U.S. patent application number 11/674068 was filed with the patent office on 2008-11-20 for method for selectively transducing pathologic mammalian cells using a tumor suppressor gene.
This patent application is currently assigned to Canji, Inc.. Invention is credited to Nancy Kan, H. Michael Shepard.
Application Number | 20080286247 11/674068 |
Document ID | / |
Family ID | 25487602 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080286247 |
Kind Code |
A1 |
Shepard; H. Michael ; et
al. |
November 20, 2008 |
METHOD FOR SELECTIVELY TRANSDUCING PATHOLOGIC MAMMALIAN CELLS USING
A TUMOR SUPPRESSOR GENE
Abstract
A method for transducing a pathologic hyperproliferative
mammalian cell is provided by this invention. This method requires
contacting the cell with a suitable retroviral vector containing a
nucleic acid encoding a gene product having a tumor suppressive
function. Also provided by this invention is a method for treating
a pathology in a subject caused by the absence of, or the presence
of a pathologically mutated tumor suppressor gene.
Inventors: |
Shepard; H. Michael; (Rancho
Santa Fe, CA) ; Kan; Nancy; (Dublin, OH) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Canji, Inc.
San Diego
CA
|
Family ID: |
25487602 |
Appl. No.: |
11/674068 |
Filed: |
February 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10912261 |
Aug 4, 2004 |
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11674068 |
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10076691 |
Feb 14, 2002 |
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10912261 |
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08403797 |
Dec 4, 1995 |
6348352 |
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PCT/US93/08844 |
Sep 17, 1993 |
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10076691 |
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07948289 |
Sep 18, 1992 |
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08403797 |
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Current U.S.
Class: |
424/93.21 ;
435/456 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 38/1709 20130101; C07K 14/47 20130101; A61P 35/02 20180101;
C12N 2740/13043 20130101; A61P 35/00 20180101; A61P 31/00 20180101;
C07K 14/4746 20130101 |
Class at
Publication: |
424/93.21 ;
435/456 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 15/87 20060101 C12N015/87; A61P 31/00 20060101
A61P031/00 |
Claims
1. A method for treating a heterogenous cell preparation of
mammalian cells wherein the preparation contains a population of
normal cells and of cells having a pathologic hyperproliferative
phenotype, comprising, transducing the cells of the heterogenous
cell preparation ex vivo in the absence of a selective medium with
a replication-incompetent retroviral vector containing a nucleic
acid encoding a p53 or retinoblastoma gene product having a tumor
suppressive function, whereby cells having the pathologic
hyperproliferative phenotype undergo apoptosis or death or express
a mature or benign phenotype.
2. The method of claim 1, wherein the gene product is a wild type
retinoplastoma gene product.
3. The method of claim 2, wherein the gene product is a wild type
p53 gene product.
4. (canceled)
5. The method of claim 1, wherein the retroviral vector lacks a
selectable marker gene.
6. (canceled)
7. The method of claim 1, wherein the cells having the pathologic
hyperproliferative phenotype are prostate cells, psoriatic cells,
thyroid cells, breast cells, colon cells, lung cells, sarcoma
cells, leukemic cells or lymphoma cells.
8. The method of claim 1, wherein the time period is less than
about ten hours.
9. The method of claim 8, wherein the time period is about four
hours.
10. The method of claim 1, wherein cells in the preparation having
the pathologic hyperproliferative phenotype come to express a
mature or benign phenotype.
11. The method of claim 1, wherein cells in the preparation having
the pathologic hyerproliferative phenotype undergo apoptosis or
die.
12-13. (canceled)
14. The method of claim 1, wherein the nucleic acid is RNA.
15. The method of claim 1, wherein the mammal is a human.
16. A method for treating a pathology in a subject caused by the
absence of a p53 or retinoblastoma tumor suppressor gene or the
presence of a pathologically mutated p53 or retinoblastoma tumor
suppressor gene wherein the absence or presence causes a cell to
pathologically hyperproliferate comprising: obtaining a
heterogenous cell preparation of mammalian cells from the subject
wherein the preparation contains a population of normal cells and
of the pathologically hyperproliferating cell, treating the
preparation according to the method of claim 1, and reinfusing the
treated preparation into the subject.
17. (canceled)
18. The method of claim 17, wherein the tumor suppressor gene is
wild type p53 gene.
19-20. (canceled)
21. The method of claim 16, wherein the cells having a pathologic
hyperproliferative phenotype are prostate cells, psoriatic cells,
thyroid cells, breast cells, colon cells, lung cells, sarcoma
cells, leukemic cells or lymphoma cells.
22. The method of claim 21, whereby cells in the preparation having
the pathologic hyperproliferative phenotype undergo apoptosis or
die.
23. (canceled)
24. The method of claim 16, wherein the nucleic acid is RNA.
25. The method of claim 16, wherein the retroviral vector lacks a
selectable marker gene.
26. The method of claim 1, wherein the cells having the pathologic
hyperproliferative phenotype are leukemic cells or lymphoma
cells.
27. The method of claim 1, wherein the pathologic
hyperproliferative phenotype is a lack of contact growth
inhibition.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/912,261, filed Aug. 4, 2004; which is a
continuation of U.S. patent No. application Ser. No. 10/076,691,
filed Feb. 14, 2002 (now abandoned); which is a continuation of
U.S. patent application Ser. No. 08/403,797, filed Dec. 4, 1995
(now issued U.S. Pat. No. 6,348,352); which is the national stage
of PCT/US93/08844, filed Sep. 17, 2003; which claims priority to
U.S. patent application Ser. No. 07/948,289, filed Sep. 18, 1992
(now abandoned); each of which is incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to a method for
selectively transducing pathologic hyperproliferative mammalian
cells in a heterogeneous cell preparation comprising
retroviral-mediated transduction of the pathologic cell with a
nucleic acid encoding a gene product having tumor suppressive
function.
[0003] Throughout this application various publications are
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0004] The human p53 gene encodes a 53 kilodalton nuclear
phosphoprotein (Lane, D. P., et al., Genes and Dev., 4:1-8 (1990);
Lee, Y--HP, Breast Cancer Res. and Trmt, 19:3-13 (1991); Rotter,
V., et al., Adv. Can. Res., 57:257-72 (1991)). The p53 protein was
first identified as a cellular protein in SV40-transformed cells
that was tightly bound to the SV40 T antigen (Lane, D. P., et al:
Nature, 278:261-3 (1979)). The wild type p53 gene has the
characteristics of a tumor suppressor gene. It is similar to the
prototype of tumor suppressor genes, the retinoblastoma gene (RB),
in that loss of heterozygosity of the p53 or RB genes characterizes
the phenotype of many types of tumor cells (Hollstein, M. et al.,
Science, 253:49-51 (1991); Levine, A. J., et al., Biochimica et
Biophysica Acta, 1032:119-36 (1990); Levine, A. J., et al., Nature
351:453-6 (1991); Weinberg, R. A. Science, 254:1138-46 (1991)). In
human malignancies associated with p53 alterations, this loss of
heterozygosity usually results from the loss of one allele (allelic
deletion), while the other allele suffers one or more somatic
mutations. Unlike RB, however, certain mutations in the p53 gene
are capable of immortalizing rodent cells and enhancing the
tumorigenicity of established cell lines (Jenkins, J. R., et al.,
Nature, 312:651-4 (1984)). Mutant but not wild type p53 can
cooperate with the activated ras oncogene in transforming primary
rat embryo fibroblasts (Eliyahu, D., et al., Nature, 312(13):646-9
(1984); Parada, L. F., et al., Nature, 312:649-51 (1984)). Other
events related to tumor progression also appear to be associated
with the expression of mutant p53. Among these is differential
modulation of the multiple drug resistance gene (MDR1) by wild type
as compared to altered p53. In this case, mutant p53 specifically
stimulates the MDR1 promoter, while wild type p53 exerts repression
(Chin, R-V., et al., Science, 255:459-62 (1992)). Another possible
way in which mutant p53 could promote tumorigenesis is by reducing
tumor cell responsiveness to transforming growth factor-.beta., a
negative regulator of cell proliferation (Gerwin, B. I., et al.,
PNAS USA, 89:2759-63 (1992)).
[0005] In addition to the in vitro data described above two animal
models have been described that implicate p53 in tumor formation.
Transgenic mice expressing a mutant p53 gene display a high
incidence of, lung, bone and lymphoid tumors (Lavigueur, A., et
al., Mol. Cell. Biol., 9(9):3982-91 (1989)). In addition, p53-null
mice (Donehower, L. A., et al., Nature, 356(19):215-21 (1992)) show
an increased risk of spontaneous neoplasms, the most frequently
observed being malignant lymphoma.
[0006] Other data which support the conclusion that mutant p53
plays an important role in tumorigenesis include re-introduction of
the wild type p53 gene into human tumor cell lines which lack p53
expression. In this case, wild type p53 can reverse the malignant
phenotype as measured by colony formation in soft agar and tumor
formation in nude mice (Chen, P. L., et al., Science, 250:1576-80
(1990); Cheng, J., et al., Can. Res., 52:222-6 (1992); Baker, S.
J., et al., Science, 249:912-15 (1990); Isaacs, W. B., et al., Can.
Res., 51:4716-20 (1991); Casey, G., et al., Oncogene, 6(10):1791-7
(1991); Shaw, P., et al., PNAS USA, 89:4495-99 (1992); Takahashi,
T., et al., Can. Res., 52:2340-3 (1992)). Tumor cell types which
have shown conversion of a non-malignant phenotype as a result of
the introduction of wild type p53 expression include prostate
(Isaacs, W. B., et al., supra), breast (Casey, G. et al. supra),
colon (Baker, S J., et al., supra; Shaw, P. et al., supra) lung
(Takahashi, T. et al., supra), and lymphoblastic leukemia (Cheng,
J. et al., supra). Other data suggest that introduction of wild
type p53 into tumor cells which have lost endogenous p53 expression
appears to be cytotoxic (Johnson, P. et al., Mol. Cell. Biol.,
11(1):1-1 (1991)). In some cases the re-introduction of wild type
p53 may result in programmed cell death, or apoptosis
(Yonish-Rouach, E. et al., Nature, 352:345-7 (1991)). The work
described above indicates strongly that alteration of the wild type
p53 gene has a role in multiple aspects of tumorigenesis and that
reintroduction of the wild type p53 coding sequence can have a
negative regulatory function or cytotoxic effect on malignant
cells.
[0007] Clinical data suggest that inactivating mutations in the p53
gene are among the most common types of mutations associated with
human malignancy (Rotten, V. et al. supra; Nigro, J. M. et al.,
Nature, 342:705-8 (1989); Gaidano, G. et al., PNAS USA, 88:5413-7
(1991); Cheng, J. et al., Mol. Cell. Biol., 10(10):5502-09 (1990)).
A classical example is the Li-Fraumeni syndrome, a familial
syndrome of several neoplasms, including breast cancer, sarcomas
and others. Specific mutations in the p53 gene are found in
affected members of the family and appear to be associated with the
predisposition to develop early cancers (Malkin, D. et al.,
Science, 250:1233 (1990); Srivastava, S. et al., Nature, 348:747
(1990)). Several laboratories have reported that alterations in the
p53 gene accompany the evolution of human CML (chronic myelogenous
leukemia) to blast crisis (acute phase) (Abuja, H. et al., J. Clin.
Invest., 87:2042-7 (1991); Foti, A. et al., Blood, 77((11):2441-4
(1991); Feinstein, E. et al., PNAS USA, 88:6293-7 (1991)). In one
CML patient who reverted briefly from the acute phase to a second
chronic phase, the inactivating point mutation in p53 which
appeared concomitantly with the acute phase disappeared and the
wild type sequence re-emerged (Foti, A. et al., supra). These data
indicate that alterations which inactivate the tumor suppressive
activity of p53 may represent pivotal events in the progression
from the chronic to the acute phase of human CML. Other clinical
data also suggest an important role for p53 mutations in disease
progression. These include a number of hematologic neoplasms as
well as solid tumors (Rotter, V. et al. supra; Abuja, H. et al., J.
Clin. Invest., supra; Abuja, H. et al., PNAS USA, 86:6783-6787
(1989); Mori, N. et al., Br. J. of Haem., 81:235-240 (1992);
Porter, P. L. et al., Am. J. Path., 140(1):145-53 1992)). Recent
reports show a dramatic association between altered p53 and
shortened survival in breast cancer (Thor, A. D. et al., J. Natl.
Can. Inst., 84(11):845-55 (1992); Isola, J. et al., J. Natl. Can.
Inst., 84(14):1109-14 (1992); Callahan, R., J. Natl. Can. Inst.,
84:826-7 (1992)).
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention generally relates to a method for
selectively transducing pathologic hyperproliferative mammalian
cells comprising retroviral mediated transduction of pathologic
cells with a nucleic acid encoding a gene product having tumor
suppressive function. The methodology provided involves the
introduction of a stably expressed tumor suppressor gene into a
heterogeneous cell preparation (containing both normal and
pathologic hyperproliferative cells) and, under suitable
conditions, selectively transducing phenotypically pathologic
hyperproliferative cells, suppressing the pathologic phenotype and
reinfusing the treated cell preparation into the patient. Also
provided by this invention is a method for treating a pathology in
a subject caused by the absence of, or the presence of a
pathologically mutated tumor suppressor gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the tumorigenicity of antibiotics-selected K562
cells in nude mice. K562 cells were infected with the p53-RV or NCV
and selected in hygromycin as described in the legend to Table 3.
(A) 5.times.10.sup.6 K562/p53 or K562JNCV (B) 1.times.10.sup.7
K562/p53 or K562/NCV were injected subcutaneously into opposite
flanks of athymic Balb/c nu/nu mice. The mice were purchased from
Simonsen Laboratories, Inc. (Gilroy, Calif.) and maintained in a
pathogen-free environment. Once tumors were formed, they were
measured weekly until the experiments were terminated.
[0010] FIG. 2 shows delayed tumor formation in nude mice induced by
K562 cells following a short-term infection with the p53-RV. K562
cells were infected with the p53-RV or NCV for 4 hours as described
in Example II. The viral supernatant was removed and the cells were
injected immediately into nude mice as described in Example
III.
[0011] FIG. 3 shows delayed tumor formation in nude mice induced by
three human cell lines following short-term infections with the
p53-RV. The three human cells lines, H69 (human small-cell lung
carcinoma), H128 (human small-cell lung carcinoma) and HTB9 (human
bladder carcinoma), were obtained from the American Type Culture
Collection (ATCC), (Rockville, Md.). The short-term infections
using either p53-RV or NCV were performed as described in Examples
II and III.
[0012] FIG. 4 shows lack of toxicity of the p53-RV viral
supernatant on normal murine bone marrow cells at a high
multiplicity. Normal mouse bone marrow cells were obtained from the
femurs of Balb/c mice. The cells were isolated by ficoll-hypaque
gradient, and infected with the p53-RV or NCV at MOI=1(A) or
MOI=10(B) for 4 hours. At the end of infection, the viral
supernatant was removed and the cells were seeded in 96-well plates
at the density of 5.times.10.sup.4 cells per well containing murine
GM-CSF ranging from 0 to 0.400 units/ml. The cells were incubated
for 3 days and the number of viable cells were determined by the
MTT assay as described by Mossman, T., J. Immunol. Methods,
65:55-63 (1983).
DETAILED DESCRIPTION OF THE INVENTION
[0013] There are approximately 5,000 bone marrow transplantations
(BMT) each year (The BBI Newsletter, 156 (1991)). Most of these are
performed on leukemia and lymphoma patients. A growing number of
BMT are being done to support more intensive therapeutic approaches
to breast and lung cancers, as well as for other indications (Droz,
J. P. Bur. J. Can., 27:831-35 (1991); Menichella, G. Br. J. Haem.,
79:444-50 (1991); Osbourne, C. R. Breast Can. Res. Trtmt.,
20:511-14 (1991)). Approximately 30% of these patients are
candidates for tumor suppressive gene therapy. This number derives
from the observation that about 30% of cancer patients either do
not express the tumor suppressor gene or express an inactivated
form of the tumor suppressor protein (Hollstein, M. et al., supra).
The preferred embodiments detailed below support the
efficaciousness of a retrovirus encoding the human wild type tumor
suppressor gene, p53-RV, in reversing the malignant phenotype of
several leukemia and lymphoma cell lines as measured by abrogation
or substantial inhibition of colony formation in soft agar assays,
and as judged by reversing/inhibiting the ability of tumor cells to
grow in nude mice following introduction of the wild type p53
gene.
[0014] For the K562 tumor cell line, which is derived from a
chronic myelogenous leukemia patient in blast crisis (Andersson, L.
C. et al., Int. J. Can., 23:143-7 (1979)) for two human small-cell
lung carcinoma cell lines (H69 and H128) (Gazdar, A. F. et al.,
Can. Res., 40(10):3502-7 (1980)), and for one transitional cell
(bladder) carcinoma cell line (HTB9) (Takahashi, R. et al., PNAS
USA, 88:5257-61 (1991)) tumor suppression by p53 can be
accomplished with a protocol involving short-term infections with
the p53-RV. This protocol is completely consistent with current
clinical methodology used in the preparation of bone marrow or
peripheral blood hematopoietic cells for autologous bone marrow
transplantation (ABMT) (Deisseroth, A. B. et al., Human Gene
Therapy, 2:359-376 (1991)).
[0015] The present invention generally relates to an improved
method of gene therapy for "negative purging" of pathologic
hyperproliferative cells that contaminate preparations of
autologous hematopoietic cells used for bone marrow reconstitution.
As used herein, the term "hyperproliferative cells" includes but is
not limited to cells having the capacity for autonomous growth,
i.e., existing and reproducing independently of normal regulatory
mechanisms. Hyperproliferative diseases may be categorized as
pathologic, i.e., deviating from normal cells, characterizing or
constituting disease, or may be categorized as non-pathologic,
i.e., deviation from normal but not associated with a disease
state. Pathologic hyperproliferative cells are characteristic of
the following disease states, thyroid hyperplasia--Grave's Disease,
psoriasis, benign prostatic hypertrophy, Li-Fraumeni syndrome
including breast cancer, sarcomas and other neoplasms, bladder
cancer, colon cancer, lung cancer, various leukemias and lymphomas.
Examples of non-pathologic hyperproliferative cells are found, for
instance, in mammary ductal epithelial cells during development of
lactation and also in cells associated with wound repair.
Pathologic hyperproliferative cells characteristically exhibit loss
of contact inhibition and a decline in their ability to selectively
adhere which implies a change in the surface properties of the cell
and a further breakdown in intercellular communication. These
changes include stimulation to divide and the ability to secrete
proteolytic enzymes. The present invention will allow for high dose
chemotherapy and/or radiation therapy, followed by autologous bone
marrow reconstitution with hematopoietic cell preparations in which
phenotypically pathologic cells have been reconstituted with a
normal tumor suppressor gene. Application of the present invention
will result in diminished patient relapses which occur as a result
of reinfusion of pathologic hyperproliferative cells contaminating
autologous hematopoietic cell preparations.
[0016] More specifically, the present invention relates to a method
for depleting a suitable sample of pathologic mammalian
hyperproliferative cells contaminating hematopoietic precursors
during bone marrow reconstitution via the introduction of a
stably-expressed wild type tumor suppressor gene into the cell
preparation (whether derived from autologous peripheral blood or
bone marrow). As used herein, a "suitable sample" is defined as a
heterogeneous cell preparation obtained from a patient, e.g., a
mixed population of cells containing both phenotypically normal and
pathogenic cells. An example of a wild type tumor suppressor gene
is the p53 gene, the coding sequence has been described by Chen et
al. supra and the amino acid sequence is shown in Table 1 (SEQ ID
NOS:1 and 2).
TABLE-US-00001 TABLE 1 50 V*SHR PGSR* LLGSG DTLRS GWERA FHDGD TLPWI
GSQTA FRVTA MEEPQ 100 SDPSV EPPLS QETFS DLWKL LPENN VLSPLY PSQAM
DDLML SPDDI EQWFT 150 EDPGP DEAPR MPEAA PPVAP APAAP TPAAP APAPS
WPLSS SVPSQ KTYQG 200 SYGFR LGFLH SGTAK SVTCT YSPAL NKMFC QLAKT
CPVQL WVDST PPPGT 250 RVRAM AIYKQ SQHMT EVVRR CPHHE RCSDS DGLAP
PQHLI RVEGN LRVEY 300 LDDRN TFRHS VVVPY EPPEV GSDCT TIHYN YMCNS
SCMGG MNRRP ILTII 350 TLEDS SGNLL GRNSF EVRVC ACPGR DRRTE EENLR
KRGEP HHELP PGSTK 400 RALPN NTSSS PQPKK KPLDG EYFTL QIRGR ERFEM
FRELN EALEL KDAQA GKEPG GSRAH SSHLK SKKGQ STSRH KKLMF KTEGP DSD* *
= Stop codon
[0017] The preferred delivery system for the wild type tumor
suppressor gene is a replication-incompetent retroviral vector. As
used herein, the term "retroviral" includes, but is not limited to,
a vector or delivery vehicle having the ability to selectively
target and introduce the coding sequence into dividing cells. As
used herein, the terms "replication-incompetent" is defined as the
inability to produce viral proteins, precluding spread of the
vector in the infected host cell. An example of such vector is
p53-RV, which has been described in detail by Chen et al. supra and
is shown in Table 2.
TABLE-US-00002 TABLE 2 General Schematic of p53-Retrovirus (p53-RV)
##STR00001##
[0018] Another example of a replication-incompetent retroviral
vector is LNL6 (Miller, A. D. et al., BioTechnigues 7:980-990
(1989)). The methodology of using replication-incompetent
retroviruses for retroviral-mediated gene transfer of gene markers
is well established (Correll, P. H. et al., PNAS USA, 86:8912
(1989); Bordignon, C. et al., PNAS USA, 86:8912-52 (1989); Culver,
R. et al., PNAS USA, 88:3155 (1991); Rill, D. R. et al., Blood,
79(10):2694-700 (1992)). Clinical investigations have shown that
there are few or no adverse effects associated with the viral
vectors (43: Anderson, Science, 256:808-13 (1992)). However, these
methods have been limited to transfers of "gene markers" such as
the neomycin gene that merely function as "tracking agents" for
marking malignant cells before, and locating malignant cells after,
reinfusion of bone marrow, however, the transduction of gene
markers confers little clinical benefit to the affected patient who
does not receive protection against subsequent relapse (Rill, D. R.
et al. Blood, supra). The subject invention eliminates the
necessity of the time consuming procedure of transducing cell
samples with a selectable marker gene, such as neomycin, to
identify pathologic cells to facilitate subsequent attempts to
remove those cells before reinfusion into the patient.
[0019] Other vectors are suitable for use in this invention and
will be selected for efficient delivery of the nucleic acid
encoding the tumor suppressor gene. The nucleic acid can be DNA,
cDNA or RNA.
[0020] The subject invention provides a "shotgun" procedure whereby
the cell sample is contacted with a retroviral vector in the
absence of selective medium that does not necessarily contain a
selectable marker gene, but notwithstanding, possesses the ability
to simultaneously selectively target and transduce only the
pathologic cell population in the heterogeneous cell preparation.
Other methods of efficient delivery or insertion of a gene of
interest into a cell are well known to those of skill in the art
and comprise various molecular cloning techniques. As used herein,
the terms "insertion or delivery" encompass methods of introducing
an exogenous nucleic acid molecule into a cell.
[0021] A variety of techniques have been employed in an attempt to
deplete marrow of pathologic hyperproliferative cells before
reinfusion, utilizing "purging" methods, e.g., monoclonal
antibodies or chemotoxins (Kaizer H. et al., Blood, 65:1504 (1985);
Gorin, N. C. et al., Blood, 67:1367 (1986); De Fabritiis, P. et
al., Bone Marrow Transplant, 4:669 (1989)). As used herein, the
term "pathologic" includes abnormalities and malignancies induced
by mutations and failures in the genetic regulatory mechanisms that
govern normal differentiation that are not the result of gene loss
or mutation. These techniques, however, have not resulted in
reduced relapse rates, and have consistently resulted in damaging
normal marrow progenitor cells (Kaizer H. et al., supra; Gorin, N.
C. et al., supra; De Fabritiis, P. et al., supra). The present
invention addresses the aforementioned inadequacies and confers
related advantages as well. These advantages include: (a) the use
of a recombinant retroviral vector that does not require a
selectable marker gene in combination with a short-term infection
in the absence of selective medium eliminating the time consuming
procedure traditionally employed to "selectively mark" the target
cells before any "purging" of such cells is attempted, thereby
dramatically reducing the time traditionally required for preparing
hematopoietic cells for transplants; and (b) the retroviral
mediated delivery methodology of the subject invention offers
selective targeting of pathologic hyperproliferative cells in
resting cultures of hematopoietic cells as a result of the higher
infection frequency by the retroviral delivery system into actively
dividing tumor cells (Miller et al., Mal. Cell. Biol.,
10(8):4239-42 (1990)).
[0022] The ex-vivo introduction of a wild type tumor suppressor
gene; via an efficient delivery system into pathologic
hyperproliferative cells contaminating peripheral blood- or
marrow-derived autologous hematopoietic cells will facilitate
suppression of the hyperproliferative phenotype, by inducing
transformation of the cell to a mature or benign phenotype or,
alternatively, by inducing apoptosis or programmed cell death,
thereby allowing patients receiving ABMT to have a longer,
relapse-free survival. As used herein, the term "mature or benign
cell" refers to the phenotypic characteristic of inability to
invade locally or metastasize.
[0023] This invention further provides a method for transducing a
pathologic hyperproliferative mammalian cell by contacting the cell
with a suitable retroviral vector containing a nucleic acid
encoding a gene product having a tumor suppressive function, under
suitable conditions such that the cell is transduced. In one
embodiment, the gene product is expressed by a tumor suppressor
gene and the tumor suppressor gene can be, but is not limited to
wild type p53 gene, retinoblastoma gene RB, Wilm's tumor gene WT1
or colon-carcinoma gene DCC. Additionally, the nucleic acid is DNA,
cDNA or RNA.
[0024] The suitable conditions for contacting can be by infecting
the sample cells in the absence of selective medium. "Suitable
retroviral vector" has been defined above. This method is
particularly useful when the pathological cells being contacted are
prostate cells, psoriatic cells, thyroid cells, breast cells, colon
cells, lung cells, sarcoma cells, leukemic cells or lymphoma
cells.
[0025] The suitable time period for contacting can be less than
about ten hours, or more specifically, about four hours.
Transduction can be known to be complete, for example, when the
hyperproliferative phenotype is characterized by the transduced
cell expressing a mature or benign phenotype or by apoptosis or
death of the transduced cell. This method has been shown to reduce
tumor formation or tumorigenicity in a subject.
[0026] This method can be practiced ex vivo or in vivo. The
practice of the ex vivo method is described above. When the method
is practiced in vivo, the retroviral vector can be added to a
pharmaceutically-acceptable carrier and systemically administered
to the subject. In one embodiment, the subject is a mammal, such as
a human patient. Acceptable "pharmaceutical carriers" are well
known to those of skill in the art and can include, but not be
limited to any of the standard pharmaceutical carriers, such as
phosphate buffered saline, water and emulsions, such as oil/water
emulsions and various types of wetting agents.
[0027] As used herein, the term "administering" for in vivo
purposes means providing the subject with an effective amount of
the vector, effective to inhibit hyperproliferation of the target
cell. Methods of administering pharmaceutical compositions are well
known to those of skill in the art and include, but are not limited
to, microinjection, intravenous or parenteral administration.
Administration can be effected continuously or intermittently
throughout the course of treatment. Methods of determining the most
effective-means and dosage of administration are well known to
those of skill in the art and will vary with the vector used for
therapy, the purpose of the therapy, the cell or tumor being
treated, and the subject being treated.
[0028] The following examples are intended to illustrate, not limit
this invention.
EXAMPLE I
Introduction of the p53-RV into Leukemia or Lymphoma Derived Cell
Lines Suppresses the Malignant Phenotype as Measured by Colony
Formation in Soft Agar
[0029] The retroviral vector carrying the human wild type p53-cDNA
has been described (Chen et al. supra). p53-RV, an amphotropic
retrovirus, is capable of infecting a wild range of human cell
types (see below). This feature provides an advantage for ex vivo
therapy of human leukemias, because the viral vector can deliver
the wild type p53-cDNA into a number of different leukemic or other
cell types, including tumor cells from solid tumors which may
metastasize to marrow. The results of soft-agar assays using three
leukemia or lymphoma cell lines following viral infection and
antibiotic selection are shown in Table 3. In all three cases
(HL-60, acute promyelocytic leukemia, p53-negative; Hut 78, acute T
cell lymphoma, p53-negative; and Molt 3, acute lymphoblastic
leukemia, mutant p53-positive) the introduction of wild type p53 by
the p53-RV resulted in either a reduction or elimination of colony
formation in soft agar.
TABLE-US-00003 TABLE 3 Cell line No. of Cells Seeded Plating
Efficiency HL-60 5 .times. 10.sup.5 TMTC HL-60/T* 5 .times.
10.sup.5 4.7% HL-60 10.sup.5 43% HL-60/T* 10.sup.5 0% HL-60 5
.times. 10.sup.4 55% HL-60/T* 5 .times. 10.sup.4 0% Hut-78 10.sup.5
9.4% Hut-78/I* 10.sup.5 0.39% Hut-78 5 .times. 10.sup.4 9.2%
Hut-78/I* 5 .times. 10.sup.4 0% Molt 3 10.sup.5 11.7% Molt 3/I*
10.sup.5 1.5%
[0030] The human leukemic cell lines, HL-60, Hut 78 and Molt 3,
were obtained from American Type Culture Collection (ATCC). The
cell lines Hut 78 and Molt 3 were infected with the p53-RV and the
HL-60 cell line was transfected with p53-RV DNA. The p53-RV
containing the wild type p53 cDNA isolated from human fetal brain
and Moloney murine leukemia viral vector has been described by Chen
et al., supra. This virus also carries the hygromycin resistant
gene whose expression is driven by the Ross sarcoma virus (RSV)
promoter sequence. The murine NIH3T3-derived packaging cell line,
PA 12 (Chen et al., supra), produces the p53-RV with titers ranging
from 1.times.10.sup.5 to 1.times.10.sup.6 virus per ml.
[0031] Viral infections were carried out overnight in the presence
of 4 .mu.g/ml polybrene. At the end of infections, 4 ml of fresh
media were added to 2 ml of each infection mixture. The cells were
selected in the presence of 400 .mu.g/ml hygromycin 2 days after
infection.
[0032] Infected cells grew to confluency in 2-3 weeks following
hygromycin selection. For the soft-agar assay, the cells were
seeded in 6-well plates at the cell densities ranging from 103 to
105 in 0.33% agar as described (Chen et al., supra). Colonies
consisting of more than 50 cells were scored 2 weeks later.
EXAMPLE II
Suppression of Colony Formation in Soft Agar by K562 (Human Chronic
Myelogenous Leukemia) Cells Following a Short-Term Infection with
P53
[0033] Mammalian cells infected with a retroviral vector carrying
an. antibiotic marker are usually pre-selected in vitro before
testing for tumorigenicity in soft agar or in nude mice (Chen et
al., supra, Cheng et al., supra). Because this process takes about
three weeks, it would be cumbersome and expensive to pursue in the
clinic. To mimic more closely the clinical situation in which tumor
suppressor gene therapy may be applied during bone marrow purging,
K562 cells were infected with p53-RV for four hours in vitro, then
immediately tested their ability to form colonies in soft agar
without any selection. A retroviral vector identical to p53-RV, but
with the p53 coding sequence deleted, was used. as a control (see
Table 4). This vector is designated NCV (Negative Control Virus).
As shown in Table 4, p53-RV decreased colony formation by infected
K562 cells in a dose-dependent manner. At a multiplicity of
infection, (MOI) of 1, the plating efficiencies of the p53 and
NCV-infected cells were similar. However, at MOI of 3 and 10, there
was a marked decrease in the plating efficiency of the p53-RV
infected cells. The plating efficiencies of the NCV-infected K562
cells were similar at all three multiplicities of infection. The
latter result suggests that the does-dependent reduction in tumor
cell colony formation observed with increasing doses of p53-RV was
due to introduction of the wild type p53. Furthermore, the result
with NCV indicates that there is little non-specific toxicity
associated with the retroviral infection-up to MOI of 10, as
measured by this assay.
TABLE-US-00004 TABLE 4 Virus No. of Cells Plating Infection Seeded
M.O.I. Efficiency(Colony No.) p53-RV 10.sup.4 1 3.10% (310) 3 0.52%
(52) 10 0% (0) Control RV 10.sup.4 1 4.30% (430) 3 5.30% (530) 10
3.40% (340) p53-RV 5 .times. 10.sup.3 1 4.40% (220) 3 1.80% (90) 10
0.25% (13) Control RV 5 .times. 10.sup.3 1 4.70% (235) 3 4.10%
(205) 10 6.50% (325)
[0034] Human chronic myelogenous leukemia (CML)-derived cell line,
K562, was obtained from ATCC, Accession No. CCL243. To perform the
short-term infections, K.sub.562 cells were infected with the
p53-RV or NCV for 4 hours as described in Example 1. Multiplicity
of infection (MOI) was determined from the titer of the viral
stocks and K562 cell number. At the end of infection, the viral
supernatant was removed by pelleting the cells, and the
concentrated cells were used immediately in the soft-agar assay as
described in Example 1.
[0035] To construct NCV, the plasmid containing the p53 viral
genome was partially digested with BamHI, ligated, and used to
transform E. coli. The plasmid with the deleted p53 gene was then
selected by restriction enzyme analysis of mini-lysate DNA. This
DNA was used to transfect/infect PsiCRIP packaging cell line as
described (Danos, O. et al., PNAS USA, 85:6460-64 (1988)). The
viral stock, termed negative control virus (NCV), was produced in
PsiCRIP packaging cell line (56: Danos et al., supra) with a titer
of about 2.times.10.sup.5 virus per ml.
EXAMPLE III
Tumorigenicity of K562 Chronic Myelogenous Leukemia Cells Following
Infection by p53-RV and Selection for Hygromycin-Resistant
Cells
[0036] To further broaden the efficacy experiments in relevant
human tumor cell lines, K562 leukemic cells were infected with the
p53-RV and hygromycin-resistant colonies (K562/p53) were tested for
tumorigenicity in nude mice. When 5.times.10.sup.6 K562/p53 cells
were injected subcutaneously into nude mice, no tumor formation was
observed. In contrast, a comparable number of K562/NCV cells
produced tumors in all five animals tested (FIG. 1A). When
1.times.10.sup.7 tumor cells were injected, the p53-RV infected
cells produced visible tumors, although much smaller than those
induced by the NCV-infected cells (FIG. 1B). It is likely that the
lesions which formed on the flank of the animal injected with
K562/p53 were induced by those cells which had lost expression of
the wild type p53 gene (Johnson et al., supra). This conclusion is
supported by the inability to detect p53 protein or transcripts in
hygromycin selected clones after only a few passages in vitro (data
not shown).
EXAMPLE IV
Tumorigenicity in Nude Mice of K562 Cells Following a Short-Term
Infection with p53-RV
[0037] To further assess the tumor suppressive activity of the wild
type p53 gene in K562 cells, and to determine whether a short-term
infection protocol would be feasible for potential therapy of
leukemias and lymphomas, K562 CML cells were co-incubated with
p53-RV for four hours before testing for the malignant phenotype as
determined by subcutaneous tumor formation in nude mice. Following
a short-term infection by the p53-RV or the NCV, K562 cells were
injected bilaterally into nude mice. In three separate experiments,
substantial suppression of tumor formation on the flank injected
with K562 exposed to the p53-RV was observed (FIG. 2).
EXAMPLE V
Growth Suppressive Activity of p53-RV on Other Human Tumor Cell
Types
[0038] While the major target for clinical trials consists of
leukemia and lymphoma patients, other cancer patients are currently
under consideration for clinical trials involving marrow
reconstitution (Miller, C. W. et al., Can. Res., 52:1695-8 (1992);
Takahashi, T. et al., Oncogene, 6:1775-8 (1991); Takahashi, T. et
al., Science, 491-4 (1989)). FIG. 3 demonstrates that short-term
infections of two small-cell lung carcinoma cell lines (H69 in FIG.
3A; H 128 in FIG. 3B) lead to substantial inhibition of tumor
growth in nude mice. In addition, a similar experiment was
performed with a human transitional cell (bladder) carcinoma cell
line (HTB-9 in FIG. 3C). In contrast, tumor cells infected with NCV
grow rapidly in this tumor model (FIG. 3A-C).
EXAMPLE VI
Preliminary In Vitro Toxicity Studies
[0039] A critical issue for clinical application of the p53-RV has
to do with whether introduction of the p53 coding sequence under
control of a murine retroviral LTR may inhibit proliferation of
normal bone marrow cells. Preliminary studies suggest that such
inhibition is not an issue in this system. To determine toxicity of
the p53-RV, it was investigated whether exposure of normal bone
marrow cells to p53-RV under conditions similar to those employed
for a short-term infection of K562 leukemic cells would have an
effect on the response of normal bone marrow cells to GM-CSF. A
three-day proliferation assay and a methylcellulose colony forming
assay using either murine or human normal bone marrow cells were
employed to ascertain such response. FIG. 5 shows that exposure of
murine bone marrow cells at either a MOI of 1.0 or 10.0 has no
effect on their proliferation in response to GM-CSF. In addition,
when either human (Table 3) or murine (data not shown) bone marrow
cells were tested in a GM-CSF dependent colony forming assay, no
effect on normal marrow progenitor colony forming units following
exposure to the p53-RV as compared to NCV or mock infected controls
was observed.
[0040] Normal human bone marrow cells were isolated by
ficoll-hypaque gradient. These cells were incubated with the
p53-RV, NCV, or growth media in the presence of 4 .mu.g/ml
polybrene for 4 hours. At the end of incubation, the cells were
pelleted, and 1.times.10.sup.6 cells per well were plated in 6-well
plates containing 0.8% methylcellulose. Colonies larger than 50
cells per colony were scored 14 days later.
TABLE-US-00005 TABLE 5 COLONY NUMBER <r8uG-CSF> rHuGMCSF
Infection None 0.02 ng/ml 0.04 ng/al Control 1 18 21 p53-RV(0.1) 0
16 28 NCV(0.1) 0 11 18 MOCK(0.1) 2 12 17 p53-RV(1.0) 2 15 23
NCV(1.0) 4 9 18 MOCK(1.00) 0 25 18
EXAMPLE VII
Negative Purging of Small Cell Lung Cancer Cells (H69) from a
Preparation of Human Bone Marrow
[0041] Increasing quantities of small-cell lung cancer cell line
H69 were added to human bone marrow cells. These cells were
subjected to 3 two hour cycles of infection with p53-RV at a M.O.I.
of 3. After infection the cells were pelleted and plated in
methylcellulose. Colony formation is shown in Table 6. Suppression
of tumor cell colony formation is evidenced in the p53-RV treated
cultures, but is absent in the mock infected cultures. There is no
evidence of suppression of bone marrow colony formation units in
either case.
TABLE-US-00006 TABLE 6 Negative Purging of Small Cell Lung Cancer
Cells (H69) From a Preparation of Human Bone Marrow 0 0.1 1.0 10.0
H69/HBMC % A. Mock Colonies H69 0 53 100 475 HMBC + Growth 199 219
235 266 Factors B. P53-RV Colonies H69 0 1 11 181 HMBC + Growth 165
223 182 273 Factors HBMC + Growth 81 48 84 171 HBMC = normal human
bone marrow cells 5 .times. 10.sup.5 HBMC per well HBMC + growth
factors = total colony counts including H69, CPU-e, BFU-E,
CFU-GEMM, and CFU-GM Hyg = Hygromycin, 100 ug/ml
EXAMPLE VIII
Mixing Experiment to Study "Bystander Effect"
[0042] Five (5).times.10.sup.7 K562 cells (obtained from the
American Type Culture Collection (ATCC)) were infected overnight
with p53RV at a MOI equals 1 in RPMI medium containing 4 ug/ml
polybrene (Sigma). Twenty-five nude mice were divided into 5
groups, with 5 animals per group. Every mouse in group 1 was
infected subcutaneously with 5.times.10.sup.6 of p53RV infected
cells. The ratio of infected to uninfected cells=1:0. Every mouse
in group 2 was injected subcutaneously with a mixture of
2.5.times.10.sup.6 infected cells and 2.5.times.10.sup.6 uninfected
cells. The ratio of infected to infected cells=1:1. Every mouse in
group 3 was injected subcutaneously with a mixture of
1.5.times.10.sup.6 infected cells and 3.times.10.sup.6 uninfected
cells. The ratio of infected to uninfected cells=1:2. Every mouse
in group 4 was injected subcutaneously with a mixture of
0.45.times.10.sup.6 infected and 4.5.times.10.sup.6 uninfected
cells (infected:uninfected-1:10). All the mice in group S were
injected subcutaneously with 5.times.10.sup.6 uninfected cells
(infected:uninfected=0:1). Nude mice were observed for tumor growth
and survival time. Results of the study are summarized below.
TABLE-US-00007 Ratio Tumor Group infected:uninfected Formation
Survival Status.sup.b 1 1:0 - Alive and healthy at 200 days 2 1:1 -
Alive and healthy at 200 days 3 1:2 - Alive and healthy at 200 days
4 1:10 - Alive and healthy at 200 days 5 0:1 +.sup.a All died
within 90 days .sup.aMeasurable tumors developed by thirtieth day.
.sup.bExperiment was terminated on day 200, when all p53RV-selected
animals were till alive and healthy.
[0043] This experiment shows that treatment with p53RV, even at a
MOI of less than 1, inhibits tumor formation or "tumorigenicity" in
nude mice.
EXAMPLE IX
Effect of Intraperitoneal Injection of p53RV in K562 Bearing SCID
Mice
[0044] Fifty (50) SCID mice were each injected i.p. with
25.times.10.sup.6 K562 cells (ATCC). Within 50 days post-injection,
leukemia developed in the mice. The mice were then randomly
separated into 3 groups and treated as outlined below:
TABLE-US-00008 Group 1: injected i.p. with RPMI media on day 50
Group 2: injected i.p. with 1 ml heat-inactivated p53RV (original
titer = 2.6 .times. 10.sup.5 virus/ml, titer below detection limit
after inactivation) on day 50 Group 3: injected i.p. with 1 ml
p53RV (2.6 .times. 10.sup.5 virus/ml) on day 50
[0045] FIG. 5 shows that mice treated with p53RV survived over
twice as long as mice treated with heat-inactivated virus or
control mice. Thus, systemic treatment with the retroviral vector
containing the tumor suppressor gene p53 reduced tumorigenicity in
the mice and prolonged survival time.
[0046] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific experiments detailed are only
illustrative of the invention. It should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
217PRTHomo sapiensp53 wild type tumor suppressor gene coding
sequence peptide 1Ser His Arg Pro Ser Gly Arg 1 52428PRTHomo
sapiensp53 wild type tumor suppressor gene coding sequence peptide
2Leu Leu Gly Ser Gly Asp Thr Leu Arg Ser Gly Trp Glu Arg Ala Phe 1
5 10 15His Asp Gly Asp Thr Leu Pro Trp Ile Gly Ser Gln Thr Ala Phe
Arg 20 25 30Val Thr Ala Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu
Pro Pro 35 40 45Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu
Pro Glu Asn 50 55 60Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp
Asp Leu Met Leu 65 70 75 80Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr
Glu Asp Pro Gly Pro Asp 85 90 95Glu Ala Pro Arg Met Pro Glu Ala Ala
Pro Pro Val Ala Pro Ala Pro 100 105 110Ala Ala Pro Thr Pro Ala Ala
Pro Ala Pro Ala Pro Ser Trp Pro Leu 115 120 125Ser Ser Ser Val Pro
Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe 130 135 140Arg Leu Gly
Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr145 150 155
160Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys
165 170 175Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr
Arg Val 180 185 190Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met
Thr Glu Val Val 195 200 205Arg Arg Cys Pro His His Glu Arg Cys Ser
Asp Ser Asp Gly Leu Ala 210 215 220Pro Pro Gln His Leu Ile Arg Val
Glu Gly Asn Leu Arg Val Glu Tyr225 230 235 240Leu Asp Asp Arg Asn
Thr Phe Arg His Ser Val Val Val Pro Tyr Glu 245 250 255Pro Pro Glu
Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met 260 265 270Cys
Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr 275 280
285Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser
290 295 300Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg
Thr Glu305 310 315 320Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His
His Glu Leu Pro Pro 325 330 335Gly Ser Thr Lys Arg Ala Leu Pro Asn
Asn Thr Ser Ser Ser Pro Gln 340 345 350Pro Lys Lys Lys Pro Leu Asp
Gly Glu Tyr Phe Thr Leu Gln Ile Arg 355 360 365Gly Arg Glu Arg Phe
Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu 370 375 380Leu Lys Asp
Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His385 390 395
400Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys
405 410 415Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp 420
425
* * * * *