have been constructed from brain, liver and bone marrow. In
some cases, cell lines created in this way retain differentiated functions
or the ability to differentiate in vivo {Snyder, E. Y., et al., Cell,
68:33-51 (1992)}. Unfortunately, in many other cases, loss of
differentiated function occurs, decreasing the usefulness of the cell line
{Jehn, B., et al., Mol. Cell. Biol., 12:3890-3902 (1992)}.
SV40 T-antigen transforms cells by multiple mechanisms including binding
and inactivation of the tumor suppressor proteins p53 and retinoblastoma
(Rb) {Andersson, A., et al., Transplantation Reviews, 6:20-38 (1992)}.
Although SV40 T-antigen has been shown to be sufficient for transformation
of rodent cells, human primary cells are more refractory to transformation
{Chang, S. E., Biochem. Biophys. Acta, 823:161-94 (1986)}. The frequency
of immortalization of human primary fibroblasts transfected with SV40
T-antigen has been estimated to be 3.times.10.sup.-7 per passage in
culture {Shay, J. W., et al., Exp. Cell Res., 184:109-18 (1989)}.
Overexpression of the epidermal growth factor (EGF) receptor is often found
in pancreatic cancers, as is overexpression of the EGF homologues c-erbB2
and c-erbB3 {Hall, P. A., et al., Cancer Surveys, 16:135-55 (1993)}. Ras
genes are among the most commonly mutated in human cancer, including
pancreatic cancer. Of the ras genes, K-ras mutations are present in 80-90%
of pancreatic ductal carcinomas {Hruban, R. H., et al., Am. J. Pathol.,
143:545-54 (1993)}. Interestingly, H-ras mutations have not been found in
pancreatic cancer {Hruban, R. H. , et al., Am. J. Pathol., 143:545-54
(1993) and Smit V. T. H. B. M., et al., Nucl. Acid Res., 16:7773-82
(1988)}. H-ras containing an activating mutation, under the control of the
elastase promoter, has been expressed in the exocrine tissue of transgenic
mice, with consequent tumor formation {Sandgren, E. P., et al., Proc.
Natl. Acad. Sci. USA, 88:93-97 (1991) and Quaife, C. J., et al., Cell,
48:1023-34 (1987)}. However, when activated H-ras was expressed
specifically in .beta.-cells using the insulin promoter, destruction of
islet cells with diabetes occurred in male mice, but not in females
{Efrat, S., et al., Mol. Cell. Biol., 10:1779-83 (1990) and Efrat S.,
Endocrinol., 128:897-901 (1991)}.
As in many other cancers, p53 is commonly mutated in pancreatic cancers.
Although c-myc overexpression has not been studied extensively in primary
human tumors, it is a potent transforming gene when expressed in the
pancreas of transgenic mice.
Gene Transfer Into Primary Cells
A problem with the development of immortalized cell lines from primary
cells, and particularly human primary cells, is that these cells are
resistant to most methods of gene transfer. Gene transfer into islet cells
has been accomplished by electroporation {German, M. S., et al., J. Biol.
Chem., 265:22063-22066 (1990)}. However, gene expression was only studied
on a transient basis and required dissociating the islets into a single
cell suspension. Such treatment is deleterious to the survival of cells
from the human pancreas {Beattie, G., et al., J. Clin. Endocr. Metab.,
78:1232-40 (1994)}. Adenovirus vectors efficiently infect pancreatic cells
{Newgard, C. B., Diabetes, 43:341-50 (1994)}, but maintaining long term
gene expression from these vectors has been a problem {Smith, T. A. G., et
al., Nature Genet., 5:397-402 (1993)}. Alternatively, transgenic
technology may be used. This usually involves expressing an oncogene,
usually SV40 T-antigen, under control of the insulin promoter in
transgenic animals, thereby generating cell tumors that can be used for
propagating insulinoma cell lines {Efrat, S., et al., Proc. Natl. Acad.
Sci. USA, 85:9037-41 (1988); Miyazaki, J. I., et al., Endocrinology,
127:127-32 (1990)}. Cell lines derived by transgenic expression of
T-antigen in .beta.-cells exhibit variable phenotypes. Some have little
glucose-stimulated insulin release or exhibit maximal responses at
subphysiological glucose concentrations, while others respond to glucose
concentrations over the physiological range. However, the near normal
responsiveness of the latter cell lines is not permanent, as continuous
cell culture results in a shift in glucose dose response such that the
cells secrete insulin at subphysiological glucose concentrations. A
detailed discussion of these cell lines is found in Newgard, C. B.,
Diabetes, 43:341-350 (1994). A human insulinoma cell line has been
obtained but it is difficult to maintain in culture and does not produce
insulin {Gueli, N., et al., Exp. Clin. Cancer Res., 6(4):281-285 (1987)}.
Retroviral-mediated gene transfer (i.e., the use of retroviruses to deliver
genes into cells) is an alternative gene transfer technology which has met
with limited success. In this technique, a desired gene is inserted into a
retroviral vector to obtain a recombinant virus which is then used to
infect target cells. Retroviruses are ribonucleic acid (RNA) viruses. In
retroviral-mediated gene transfer, the viral RNA is first converted to
deoxyribonucleic acid (DNA) after an RNA virus penetrates a target cell.
If the target cell penetrated is a replicating cell (i.e., mitotically
active), the DNA will enter the nucleus and integrate into the genome of
the target cell. In this integrated form, the vital genes are expressed.
Integration of the viral genome into the target cell's genome is an
essential part of its replication. Retroviral vectors are extremely
efficient at infecting a wide variety of cell types, including primary
cells from many tissues {McLachlin, J. R., et al., Prog. Nuc. Acid Res.
Mol. Biol., 38:91-135 (1990)}. The major drawback of retroviral vectors is
that mitotically active cells are required in order for the retroviral
preintegration complex to enter the nucleus and integrate into the genome.
U.S. Pat. No. 5,256,553 to Overell discloses a retroviral vector containing
three inserted genes (two oncogenes and at least one heterologous gene)
each of which is independently transcribed in an infected cell under the
control of its respective transcriptional control sequence. In its Example
1, the patent discloses primary rat embryo fibroblasts (REFs) Balb/3T3 and
.psi.2 (.psi.2 is a retroviral packaging cell line derived from 3T3 cells)
transformed by two triple promoter retroviral vectors each containing a
v-Ha-ras oncogene, a v-myc oncogene, and a neomycin phosphotransferase
(neo) gene which confers resistance to G418 antibiotic resistance. Example
2 of the patent discloses two other triple-promoter vectors, similar to
those of Example 1 except that instead of the neo gene, these vectors
contained hygro (hph) gene which conferred resistance to hygromycin B. The
Example 2 vectors were used to transform Balb/3T3 and .psi.2 cells. In
Example 3 of the patent, the vectors of Examples 1 and 2 were transfected
into .psi.2 cells. Viruses harvested from the virus-producing clones were
incubated with Balb/3T3 cells and found to be capable of infecting the
cells. However, it must be noted that cellular transformation is a
multistep genetic process in all species, but the process differs between
human and rodents in the relative refractoriness of human cells to
transformation. The reason for this difference is not known. Additionally,
primary human cells are often relatively refractory to many methods of
stable gene transfer. Together, these facts make the development of human
cell lines in vitro difficult. Thus, most human cell lines have been
derived from primary cancers that have been adapted to culture in vitro.
SUMMARY OF THE INVENTION
One aspect of the invention presents vectors containing two or more
oncogenes under the control of one or more inducible promoters and/or
genetic elements. The preferred vector contains two or more, preferably
two or three, oncogenes under the control of one inducible promoter or two
genetic elements. The inducible promoter provides a means for activating
or suppressing the transcription and thus the expression of the oncogenes.
The genetic element, preferably a pair of genetic elements flanking the
oncogenes, allows for the excision (removal) of the oncogenes from the
vector or the genome or genetic sequence into which the vector has
integrated. These vectors are preferably viral vectors capable of
producing infectious, but replication deficient, viruses. The most
preferred vectors are retroviruses. The vectors may further comprise genes
coding for repressor(s) or activator(s) for the inducible promoter(s).
These genes are hereinafter referred to repressor or activator genes,
respectively. Alternatively, the vectors may contain binding site(s) in
the inducible promoter(s) for such repressor or activator gene(s). The
vectors may each additionally contain one or more desired genes which are
expressed in the genetically modified cells.
Another aspect of the invention presents a method for producing cells
useful for transplantation. The method uses the above vectors to transform
target cells. In the genetically modified cells, the oncogenes are
expressed and the cells allowed to multiply to establish a cell line. Once
a sufficient number of cells are obtained, the inducible promoter(s) are
repressed to suppress expression of the oncogenes or the oncogenes are
removed. If the cells are precursor cells, they are then allowed to
differentiate. The genetically modified, oncogene-suppressed or -removed,
and/or differentiated cells are useful for transplantation into patients.
Another aspect of the invention presents cell lines produced by the above
method.
Another aspect of the invention presents cell transplantation therapies by
means of transplanting the above genetically modified, oncogene-suppressed
or -removed, and differentiated cells into patients.
Another aspect of the invention presents non-naturally occurring human cell
lines, with extended lifespan in vitro, transformed by one or more
exogenous oncogenes under the control of one or more, preferably
exogenous, inducible promoters. More preferably, the cell lines are
transformed by at least two oncogenes. The preferred cell lines are human
pancreatic cell lines. Most preferably, the cell lines produce insulin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows plasmid pGEM-PMPR. Open boxes represent regulatory elements.
Hatched boxes represent coding sequences. All circular plasmids are drawn
in linear form and only the subcloned genes and their flanking elements
are shown. The notations are as follows: SV-T or SV T-Ag (SV40 T antigen),
myc (human c-myc), ras (H-ras.sup.val12), neo or Neo.sup.r (neomycin
resistance gene), LTR (retroviral long terminal repeat), LTRo (modified
LTR containing lac operator sequence), SVo (modified SV40 promoter
containing lac operator sequence), RSV (Rous sarcoma virus LTR promoter)
and PO (poliomyelitis virus ribosomal internal entry sequence). Letters
above the structure represent restriction enzyme sites: N (Not I), H (Hind
III) and E (EcoR I). Arrows indicate expected transcription initiation
sites. Scheme is not drawn to scale.
FIG. 1B shows plasmid pG-TPMPR. Notations are as in FIG. 1A.
FIG. 1C shows retroviral vectors pLSNVoL and pLoRNLo. Notations are as in
FIG. 1A.
FIG. 1D shows retroviral vectors pLNSVoTPMPRL and pLoTPMPRRRNLo. Notations
are as in FIG. 1A.
FIG. 2 schematically presents the restriction map of pLNSVLacOCatL.
FIG. 3 schematically presents the restriction map of pLoCRNLo.
FIG. 4 schematically presents the development of pseudotyped retroviruses
LNSVoTPMPRL and LoTPMPRRNLo
FIG. 5 schematically presents the lac operator (O)-lac repressor (I)
system.
FIG. 6 schematically presents the retroviral vector pLISVHygL containing
LacI (lacI gene), SV (SV40 early promoter), hyg (hygromycin dominant
selectable marker), and LTR (long terminal repeats).
FIG. 7 shows the insulin-positive cells in TRM-6.
FIG. 8 schematically presents the provirus structure, drawn in linear form,
in the producer cell line #4-11 and TRM-1 cells. Open and hatched boxes
represent regulatory elements and genes to be expressed, respectively. The
notations are as in FIG. 1. Letters above the structure represent
restriction enzyme sites used in Southern blot analyses, H (Hind III), E
(EcoR I), N (Not I) and P (PflM I).
DETAILED DESCRIPTION OF THE INVENTION
Gene Transfer
As used in this application, the term "vector" refers to DNA or RNA
vehicle, such as a plasmid, comprising nucleotide sequences enabling
replication of the DNA or RNA in a suitable host cell, such as a bacterial
host. In this invention, a vector includes a recombinant retrovirus
containing oncogenes which are transcribed into mRNA and translated into
proteins when the proviral sequence is expressed in the genetically
modified target cell.
"Transfection" refers to the introduction of an exogenous nucleotide
sequence, such as DNA vectors in the case of mammalian target cells, into
a target cell whether or not any coding sequences are ultimately
expressed. Numerous methods of transfection are known to those skilled in
the art, such as: chemical methods (e.g., calcium-phosphate transfection),
physical methods (e.g., electroporation, microinjection, particle
bombardment), fusion (e.g., liposomes), receptor-mediated endocytosis
(e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) and by
biological infection by viruses such as recombinant viruses {Wolff, J. A.,
ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)}. In the case of
infection by retroviruses, the infecting retrovirus particles are absorbed
by the target cells, resulting in reverse transcription of the retroviral
RNA genome and integration of the resulting provirus into the cellular
DNA. Genetic modification of the target cell is the indicia of successful
transfection. "Genetically modified cells" refers to cells whose genotypes
have changed as a result of cellular uptakes of exogenous nucleotide
sequence by transfection. "Primary cells" are cells that have been
harvested from the tissue of an organism.
One aspect of the invention presents vectors containing two or more
oncogenes under the control of one or more inducible promoters and/or
genetic elements, capable of expression in the cells they genetically
modified. For example, each vector may contain two to five oncogenes under
the control of one or more inducible promoters or genetic elements. More
preferably, all the oncogenes are under the control of one inducible
promoter or a pair of genetic elements. The most preferred vector contains
two or three oncogenes under the control of one inducible promoter or a
pair of genetic elements. The vectors also preferably contain repressor or
activator gene(s) which interact with the promoter(s). Alternatively, the
vectors may contain site(s) for the introduction of the repressor or
activator gene(s). These vectors are preferably viral vectors, in which
case the present invention also presents their recombinant viruses.
Preferably, the oncogenes are dominant oncogenes. The recombinant viruses
are preferably infectious but replication defective. The vectors are
preferably capable of transfecting cells and stably expressing the
oncogenes to enable growth of the cells for an extended period of time in
vitro. The present invention is preferably directed to genetically
modifying eukaryotic cells that are otherwise incapable of extended growth
in vitro. The latter eukaryotic cells are preferably mammalian and more
preferably human cells. In a one vector system, the vector may further
comprise one or more genes coding for one or more proteins which repress
or activate the inducible promoters. Alternatively, in a two-vector
system, the vector may contain a site for such repressor or activator
genes. The repressor or activator genes are subsequently introduced into
the genetically modified cells by transfection by a second vector
containing the repressor or activator genes. Specific examples of a one
vector and two-vector systems are discussed in the section "Inducible
Promoters And Genetic Elements", below. The vectors may each additionally
contain one or more desired gene(s) which can be stably expressed in the
cells genetically modified by them. The vectors can be introduced
(transfected) into the target cells by any methods known in the art, such
as those described above. The preferred vectors are viral vectors and the
cells are preferably genetically modified by infection with infectious,
but replication deficient, recombinant viruses. Retroviral vectors and
retroviral-mediated gene transfers are the most preferred.
In the present invention, the vector may contain one oncogene. However, by
using a vector containing two or more oncogenes under the control of
preferably a single inducible promoter or a pair of genetic elements, the
present invention possesses advantages over the prior art. Multiple
genetic alterations may be needed for complete transformation. Efficient
transformation may be achieved by oncogene cooperation {Hunter, T., Cell,
64:249-270 (1991)}. Transfer of oncogenes in separate vectors, especially
in the form of plasmid transfection {Taylor, W. R., et al., Oncogene,
7:1383-1390 (1992); Spandidos, D. A., et al., Anticancer Res., 9:1149-1152
(1989)}, is much less efficient than simultaneous transfer of multiple
oncogenes in a single retroviral vector. Previously, simultaneous transfer
of oncogenes in retroviral vectors used separate promoters to drive each
oncogene {Overell, R. W., et al., Mol. Cell. Biol., 8:1803-1808 (1988)}.
However, this may lead to promoter interference {Emerman, M., et al.,
Nucl. Acid. Res., 14:9381-9396 (1986)}. In addition, no inducible promoter
in two-oncogene vectors were available although such promoters were used
in single oncogene system {Efrat, S., et al., Proc. Natl. Acad. Sci. USA,
92:3576-3580 (1995); Epstein-Baak, R., et al., Cell Growth Diff.,
3:127-134 (1992)}. In the present invention, a single oncogene may be
used, such as p53, preferably if it will trigger the formation of
oncogenes in the genes of the transfected cell.
Another aspect of the invention presents cell transplantation therapies
using cells genetically modified by the above vectors. These cells are
transplanted into a patient, e.g., to replace the destroyed or
malfunctioning cells in the patient or to produce the desirable gene
products. The genetically modified cells are preferably of the same
species as the host into which they will be transplanted. Generally,
mammalian target cells are used for treating mammalian subjects. Thus, in
the case of a human patient, the cells are preferably human.
The target cells can be adult or precursor cells. Precursor cells are cells
which are capable of differentiating, e.g., into an entire organ or into a
part of an organ, such as cells which are capable of generating or
differentiating to form a particular tissue (e.g., muscle, skin, heart,
brain, uterus, and blood). Examples of precursor cells are endocrine
precursor cells and fetal cells. Fetal cells are readily obtained and
capable of further growth. In the case of recombinant retroviruses, fetal
cells are still capable of division and can therefore serve as targets for
these viruses. Adult cells can be coaxed to grow, for example, by growing
them in the extracellular matrix from 804G cells and HGF/SF, or by
exposing them to mitotic agents, such as collagenase, dexamethasone,
fibroblast growth factor, before infecting them with the recombinant
retroviruses. The expression of the oncogenes in the genetically modified
target cells spur further cell growth for an extended period of time.
The present invention deals in particular with the novel infection of human
cells and production of infected human cell lines that can grow in vitro
for an extended period of time, such as for 50 cell divisions or for at
least six months, more preferably for at least 150 cell divisions or 10
months, and most preferably at least a year. These cell lines are
preferably transformed by the above vectors. In particular, the present
invention discloses the first cell lines to be generated from the
endocrine precursor cells of the human pancreas, and the first
insulin-producing cell lines directly derived from human fetal pancreas,
or fetal pancreas of any species. These insulin-producing cell lines are
preferably derived from cells infected by retroviral vectors containing at
least two oncogenes under the control of an inducible promoter. The
preferred retroviral vector expresses SV40 T antigen and H-ras.sup.val12,
in the infected cells, under the control of a lac repressor-responsive
promoter.
The inducible promoters and genetic elements in the vectors inducibly
regulate the oncogene expression since the expression of multiple
oncogenes in primary cells, e.g., endocrine precursor cells, would be
likely to interfere with the ability of those cells to differentiate.
Moreover, expression of the oncogenes in the host may cause tumor. Thus,
once the number of the genetically modified cells have reached the desired
amount for harvest, the oncogenes in the cells are then suppressed or
removed, and precursor cells if present are allowed to differentiate into
mature cells. These differentiated mature cells are then transplanted into
the patient. Thus, regardless of the in vitro lifespan of the cell lines,
the most preferred cell line presents non-dividing, preferably
differentiated, human cell lines useful for transplantation, preferably
because they produce a desired product.
There are two aspects to the cell transplantation. In the first aspect, the
transplanted cells serve to supplement the cells that are destroyed,
malfunctioning, or absent in the transplant patient. In the second aspect,
the vector may contain a foreign gene expressing a desired product that is
missing, malfunctioning or expressed at a low level in the transplant
patient. In the second case, the transplanted cells express the desired
gene product in the transplant patient.
In the practice of the first aspect of the cell transplantation therapy,
the target cells are preferably those that are not regenerated in the
patient. Thus, for example, human fetal neurons can be grown and
multiplied in vitro by the above method and the oncogenic-suppressed or
-removed, differentiated neurons transplanted into human patients. The
patients are those suffering from loss of or dysfunctional neurons, such
as patients suffering from: Alzheimer, Parkinson, and other
neurodegenarative diseases. Similarly, human bone marrow or stem cells may
be produced and transplanted into patients suffering from depressed immune
response. These patients include those suffering from inherited defects,
cancer, immunodeficiency syndrome (AIDS) or patients undergoing cancer
therapy. Once in circulation, the transplanted bone marrow or stem cells
travel to the bones where the immature cells grow into functioning B and T
cells. Other fetal cells that may be used are endocrine secreting cells
such as pituitary and hypothalamus cells, in particular, endocrine
precursor cells, such as human fetal pancreatic (HFP) cells. The
genetically modified and transplanted cells preferably supplement the
transplant host's cells in the production of the needed endocrine
hormones. Myoblasts can also be genetically modified, differentiated, and
transplanted into patients suffering from loss of, malfunctioning, or
degenerating muscle, such as patients suffering from cardiac disorder and
muscular dystrophy. Other examples include transplantation of genetically
modified, oncogene-suppressed or -removed, differentiated fetal pancreatic
cells into human patient. Preferably, the transplanted cells secrete
insulin in response to glucose level in the patient, in an amount and with
the precise timing that is needed for control of glucose homeostasis. The
vector may additionally contain one or more genes which encode a desired
gene product. The desired gene product may be lacking, absent or defective
in the transplant host. Thus, the transplanted cells, by expressing the
gene product, supplement or overcome the transplant host's lack of the
normal gene product. For example, the vector may additionally contain
Factor IX gene which encodes a blood clotting factor. Once transplanted
into a hemophilic patient, the resulting genetically modified cells
produces the blood clotting factor in vivo to supplement the patient's
blood clotting factor. In another example, the vector may contain a gene
encoding dystrophin which is then used to genetically modified myoblasts
or other cells for transplant into patients suffering from muscular
dystrophy. In yet another example, to increase the production of
neurotransmitters, neuronal cells are infected with the recombinant
viruses containing the oncogenes, inducible promoter and one or more genes
coding for neurotransmitters. Other examples of desirable genes are those
which produce: immunoglobulins, serum proteins, viral or tumor cell
antigens, or biologically active molecules such as enzymes, hormones,
growth factors, or receptors for hormones or growth factors, or homologues
of the foregoing. Examples of the desired genes also include non-mammalian
genes, such as bacterial sequences encoding for cholesterol-metabolizing
enzymes.
The present method allows for the establishment and extended growth of cell
lines, particularly fetal cell lines, of genetically modified,
oncogene-suppressed or -removed and differentiated cells that are well
characterized and can thus be used on many human patients, without
requiring a cell line tailored to each individual patient. Preferably,
these cells lines are immortal.
To reduce immunorejection by the transplant patient, the preferred vector
and virus may additionally contain genes which reduces immunogenecity in
the genetically modified cell lines. An example of such a gene is the
adenoviral P19 gene which encodes a transmembrane glycoprotein (gp19K).
gp19K is localized in the endoplasmic reticulum and binds to class I
antigen (Ag) of the major histocompatibility complex (MHC). This binding
blocks the transport of class I Ag to the surface of the infected cell and
prevents class-I-restricted cytolysis by cytotoxic T lymphocyte (CTL)
{Paabo, S., et al., Cell, 50:311-317 (1987) and references within; Wold,
W. S. M., and Gooding, L. R., Mol. Biol. Med., 6:433-452 (1989)}. With
reduced immunogenicity, genetically modified cell line banks can be
established to supply these cells for transplantation into e.g., human
patients at treatment centers remote from the cell line banks. The
availability of the cell lines and cell line banks also provide ready
sources of the cells for use for other purpose known in the art, replacing
scarce sources such as cadavers and fetal tissues.
Alternatively, to further reduce host versus graft immune rejection, one
may use the patient's cells and coaxed their growth by exposing them to
mitotic agents, such as collagenase, dexamethasone, fibroblast growth
factor, before genetically modifying them using the methods of the present
invention.
Besides transplantation, the genetically modified cell lines can be
cultured and used to produce the desired gene products in vitro which are
harvested and purified according to methods known in the art. If the
genetically modified cells are used to produce the desired gene products
in vitro, it is not necessary to incorporate inducible promoter(s) in the
vectors as tumorigenicity, a concern for a transplant host, will not be a
concern in this case.
The cell lines described herein also provide well characterized cells for
other purposes such as for screening of chemicals which interact with
proteins on the cells' surface, e.g., for therapeutic uses.
Viral Vector Selection
Retroviral vectors are the preferred vectors of this invention, though
other viral vectors may be used, such as adenoviral vectors. Though
adenoviral vectors have the advantage of not requiring dividing cells for
transfection, they have a disadvantage in that they do not integrate into
the genome, possibly making it more difficult to derive stable cell lines.
Adeno-associated viral (AAV) vectors might also be used but have the
disadvantage of a smaller packaging limit than retroviral vectors.
The retroviral vector can be any that are known in the art. Retroviruses to
be adapted for use in accordance with this invention can be derived from
many avian or mammalian hosts. However, a requirement for use is that the
virus be capable of infecting cells which are to be the recipients of the
new genetic material (oncogene and/or desired gene) to be transduced using
the retroviral vectors. Examples of retroviruses include avian
retroviruses, such as avian erythroblastosis virus (AMV), avian leukosis
virus (ALV), avian myeloblastosis virus (ABV), avian sarcoma virus (ACV),
Fujinami sarcoma virus (FuSV), spleen necrosis virus (SNV), and Rous
sarcoma virus (RSV). Non-avian viruses include: bovine leukemia
virus(BLV); feline retroviruses such as feline leukemia virus (FeLV) or
feline sarcoma virus (FeSV); murine retroviruses such as murine leukemia
virus (MuLV), mouse mammary tumor virus (MMTV), and murine sarcoma virus
(MSV); rat sarcoma virus (RaSV); and primate retroviruses such as human
T-cell lymphotropic viruses 1 and 2 (HTLV-1, 2), and simian sarcoma virus
(SSV). Many other suitable retroviruses are known to those skilled in the
art. A taxonomy of retroviruses is provided by Teich, in Weiss, et al.,
eds., RNA Tumor Viruses, 2d ed., Vol. 2 Cold Spring Harbor Laboratory, New
York, pp. 1-16 (1985). Particularly preferred retroviruses for use in
connection with the present invention are the murine retroviruses known as
Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMSV)
and Kirsten murine sarcoma virus (KiSV). The MoMSV genome can be obtained
in conjunction with a pBR322 plasmid sequence pMV (ATCC37190), while a
cell line producer of KiSV in K-BALB cells has been deposited as ATCC
163.3. A deposit of a plasmid (pRSVneo) derived from pBR322 including the
RSV genome and a neo marker is available as ATCC 37198. A plasmid (pPBI01)
comprising the SNV genome is available as ATCC 45012. For example, a
retroviral vector may be constructed so as to lack one or more of the
replication genes such as gag (group-specific antigen), pol (polymerase)
or env (envelope) protein encoding genes. The resulting recombinant
retrovirus would thus be capable of integration into the chromosomal DNA
of an infected host cell, but once integrated, be incapable of replication
to provide infective virus, unless the cell in which it is introduced
contains another proviral insert encoding functionally active trans-acting
viral proteins. Methods for producing infectious but replication deficient
viruses are known in the art such as described in Mann, et al., Cell,
33:153 (1983) and Miller, et al., Mol. Cell Biol., 6:2895 (1986), hereby
incorporated by reference in their entirety.
Oncogene Selection
The multiple, preferably dominant, oncogenes can be any that are known in
the art. The oncogenes are preferably chosen according to the synergy
amongst them in cellular transformation, and their ability to transform
the target cells. Further, the large sizes of some oncogenes may affect
their inclusion on the same vector. In order to provide transforming
capability, the RNA or DNA constructs of the present invention incorporate
at least two or three oncogenes, which can be derived from viral, cellular
genomes, mammalian or avian chromosomal RNA or DNA. Partial lists of
oncogenes are provided by Bishop, et al., in Weiss, et al., eds., RNA
Tumor Viruses, Vol. 1, Cold Spring Harbor Laboratory, New York, pp.
1004-1005 (1984), and Watson et al., Molecular Biology of the Gene, 4th
Ed., Vol II (Benjamin Cummings, Menlo Park, Calif., USA) p. 1037. Included
are the known oncogenes such as src, yes, abl, fps, fes, fms, ros, kit,
mos, raf, H-ras, K-ras, sis, SV40 T-antigen (SV40 T-Ag), Her2/neu,
C-erbB2, C-erB3, myc, myb, fos, ski and erbA. Many oncogene products have
tyrosine-specific protein kinase or serine/threonine protein kinase
activity, or appear to be homologues of growth factors, growth factor
receptors, or are nuclear proteins with unknown function. Many oncogenes
can be obtained from public collections of deposited biological materials.
Thus, v-raf is present in the plasmid pF4 deposited as ATCC 45010 {Rapp,
et al., Proc. Natl. Acad. Sci. USA, 80:4218 (1983)}; v-myc.sup.mc29 is
available as ATCC 45014; and v-Ha-ras is a genetic component of ATCC
41047.
Inducible Promoters and Genetic Elements
The oncogenes in each vector are under the control of one or more and
preferably at most two, inducible promoters or inducible genetic elements.
More preferably, multicistronic transcriptional units are used to express
all the oncogenes under the control of the same promoter.
Inducible promoters and inducible genetic elements are known in the art and
can be derived from viral or mammalian genomes. Examples of inducible
promoters are: lacO-containing SV40 promoter, lacO-containing LTR
promoter, metallothionein promoter, and the TET promoter. There are
numerous sources of SV40 DNA, including commercial vendors such as New
England Biolabs, Inc., Beverly, Mass., USA. In the inducible system which
uses inducible genetic elements, the oncogenes are suppressed by excising
them from the transfected cells. For example, in a two-vector system, the
first vector contains the oncogenes flanked by the genetic elements
consisting of recombination sites from the bacteriophage P1 Cre/lox
recombination system. After the first vector has transformed the target
cells and the cells have multiplied to a desired number, a second vector
is used to transfect the cells. The second vector contains a Cre
recombinase gene which when expressed in the cells, will excise the
oncogenes from the genome of the cells. The P1 Cre/lox system is described
in Dale, E. C., et al., Proc. Natl. Acad. Sci. USA, 88:10558-10562 (1991),
hereby incorporated by reference in its entirety. Alternatively, the
vector may contain both inducible promoter(s) and genetic element(s). In
the simplest example, the vector contains an inducible promoter and a pair
of genetic elements flanking the oncogenes. In this system, the inducible
promoter may be used to gradually reduce the expression of the oncogenes,
e.g., to gradually adapt the cells to the absence of oncogenic activities,
before the genetic elements are manipulated to excise the oncogenes.
Construction of suitable vectors containing the desired oncogenes and
inducible promoter or genetic element system employs standard ligation
techniques. Isolated plasmids or nucleotide sequences are cleaved,
tailored, and religated in the form desired to form the plasmids required.
For example, useful plasmid vectors for amplifying the retroviral genetic
elements in bacterial hos | | |
