U.S. patent application number 11/981179 was filed with the patent office on 2008-04-24 for methods for treating cancers and restenosis with p21.
Invention is credited to Elizabeth G. Nabel, Gary J. Nabel, Zhi-yong Yang.
Application Number | 20080095835 11/981179 |
Document ID | / |
Family ID | 36758550 |
Filed Date | 2008-04-24 |
United States Patent
Application |
20080095835 |
Kind Code |
A1 |
Nabel; Gary J. ; et
al. |
April 24, 2008 |
Methods for treating cancers and restenosis with P21
Abstract
The p21 gene encodes a cyclin dependent kinase inhibitor which
affects cell cycle progression, but the role of this gene product
in altering tumor growth has not been established. The present
inventors have now discovered that the growth of malignant cells in
vivo is inhibited by expression of p21. Expression of p21 resulted
in an accumulation of cells in G.sub.0/G.sub.1, alteration in
morphology, and cell differentiation.
Inventors: |
Nabel; Gary J.; (Ann Arbor,
MI) ; Yang; Zhi-yong; (Ann Arbor, MI) ; Nabel;
Elizabeth G.; (Ann Arbor, MI) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
36758550 |
Appl. No.: |
11/981179 |
Filed: |
October 31, 2007 |
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11981179 |
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Current U.S.
Class: |
424/450 ;
514/44R |
Current CPC
Class: |
A61K 48/005 20130101;
A61K 38/1774 20130101; A61P 35/00 20180101; A61K 31/711 20130101;
A61K 38/1709 20130101; A61K 38/1774 20130101; A61K 9/0019 20130101;
A61K 2300/00 20130101; A61K 38/45 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 38/1709 20130101; C12N 2710/10343
20130101; A61P 9/00 20180101; C12N 15/86 20130101; A61K 31/70
20130101; A61K 38/45 20130101 |
Class at
Publication: |
424/450 ;
514/044 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61K 9/127 20060101 A61K009/127; A61P 35/00 20060101
A61P035/00; A61P 9/00 20060101 A61P009/00 |
Claims
1. A method of treating a cancer in a patient in need thereof
comprising administering in vivo a therapeutically effective amount
of a composition comprising: (i) an expression vector containing a
gene encoding p21; and (ii) a pharmaceutical carrier.
2. The method of claim 1, wherein said expression vector is a
eukaryotic or viral vector.
3. The method of claim 2, wherein said viral vector is an
adenoviral vector.
4. The method of claim 1, wherein said cancer is melanoma.
5. The method of claim 1, wherein said cancer is renal cell
carcinoma.
6. The method of claim 1, wherein said expression vector is
encapsulated in a liposome.
7. The method of claim 1, wherein said patient is human.
8. The method of claim 1, wherein said composition comprises
10.sup.10 expression vectors per ml.
9. The method of claim L, wherein said composition further
comprises an immunotherapeutic agent, genetic therapeutic,
cytokine, prodrug converting enzyme or anticancer agent.
10. The method of claim 1, wherein said composition further
comprises a second expression vector comprising a gene encoding an
immunotherapeutic agent, genetic therapeutic, cytokine or prodrug
converting enzyme.
11. The method of claim 1, wherein said expression vector further
comprises a second gene encoding an immunotherapeutic agent,
genetic therapeutic, cytokine or prodrug converting enzyme; wherein
said second gene is in the same reading frame as said gene encoding
p21.
12. A method of treating restenosis in a patient in need thereof
comprising administering in vivo a therapeutically effective amount
of a composition comprising: (i) an expression vector containing
the gene which encodes p21; and (ii) a pharmaceutical carrier.
13. A method of treating a cancer in a patient in need thereof
comprising administering in vivo a therapeutically effective amount
of a composition comprising: (i) an expression vector comprising a
gene encoding p21 fused to a gene encoding a prodrug converting
enzyme.
14. The method of claim 13, wherein said prodrug converting enzyme
is thymidine kinase, cytosine deaminase or
.beta.-glucurodinase.
15. The method of claim 14, wherein said composition further
comprises a pharmaceutically acceptable carrier.
16. The method of claim 14, wherein said expression vector is a
viral vector.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention provides methods for treating or
preventing restenosis and cancer in vivo by administration of a
composition comprising an expression vector containing a gene
encoding p21 and a pharmaceutical carrier.
[0003] 2. Discussion of the Background
[0004] The identification of cell cycle regulatory proteins has
been greatly facilitated by studies of mutant yeast strains with
abnormalities related to cell proliferation. Among the gene
products defined in yeast is Far 1 (1), whose mammalian homologue,
p21, alters the activity of cyclin-dependent kinases and is
implicated in cell cycle progression and senescence (2-13). p21,
also known as WAF1, CIP1 or SDI1 (11, 12, 14, 15), is a downstream
target of the p53 tumor suppressor gene and has thus been
implicated indirectly in malignant transformation (15-18).
Induction of p53 in response to DNA damage results in G1 checkpoint
arrest (16-19), at which point. DNA repair is accomplished prior to
DNA replication in S phase Consistent with its presumed role as a
downstream effector for p53, p21 has been shown to inhibit
proliferating cell nuclear antigen (PCNA) dependent DNA replication
but not DNA repair in vitro (20).
[0005] Zhang et al, Genes & Development (1994) 8:1750) studied
p21 in vitro. As p21 functions as a kinase inhibitor, it had been
predicted that normal cells should contain virtually no active
cyclin kinases. By demonstrating that p21-containing cyclin kinases
exist in both active and inactive states, Zhang et al rationalized
that p21 was involved in controlling cell cycle progression in
normal cells. Zhang et al found that in fibroblasts transformed
with a variety of tumor viral oncoproteins, cyclin kinases exist in
a binary state [cylcin/CDK]; whereas in normal fibroblasts multiple
cyclin kinases exist in quaternary complexes containing p21
[cyclin/CDK/proliferating cell nuclear antigen (PCNA)/p21]. Active
complexes contain a single p21 molecule. In contrast inactive
complexes posses multiple p21 subunits. Although changes in p21
stoichiometry were sufficient to account for the conversion of
active to inactive complexes in vitro, Zhang et al believed that
"association of cyclin knases with p21 must be intertwined with
other modes of regulation in vivo." Zhang et al noted that "it is
not known what effect association with noninhibitory levels of p21
might have on the function of these CDK-modifying enzymes in
vivo."
[0006] WO 94/09135 describes methods and diagnostic kits for
diagnosing transformation of a cell, involving detection of the
subunit components of cyclin complexes. In particular, the method
pertains to the interaction of cyclins, PCNA, CDKs and low
molecular weight polypeptides such as p21, p19 and p16.
[0007] Despite the evidence of cyclin kinase inhibitory activity in
vitro, the role of p21 in tumor formation and its ability to
reverse the malignant phenotype in vivo has not been defined.
SUMMARY OF THE INVENTION
[0008] Accordingly, one object of the present invention is to
provide methods for treating and preventing cancer (tumor
formation) in vivo.
[0009] A second object of the present invention is to provide
methods for treating and preventing restenosis in vivo.
[0010] A third object of the present invention is to provide
methods to induce antitumor effects in cells through induction of
terminal differentiation. This method is useful for altering
expression of cell surface proteins which might potentially
facilitate immune recognition of tumors or causing the secretion of
factors which might secondarily inhibit cell growth.
[0011] The present inventors have now determined the role of the
p21 cyclin-dependent kinase inhibitor on tumor cell growth and
restenosis. p21 is induced by p53 (6,7,15-18) and has thus been
implicated as a downstream effector of p53 tumor suppression (23).
The present inventors provide the first direct demonstration that
p21 expression is sufficient to produce these tumor and restenosis
suppressor effects in vivo. p21 expression was also found to
facilitate transcriptional activation by NF-.kappa.B providing a
mechanism whereby p21 can directly influence the expression of
genes, such as adhesion molecules, associated with differentiation.
The suppression of tumor growth and restenosis as well as the
induction of the differentiated phenotype arises from altered
patterns of gene expression, mediated in part by NF-kB, resulting
from p21 induced transcriptional regulation leading to terminal
differentiation and growth arrest. Previous attempts to induce
antitumor effects through induction of terminal differentiation
have involved the use of cytotoxic drugs or hormones (25-28) which
have had variable success in achieving this effect.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 (A) are graphs depicting the cell cycle analysis in
malignant cell lines and expression of p21 and (B) are western
blots of Renca cell lines transduced with adenoviral and eukaryotic
expression vectors.
[0013] FIG. 2 are graphs depicting the inhibition of tumor growth
following introduction of ADV p21 into Renca tumor cells followed
by inoculation. The presence of tumor (A,C) and tumor diameter
(B,D) were evaluated.
[0014] FIG. 3 are graphs depicting the effects of introduction of
ADV p21 into established Renca tumor cells in vivo inhibits tumor
growth. Tumor diameter was measured in two perpendicular dimensions
using calipers.
[0015] FIG. 4 are photographs depicting the in vitro effects of p21
on malignant cell growth and differentiation. Phase contrast
microscopy was performed on the indicated cells 5 days after the
indicated treatments. Magnification (20.times.).
[0016] FIG. 5 are graphs depiciting survival of mice with
established tumors treated with ADV p21 or control vectors. BALB c
mice (a,b) or nu/nu CD-1 mice (c,d) were injected with Renca cells
incubated in vitro with PBS (.quadrature.,.box-solid.), ADV-p21
(.diamond.,.diamond-solid.) ADV-.DELTA.E1
(.DELTA./.tangle-solidup.) at an MOI of 300.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] The present invention provides a method for treating cancer
or restenosis comprising administering to a patient in need thereof
a tumor inhibiting amount of a composition comprising:
[0018] (i) an expression vector containing the gene which encodes
p21 and
[0019] (ii) a pharmaceutically acceptable carrier.
[0020] The cDNA encoding p21 has been described by Xiong et al,
Nature 366:701 (1993), incorporated herein by reference.
[0021] Suitable expression vectors useful in accordance with the
present invention include eukaryotic and viral vectors. Useful
eukaryotic vectors include pRcRSV and pRcCMV or other RSV, CMV or
cellular enhancers and promoters driving expression of p21 with
various polyadenylate sequences. Preferably viral vectors are
used.
[0022] Viral vector systems have been indicated as highly efficient
in transferring genes to mammals containing deficient genes. See,
for example, Crystal Am. J. Med. 92(6A): 44S-52S (1992); Lemarchand
et al., Proc. Nat'l Acad. Sci. USA. 89(14):6482-6486 (1992),
incorporated herein by reference. Preferably, retroviral vectors
with impaired ability to replicate and transform are used. Suitable
viral vectors which express p21 useful in accordance with the
present invention include adenoviral vectors, Ad5-360 in
combination with pAd-BglII as described by Davidson et al, Nature
Gen. 3:219 (1993), (incorporated herein by reference). Preferably,
adenoviral vectors are used.
[0023] Preferred adenoviral vectors include: ADV described by
Davidson et al, Nature Gen. 3:219 (1993), (incorporated herein by
reference); or other adenovirus types, including types 7001, or
types 1 or 12 (as described by Ranheim et al, J. Virol. 67:2159
(1993); Green et al, Ann. Rev. Biochem. 39:701 (1970)).
[0024] The p21 can be inserted into these expression vectors and
used for cell transfection using conventional recombinant
techniques such as described by Sambrook, Fritsch, & Maniatis,
in "Molecular Cloning, A Laboratory Manual" (2d ed): pp. E.5. (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), the
disclosure of which is hereby incorporated by reference.
Alternatively, the expression vectors can be prepared using
homologous recombination techniques as described by Davidson et al,
1993, Nature Gen. 3:219-223 or Lemarchand et al. Proc. Nat'l Acad.
Sci. USA 89(14):6482-6486 (1992).
[0025] The expression vectors of the present invention can
additionally contain regulatory elements such as promoters and
selection markers such as antibiotic resistance genes.
[0026] It is well established that viral vectors will be taken up
in and integrated into cells in vivo and express the viral DNA,
including inserted constructs. See, e.g., Yoshimura et al. J. Biol.
Chem. 268(4):2300-2303 (1993); Crystal Am. J. Med. 92(6A):445-525
(1992); Lemarchand et al. Proc. Nat'l Acad. Sci. USA
89(14):6482-6486 (1992) the disclosures of which are hereby
incorporated by reference.
[0027] In an alternate embodiment, it is also understood that other
delivery systems besides expression vectors can be used to deliver
p21 protein. Principally, these techniques, including the use of
liposomes and DNA conjugates, are expected to provide similar
delivery yields as those provided by the expression vectors
discussed above. That is, rather than expressing the p21 gene via
an expression vector, it is also possible to incorporate a
therapeutic amount of p21 in a vehicle.
[0028] In a second alternate embodiment, p21 can be expressed as a
fusion protein. In this embodiment, the gene encoding p21 is fused
to a gene encoding an immunotherapeutic agent, genetic therapeutic
(such as HLA-B7), protein (such as cytokines, preferably, GM-CSF,
IL-2 and/or IL-12), prodrug converting enzymes (such as thymidine
kinase, cytosine deaminase and .beta.-glucurodinase) or anticancer
drug such as cis-platinum.
[0029] Fusion genes are proteins produced therefrom are described
in Molecular Cloning: A Laboratory Manual, Sambrook et al, 2nd
edition, Cold Spring Harbor Laboratory Press, 1989 (in particular,
chapter 17) incorporated herein by reference.
[0030] Thymidine kinase can be obtained as described in AU8776075,
incorporated herein by reference. .beta.-glucuronidase and fusion
proteins thereof are described in U.S. Pat. No. 5,268,463 and U.S.
Pat. No. 4,888,280, incorporated herein by reference. Cytosine
deaminase and fusion proteins thereof are described in WO 9428143,
incorporated herein by reference.
[0031] In addition combination therapies of viral vectors and
liposomes have also shown tremendous promise and are also
contemplated for use in the invention. Yoshimura et al, J. Biol.
Chem., 268(4):2300-2303 (1993), incorporated herein by
reference.
[0032] Liposomes are known to provide highly effective delivery of
active agents to diseased tissues. For example, pharmacological or
other biologically active agents have been effectively incorporated
into liposomes and delivered to cells. Thus, constructs in
accordance with the present invention can also be suitably formed
in liposomes and delivered to selected tissues. Liposomes prepared
from cationic lipids, such as those available under the trademark
LIPOFECTIN (Life Technologies, Inc., Bethesda, Md.) are preferred.
Particularly appealing to liposome based treatments is the fact
that liposomes are relatively stable and possess relatively long
lives, prior to their passage from the system or their metabolism.
Moreover, liposomes do not raise major immune responses.
[0033] Thus, in one aspect of the present invention a vector
Containing a gene encoding p21 is incorporated into a liposome and
used for the delivery of the construct to a specific tissue. The
liposome will aid the construct in transfecting a cell and becoming
expressed by the cell, ultimately generating p21 protein.
[0034] The composition of the present invention is a
therapeutically effective amount of a vector which expresses p21
and a pharmaceutically acceptable carrier. In order to administer
the viral vectors, suitable carriers, excipients, and other agents
may be incorporated into the formulations to provide improved
expression of p21.
[0035] A multitude of appropriate formulations can be found in the
formulary known to all pharmaceutical chemists: Remington's
Pharmaceutical Sciences, 15th Edition (1975), Mack Publishing
Company, Easton, Pa. 18042. (Chapter 87: Blaug, Seymour). These
formulations include for example, powders, pastes, ointments,
jelly, waxes, oils, lipids, anhydrous absorption bases,
oil-in-water or water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of a variety of molecular weights),
semi-solid gels, and semi-solid mixtures containing carbowax.
[0036] Any of the foregoing formulations may be appropriate in the
treatment with the viral vectors, provided that the viral particles
are inactivated in the formulation and the formulation is
physiologically compatible.
[0037] The amount of p21 to be administered will depend on the size
of the patient and the state to which the cancer has progressed. By
modifying the regulatory elements of the Vector using conventional
techniques or by varying the amount of viral vector titre
administered, the amount of p21 expression can be adjusted to the
patients needs. Typically, it is desirable to deliver approximately
50 viral vectors per cell to be treated. With the adenovirus,
formulations should generally contain on the order of 10.sup.10
viral infectious units per ml. With retrovirus, slightly different
titers may be applicable. See Woo et al, Enzyme 38:207-213 (1987),
incorporated herein by reference. Additional assistance in
determining appropriate dosage levels can be found in Kay et al,
Hum. Gene Ther. 3:641-647 (1992); Liu et al, Somat. Cell Molec.
Genet. 18:89-96 (1992); and Ledley et al, Hum. Gene Ther. 2:331-358
(1991), incorporated herein by reference.
[0038] Depending upon the particular formulation that is prepared
for the administration of the expression vectors, administration of
the compositions of the present invention can be accomplished
through a variety of methods. The composition of the present
invention are preferably administered by direct injection of the
expression vector (or liposome containing the same) into the tumor
such as described in U.S. Pat. No. 5,328,470, incorporated herein
by reference.
[0039] Breast, renal, melanoma, prostate, glioblastoma,
heptocarcinoma, colon and sarcoma cancer types can be treated in
accordance with the present invention. Methods of diagnosis and
monitoring these cancer types are well known in the art.
[0040] Arterial injury from angioplasty induces a series of
proliferative, vasoactive, and inflammatory responses which can
lead to restenosis. Although several factors have been defined
which stimulate this process in vivo, the role of specific cellular
gene products in limiting the response is not well understood. The
present inventors have now found that p21 acts to limit the
proliferative response to balloon catheter injury. Vascular
endothelial and smooth muscle cell growth was arrested through the
ability of p21 CKI to inhibit cyclin-dependent kinases and
progression through the G.sub.1 phase of the cell cycle. Restenosis
is a clinical condition which can be diagnosised and monitored as
described in Epstein et al, JACC 23(6):1278 (1994) and Landau et
al, Medical Progress 330(14):981 (1994), incorporated herein by
reference.
[0041] The compositions of the present invention can be used to
treat all mammals, in particular humans.
[0042] The compositions of the present invention can be
administered in combination with immunotherapeutic agents, genetic
therapeutics (such as HLA-B7), proteins (such as cytokines,
preferably, GM-CSF, IL-2 and/or IL-12), prodrug converting enzymes
(such as thymidine kinase, cytosine deaminase and
.beta.-glucurodinase) and anticancer drugs such as cis-platinum.
Alternatively, the compositions of the present invention can be
administered in combination with expression vectors comprising
genes encoding the above immuno-therapeutics, genetic therapeutics,
proteins, prodrug converting enzymes and anticancer drugs.
[0043] Alternatively, the compositions can be administered during
adoptive cell transfer therapy.
[0044] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Example 1
Use of P21 Cycin-Dependent Kinase Inhibitor to to Treat Restemosis
in vivo
[0045] In this study, the effect of p21 expression on endothelial
and smooth muscle cells in vitro and in a porcine model of arterial
balloon injury in vivo was analyzed.
Cell Culture and Transfection
[0046] Primary porcine vascular endothelial and smooth muscle cells
were derived from the aorta of 6-month-old domestic Yorkshire pigs
and were used between the second and fifth passage. Endothelial and
smooth muscle cells were grown to 70% confluence in medium 199 with
10% FBS. Cells were infected with ADV-p21 or ADV-.DELTA.E1 (MOI
300/cell) for 1 hour in DMEM and 2% FCS, and normal media was added
after 1 hour. Control cells were uninfected and carried in M199
with 10% FBS. Twenty-four hours later, the cells were split into 6
well dishes at 6.times.10.sup.4 cells per well. Cells were
harvested at 0, 2, 5, 7, and 10 days, and cell numbers were
determined by a hemocytometer. Cell viability was assessed by
trypan blue exclusion.
Cell Cycle Analysis
[0047] Cells were infected at an MOI of 300/cell with the
ADV-.DELTA.E1 or ADV-p21 vectors as described above, harvested,
washed with PBS twice, and then fixed in 701 ethanol (EtOH) (King
et al, Cell 79, 563-571 (1994)) for 30 minutes at 4.degree. C. The
cells were treated with 1U DNase-free RHase in 1 ml of PBS for 30
minutes at 37.degree. C., and resuspended in 0.05 mg/ml propidium
iodide (made as a 10.times. stock in PBS). Cells were analyzed by
flow cytometry using a FACScan model (Becton Dickinson).
Fluorescence measurements were accumulated to form a distribution
curve of DNA content. Fluorescence events due to debris were
subtracted before analysis.
Adenoviral Vectors
[0048] The recombinant adenoviral vector, ADV-p21, was constructed
by homologous recombination between sub360 genomic DNA, an Ad5
derivative with a deletion in the E3 region, and a p21 expression
plasmid, pAd-p21. Briefly, the pAd-p21 plasmid was prepared by
introducing the Hind III-XbaI fragment of a p21 expression vector
utilizing the Rous sarcoma virus promoter (RSV) to regulate
expression of p21 into the Bgl II site of pAd-Bgl II (Heichman
& Roberts, Cell 79, 557-562 (1994)). The structure of these
replication defective E1A, E1B deleted viruses was confirmed by
Southern blotting. All recombinant viruses were propagated in 293
cells and purified as described (Davidson et al., 1993, Nature Gen.
3:219-223). Cesium chloride purified virus was dialysed against
PBS, and diluted for storage in 13% glycerol-PBS solution to yield
a final concentration of 1-1.times.10.sup.12 viral particles/ml
(0.8-5.times.10.sup.10 pfu/ml). All stocks were sterilized with a
0.45 .mu.m filter and evaluated for the presence of replication
competent adenovirus by infection at a MOI of 10 onto 3T3 cells.
None of the stocks used in these experiments yielded
replication-competent virus.
Porcine Arterial Injury
[0049] After anesthesia and intubation, domestic Yorkshire pigs
(12-15 kg) underwent sterile surgical exposure of the iliofemoral
arteries, and a double-balloon catheter (C.R. Bard, Inc.) was
inserted into the iliofemoral artery. The proximal balloon was
inflated to a pressure of 500 mmHg, measured by an on-line pressure
transducer, for 5 minutes. Animals were sacrificed 1, 7, and 21
days after injury.
In Vivo Gene Transfer
[0050] Direct gene transfer was performed in the iliofemoral
arteries of Yorkshire pigs using a double balloon catheter as
described (Nabel et al, 1990, Science 249:1285-1288). In each
animal, both iliofemoral arteries were infected with the same
vector at a titer of 1.times.10.sup.10 pfu/ml, and 0.7 ml was used
in each animal (final dose of 7.times.10.sup.9 pfu) (Ohno et al,
1994, Science 265:781-784; Chang et al, 1995, Science
267:518-522).
[0051] The vessel segments infected with ADV-p21 (n=28 arteries) or
ADV-.DELTA.E1 (n=28 arteries) vectors were excised 7 or 21 days
later. To evaluate intimal cell proliferation, animals sacrificed
at 7 days received an intravenous infusion of
5-bromo-2'-deoxycytosine (BrdC) (Sigma, St. Louis, Mo.) 25 mg/kg
total dose, 1 hour prior to death. Each artery was processed in an
identical manner as described (Ohno et al, 1994, Science
265:781-784). All animal experiments were performed in accordance
with NIH guidelines and with approval of the University of Michigan
Committee in the Use and Care of Animals.
RT-PCR Analysis
[0052] Total RNA was prepared using Trizol reagents (GIBCO/BRL)
according to the manufacturer's protocol. Briefly, artery samples
were homogenized in Trizol reagent. RHA was precipitated with
ethanol (EtOH), washed in cold 75% EtOH three times, dried and
resuspended in RNAse-free TE buffer. PCR for the p21 gene was
performed (Muller et al, 1994, Circ. Res. 75:1039-1049) in the
presence or absence of reverse transcriptase (RT) with the primers:
5'-GAG ACA CCA CTG GAG GGT GAC TTC G-3' (sense); and 5'-GGG CAA ACA
ACA GAT GGC TGG CAA C-3' (antisense). The antisense primer was
specific for recombinant p21 RNA and not endogenous porcine p21
RNA.
Measurement of Cell Proliferation and Morphometry
[0053] Measurements of cell proliferation were made 7 days after
balloon injury and adenoviral infection using a monoclonal antibody
to BrdC. Arterial sections were fixed, embedded, and sectioned, and
immunohistochemistry using a monoclonal
anti-5-bromo-2'-deoxycytidine antibody was performed (Ohno et al,
1994, Science 265:781-784) to label nuclei in proliferating cells.
For each artery, the number of labeled and unlabeled nuclei in the
intima were quantitated using a microscope based video image
analysis system (Image One Systems, Universal Imaging Corporation,
Westchester, Pa.). A proliferation index was calculated as the
ratio of labeled cells to total number of cells.
[0054] Intimal and medial cross sectional areas were measured in 4
sections from each artery spanning the 2 cm region of arterial
injury and adenoviral infection with the image analysis system
(Ohno et al, 1994, Science 265:781-784). An intima to media (I/M)
area ratio for each artery was determined as the average I/M area
ratio of the 4 sections.
Immunohistochemistry
[0055] Immunohistochemical studies were performed with antibodies
to BrdC, smooth muscle .alpha.-actin, and p21, using methods as
described (Ohno et al, 1994, Science 265:781-784; Muller et al,
1994, Circ. Res. 75:1039-1049). The following primary antibodies
were used: a monoclonal mouse anti-BrdC antibody, 1:1000 dilution
(Amersham Life Sciences); a monoclonal mouse anti-smooth muscle a
actin antibody, 1:500 dilution (Boehringer Mannheim Biochemical);
and a polyclonal mouse anti-human p21 antibody, 1:1500 dilution
(Santa Cruz). Control experiments were performed using a purified
mouse IgG.sub.2b, antibody, 1:100 dilution (Promega), which did not
stain the arterial specimens. Slides were developed with either a
streptavidin-horseradish peroxidase complex (Vector Laboratories)
or a Vectastain ABC-alkaline phosphatase reagent (Vector
Laboratories), and counterstained in methyl green.
Statistical Analysis
[0056] Comparisons of intimal BrdC labeling index and I/M area
ratios between ADV-p21 and ADV-.DELTA.E1 arteries were made by
two-tailed, unpaired t-test. Statistical significance was assumed
if a null hypothesis could be rejected at the 0.05 level.
Results
Expression of p21 Inhibits Vascular Cell Proliferation and Induces
Cell Cycle Arrest in vitro
[0057] To study the effects of p21 on vascular cell growth and cell
cycle distribution, quiescent porcine vascular endothelial and
smooth muscle cells were infected in vitro with an adenoviral
vector, ADV-p21 or a control vector containing an E1 deletion,
ADV-.DELTA.E1 and then stimulated to proliferate by incubation in
10% FBS. Exposure of uninfected or ADV-.DELTA.E1 infected cells to
serum resulted in rapid proliferation of endothelial and smooth
muscle cells. In contrast, expression of p21 in vascular
endothelial and smooth muscle cells resulted in inhibition of cell
proliferation by >90%; these cells were still viable (>95%)
as assessed by trypan blue exclusion. Expression of p21 in vascular
endothelial and smooth muscle cells also resulted in accumulation
of cells in G.sub.0/G.sub.1, as assessed by propidium iodine
staining. These data suggest that cells were arrested in cell cycle
by p21 expression rather than p21 causing cell death.
p21 is Induced in Balloon Injured Arteries in vivo.
[0058] To investigate the potential of p21 to regulate vascular
cell growth in vivo, we first determined whether p21 expression is
induced in injured arteries. Porcine iliofemoral arteries were
either uninjured or injured by balloon angioplasty, and injured
segments were analyzed 1, 7, and 21 days later for p21 expression,
assessed by immunohistochemistry with a p21 antibody. This porcine
model of arterial injury results in intimal thickening by 3 weeks
(Ohno et al, 1994, Science 265:781-784). The lesion is
characterized by rapid smooth muscle cell proliferation during the
first 7 days after arterial injury, followed by expansion of the
intima due to elaboration of extracellular matrix during the
subsequent 2 weeks. Normal, uninjured porcine arteries expressed no
p21. One day following arterial injury, p21 protein was not present
in the intima; however, at 7 days, there was p21 protein in
approximately 50% of intimal smooth muscle cells. At 21 days, p21
expression was present in lower regions of the intima, next to the
internal elastic lamina, in regions where cell proliferation was
not present (Ohno et al, 1994, Science 265:781-784). Indeed, p21
expression in general was inversely correlated with smooth muscle
cell proliferation. These findings suggest that p21 expression is
associated with arrest of vascular cell proliferation in injured
arteries.
Expression of p21 in Injured Arteries Limits the Development of
Intimal Hyperplasia.
[0059] To assess the direct effect of p21 on vascular cell growth
in vivo, p21 vectors were introduced into porcine arteries
immediately following injury. The right and left iliofemoral
arteries of domestic pigs were balloon injured and infected with
ADV-p21 or ADV-.DELTA.E1 using a double-balloon catheter
(1.times.10.sup.10 pfu/ml, 0.7.times.100 pfu total dose). In vivo
gene transfer of ADV-p21 was demonstrated in injured porcine
arteries 7 days after infection by RT-PCR analysis. p21 RNA was
detected by RT PCR in infected left and right iliofemoral arteries
but not in a noninfected carotid artery from the same animal or in
ADV-.DELTA.E1 noninfected and infected arteries.
[0060] The effect of p21 expression on intimal cell growth in vivo
was next assessed by two methods, quantitating incorporation of
BrdC into intimal cells 7 days after gene transfer and measuring
I/M area ratios at 3 weeks. A 35% reduction in intimal BrdC
incorporation was observed in ADV-p21 infected arteries, compared
with ADV-.DELTA.E1 arteries, 7 days after gene transfer
(5.3.+-.0.9% vs. 8.1.+-.0.4%, p=0.035). These BrdC labeled intimal
cells costained with a monoclonal antibody to smooth muscle
.alpha.-actin, suggesting that inhibition of intimal smooth muscle
cell proliferation was present in ADV-p21 animals. A significant
reduction in I/M area ratio of 37% was observed in ADV-p21 infected
arteries, compared with ADV-.DELTA.E1 infected arteries
(0.37.+-.0.06 vs. 0.59.+-.0.06, p=0.015). These results suggest
that infection of arteries with ADV-p21 at the time of balloon
injury inhibits the proliferation of intimal smooth muscle cells
and significantly limits the development of a neointima.
Example 2
Use of P21 Cycin-Dependent-Kinase Inhibitor to Suppress
Tumorigenicity in vivo
[0061] In this study, the effect of p21 expression on tumor growth
in vitro and in a murine model in vivo was analyzed.
Cell Cycle Analysis
[0062] Cells were infected at an MOI of 200-300 with the
ADV-.DELTA.E1 or ADV-p21 vectors or transfected with the p21
expression vector by DNA/liposome complexes. The cells were
infected as above and harvested, washed with PBS twice, then fixed
in 70% EtoH for 30 minutes of 4.degree. C. The cells were treated
with 1U Dnase-free RNase in 1 ml of PBS for 30 minutes at
37.degree. C., and finally, resuspended in 0.05 mg/ml propidium
iodide (made as a 10.times. stock in PBS, and cells were analyzed
by flow cytometry using a FACScan model (Becton Dickinson)
Fluorescence measurements were accumulated to form a distribution
curve of DNA content. Fluorescence events due to debris were
subtracted before analysis.
Western Blot Detection of p21
[0063] 3-5.times.10.sup.6 cells were harvested at the time points
indicated, lysed with 1 ml of 50 mM Tris-Hcl (pH 6.8), 100 mM DTT,
2% SDS, 0.1% bromophenol blue, 10% glycerol, and boiled for 0.5
minutes. The samples were finally spun at 10,000 rpm for 5 minutes,
and supernatants were collected. 20 .mu.l were loaded into 15%
SDS-PAGE and blotted into nitrocellulose membrane. p21 protein was
visualized using an antipeptide rabbit polyclonal antibody (Santa
Cruz) together with an antirabbit horseradish peroxidase secondary
antibody and subsequent ECL chemiluminescent detection
(Amersham).
Gene Transfer of p21
[0064] Cells were maintained in Dulbecco's modified eagle medium
(DMEM) containing 10% fetal calf serum. The recombinant adenoviral
vector, ADV-p21, was constructed by homologous recombination
between sub360 genomic DNA, an Ad5 derivative with a deletion in
the E3 region, and a p21 expression plasmid, pAd-p21. These
recombinant adenoviral vectors have sequences in the E1A and E1B
region deleted, impairing the ability of this virus to replicate
and transform nonpermissive cells. Briefly, the pAd-p21 plasmid was
prepared by introducing the Nru I and Dra III fragment from
pRc/CMV-p21, kindly provided by Drs. D. Beach and G. Hannon (Xiong
et al, Nature 366, 701 (1993); Serano et al, Nature 366, 704
(1993)) into the Bgl II site of pAd-Bgl II (Davidson et al, Nature
Genet. 3, 219 (1994)) which had the left hand sequence of Ad5
genome, but not E1A and E1B. Virus was prepared as described
previously (Ohno et al, Science 265, 781 (1994). The structure of
these viruses was confirmed by Southern blotting. All recombinant
viruses were propagated in 293 cells and purified as described
(Davidson et al, Nature Genet. 3, 219 (1994)). Cesium chloride
purified virus was dialysed against PBS, and diluted for storage in
13% glycerol-PBS solution to yield a final concentration of
1-3.times.10.sup.12 viral particles/ml (0.8-5.times.10.sup.10
pfu/ml). All stocks were sterilized with a 0.45 .mu.m filter and
evaluated for the presence of replication competent adenovirus by
infection at a MOI of 10 onto 3T3 cells. None of the stocks used in
these experiments yielded replication-competent virus.
[0065] The eukaryotic expression plasmid, pRc/RSV p21, was prepared
by introduction of the p21 cDNA from pRc/CMV-p21 into pRc/RSV
(Invitrogen), and transfection of 293 cells performed by using
calcium phosphate transfection (Perkins et al., manuscript
submitted).
Bystander Assay
[0066] U373 human glioblastoma cells, kindly provided by Dr. K.
Murazko, were infected with ADV-p21 (MOI 200). One day later, cells
were trypsinized, counted, and mixed with the indicated number of
uninfected U373 cells. 10,000 cells for each mixed population were
plated into a 96 well disk. Five days later, the MTT assay (Mosman,
J. Immunol. Methods 65, 55 (1983)) was performed to determine the
proliferation rate of these cell populations.
Gene Transfer of p21 and Effect on Cell Cycle Progression in
Malignant Cells.
[0067] The effect of p21 on cell cycle distribution was determined
in tumor cell lines by infection with an adenoviral vector,
ADV-p21, or a similar E1 deletion virus with no recombinant p21,
ADV-.DELTA.E1. Expression of p21 in the adenoviral vector was
regulated by the CMV enhancer/promoter and bovine growth hormone
polyadenylation sequence. Expression of p21 within a representative
malignant cell line, the B16BL6 melanoma, resulted in an
accumulation of cells in the G.sub.0/G.sub.1 phase of the cell
cycle, suggesting arrest predominantly at the G1/S boundary (FIG.
1a). Recombinant p21 expression was confirmed in murine (Renca) or
human (293) renal cell carcinoma lines, and the murine (B16BL6)
melanoma cell line by using Western blot analysis. Readily
detectable protein expression from the adenoviral vector was
achieved -1 day after introduction of the gene (FIG. 1b, lanes 4,
5, 13, 14 vs. 1-3,10-12). In addition, a eukaryotic expression
plasmid regulated by the Rous sarcoma virus (RSV) enhancer/promoter
and bovine growth hormone polyadenylation site showed comparable
expression in 293 cells (FIG. 1b, lanes 7,9 vs. 6,8). In both
cases, expression of the recombinant protein correlated with
inhibition of cell division and other vectors with the same
regulatory elements did not show the effects of p21 described
here.
Differentiation and Morphologic Effects of p21.
[0068] When the effect of p21 on cell growth was examined in vitro,
tumor cells infected with ADV-p21 showed morphological changes,
such as an increased nuclear to cytoplasmic ratio, an increase in
adherence and growth arrest, consistent with a differentiated
phenotype (FIGS. 2,3). Human melanoma cells, UM-316, showed nuclear
condensation and a >4-fold increase in melanosome formation by
electron microscopy after infection with ADV-p21 (FIG. 2;
p<0.005 by the Wilcoxon rank sum test). In these cells, an
.about.5-fold increase in melanin production was observed within 2
days after gene transfer in cells and supernatant fractions in
vitro (FIG. 3).
[0069] In some lines, cell death was observed to follow terminal
differentiation after extended cell culture, but there was no
evidence of apoptosis, as determined by the pattern of DNA
fragmentation (FIG. 4a), propidium iodine staining or TdT
immunostaining. In addition, mixtures of uninfected and infected
cells showed a lack of bystander effect (FIG. 4b), suggesting that
gene transfer and expression in recipient cells was required and
that efficient infection of p21 is required to eradicate growth of
established tumors.
Inhibition of Tumor Cell Growth in vivo.
[0070] To assess the effect of p21 on the growth of malignant cells
in vivo, Renca cells were infected with ADV-p21, an ADV-.DELTA.E1
control, or incubated with phosphate buffered saline (PBS), and
inoculated into recipient mice. p21 expression completely
suppressed the growth of tumors in all animals inoculated with
2.times.10.sup.5 cells (FIGS. 5a,b). Because it remained possible
that expression of p21 could alter the immunogenicity of infected
cells and thus work through an immune mechanism, similar studies
were undertaken in CD-1 nu/lu immunodeficient mice. Similar
inhibition of tumor growth was observed in these animals (FIGS.
5c,d), consistent with a direct effect on cell proliferation.
[0071] To determine whether ADV-p2.1 could alter the growth of
established tumors, Renca tumor nodules (-0.5 cm) were injected
with either PBS, ADV-.DELTA.E1, or ADV-p21. Direct transfer of
adenoviral vectors encoding a human placental alkaline phosphatase
reporter into established tumors caused infection of up to 95% of
cells estimated by quantitative morphometry after 5 repeated daily
injections of 10.sup.9 PFU. This treatment also inhibited tumor
growth, and when injections were performed repetitively (5 daily
injections, repeated after one week), could lead to long-term cure
as determined by survival (>40 days) and the inability to detect
macroscopic tumor in mice with previously detectable nodules. In
both cases these results were statistically significant.
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[0099] Having now fully described the invention, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention as set forth herein.
Sequence CWU 1
1
2 1 25 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 gagacaccac tggagggtga cttcg 25 2 25 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 gggcaaacaa
cagatggctg gcaac 25
* * * * *