U.S. patent application number 11/316482 was filed with the patent office on 2006-10-26 for methods and compositions for the treatment of ocular diseases.
This patent application is currently assigned to Canji, Inc.. Invention is credited to G. William Demers.
Application Number | 20060239973 11/316482 |
Document ID | / |
Family ID | 22126207 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060239973 |
Kind Code |
A1 |
Demers; G. William |
October 26, 2006 |
Methods and compositions for the treatment of ocular diseases
Abstract
Methods and compositions for the tretament of ocular disease
with a cyclin dependent kinase inhibitor are provided.
Inventors: |
Demers; G. William; (San
Diego, CA) |
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: |
22126207 |
Appl. No.: |
11/316482 |
Filed: |
December 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10247136 |
Sep 18, 2002 |
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11316482 |
Dec 21, 2005 |
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09075505 |
May 8, 1998 |
6489305 |
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10247136 |
Sep 18, 2002 |
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Current U.S.
Class: |
424/93.2 ;
435/456; 514/44R |
Current CPC
Class: |
A61K 48/0008 20130101;
C12N 15/86 20130101; C12N 2710/10343 20130101; A61K 48/00 20130101;
A61K 48/005 20130101; C12N 7/00 20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/861 20060101 C12N015/861 |
Claims
1. A method for the treatment of an ocular disease comprising the
administration of a nucleotide sequence encoding a cyclin dependent
kinase inhibitor.
2. The method of claim 1, wherein the cyclin dependent kinase
inhibitor is p16 or p21.
3. The method of claim 2, wherein the cyclin dependent kinase
inhibitor is p21.
4. The method of claim 3, wherein p21 is provided in an viral
vector.
5. The method of claim 4 wherein the viral vector is an adenoviral
vector.
6. The method of claim 5 wherein the adenoviral vector is a human
adenovirus type 5 vector.
7. The method of claim 6 wherein the human adenovirus vector is a
replication deficient adenoviral vector.
8. The method of claim 7 wherein the replication deficient vector
is deleted in the E4 region.
9. The method of claim 8 wherein the vector retains E4 orf 6 and E4
orf 6/7 but does not encode functional E4 peptide.
10. The method of claim 1 wherein the ocular disease is selected
from the group consisting of glaucoma surgery failure,
proliferative vitreoretinopathy and age related macular
degeneration, retinopathy of prematurity, and diabetic
retinopathy.
11. The method of claim 1 wherein the cyclin dependent kinase
inhibitor is p21 or p16.
12. The method of claim 1, wherein said cyclin dependent kinase
inhibitor is p21.
Description
BACKGROUND OF THE INVENTION
[0001] Gene therapy has been proposed as an approach to the
treatment of ocular diseases by a number of investigators. Hermens
et al. (J. Neurosci. Methods 71:85-98 (1997)) disclosed the
injection of an adenoviral vector containing a lacZ gene as a
reporter gene into the central and peripheral nervous system of the
rat. In that system areas with a laminar structure such as the eye
demonstrated more widespread transgene expression. Ali et al. (Hum.
Mol. Genet. 5:591-5949 (1996)) disclosed the use of an
adeno-associated virus (AAV) as a vector carrying lacZ to transduce
all layers of the neuroretina as well as the retinal epithelium
following subretinal injection. Mashhour (Gene Ther. 1:122-126
(1994)) disclosed that injection of an adenovirus vector carrying
lacZ into the vitreous body, the anterior chamber, or the
peribulbar body of mice did not result in any detectable cytopathic
effect and was associated with endocytosis of viral particles in
corneal, photoreceptor, bipolar, ganglionic, and oculomotor muscle
cells, depending on the administration route.
[0002] The use of gene therapy to treat heritable diseases of the
eye in particular has been proposed by several researchers. For
example, Li et al. (Proc. Natl. Acad. Sci. U.S.A. 92:7700-7704
(1995)) disclosed the use of adenovirus-mediated transfer of human
beta-glucuronidase cDNA expressed under the control of a non-tissue
specific promoter injected intravitreally or subretinally to
reverse the pathological changes of lysosomal storage disease in
the eyes of mice with mucopolysaccharidosis VII.
[0003] Rescue of photoreceptors by gene therapy has been
demonstrated in several experimental systems. Cayouette and Gravel
(Hum. Gene Ther. 8:423-430 (1997)) disclosed adenovirus-mediated
gene transfer of ciliary neurotropic factor prevented photoreceptor
degeneration in the retinal degeneration (rd) mouse, an animal
model of retinitis pigmentosa. Bennett et al. (Hum. Gene Ther.
7:1763-1769 (1996)) disclosed the rescue of photoreceptor cells in
rd mice using a recombinant replication defective adenovirus
containing murine cDNA for beta phosphodiesterase. Dunaeif et al.
(Hum. Gene Ther. 6:1225-1229 (1995)) disclosed retroviral gene
transfer into retinal pigment epithelial cells followed by
transplantation into rat retina in an experimental rat model to
preserve photoreceptors.
[0004] The use of suicide genes delivered by recombinant viral
vectors to kill ocular cells has focused mainly on the use of the
herpes thymidine kinase gene. For example, Sakamoto, et al.
(Ophthalmology 102:1417-1424 (1995) described the inhibition of
experimental proliferative vitreoretinopathy by retroviral
vector-mediated transfer of the herpes simplex thymidine kinase
gene. Murata et al. (Ophthalmic Res. 29:242-251 (1997) described
the use of retroviral vectors to transfer the herpes simplex virus
thymidine kinase gene in a rabbit model of proliferative
vitreoretinopathy.
[0005] The role of tumor suppressor genes such as p16, p21, p53, or
RB in hyperproliferative diseases of the eye has been challenging
to elucidate. Studies of cell cycle regulation in the ocular lens
using transgenic mice have shown that inactivation of RB can cause
postmitotic lens fiber cells to enter the cell cycle. However, when
p53 is present, inactivation of RB in this cell type results in
cell death rather than continued proliferation. Although p53 is
known to upregulate expression of the cyclin-dependent kinase
inhibitor p21, overexpression of p21 in transgenic lens is not
sufficient to cause apoptosis in transgenic mouse lens (Fromm et
al. Dev. Genet. 20:276-287 (1997)). In vascular tissue, Chang et
al. (J. Clin. Invest. 96:2260-2268 (1995)) discloses that
adenovirus mediated overexpression of p21 inhibits vascular smooth
muscle cell (VSMC) proliferation in vitro by arresting VSMCs in the
G1 phase of the cell cycle. In addition, Chang, et al. demonstrated
that localized adenoviral delivery of p21 in conjunction with
balloon angioplasty significantly reduced neointima hyperplasia in
the rat carotid artery model of restenosis.
SUMMARY OF THE INVENTION
[0006] The present invention discloses methods and compositions for
the treatment of ocular diseases. In particular, the present
invention provides a method for the treatment of ocular
hyperproliferative diseases by the administration of cyclin
dependent kinase inhibitors. The present invention further provides
pharmaceutical formulations for the intracellular delivery of
cyclin dependent kinase inhibitors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 comprises FIGS. 1A and 1B, which depict the response
of ocular fibroblasts to infection with recombinant adenoviruses as
described in Example 3 herein. FIG. 1A indicates the response of
hofgon cells to a dose of 5.times.10.sup.8 TOCC viral particles
(rAd-p21). FIG. 1B represents the response of hofgon cells to a
dose of 5.times.10.sup.8 ZZCC viral particles (rAd-null).
[0008] FIG. 2 is a graph representing the 3H-thymidine
incorporation in fibroblasts exposed to recombinant adenoviruses
incorporating p16 (XTCC), p21 (TOCC) and RB56 (QLCC) sequences and
null control vector (ZZCC).
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0009] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entirety for all purposes as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference.
[0010] The present invention provides a method of treating ocular
disease by the administration of cyclin dependent kinase
inhibitors. Cyclin dependent kinase inhibitors may be administered
as proteins or by the administration of a recombinant vector
containing the cyclin dependent kinase inhibitor coding sequence
permitting intracellular expression of the cyclin dependent kinase
inhibitor coding sequence in the target cell. The present invention
further provides pharmaceutical formulations of cyclin dependent
kinase inhibitors and vectors containing the coding sequences for
cyclin dependent kinase inhibitors. Particularly preferred cyclin
dependent kinase inhibitors are p16 and p21. Particularly preferred
vectors include recombinant adenoviral vectors, plasmid vectors,
retroviral and herpes viral vectors.
[0011] The present invention demonstrates that the administration
of pharmaceutical formulations comprising cyclin dependent kinase
inhibitors are particularly effective in the treatment of diseases
of the eye associated with hyperproliferation where other
hyperproliferative agents, such as pRB56, are ineffective. The
present invention demonstrates the utility of cyclin dependent
kinase inhibitors, such as p21 and/or p16, are useful in the
treatment of ocular diseases, especially those associated with
hyperproliferation of fibroblasts and retinal pigmented epithelial
cells, as well as angiogenic diseases associated with the
proliferation of endothelial cells.
A. Preclinical Model
[0012] In the practice of the invention as exemplified herein,
recombinant adenoviral vectors encoding p16 (XTCC), p21 (TOCC) and
p56RB (QLCC) and a null control vector (ZZCC) prepared in
substantial accordance with the teaching of Example 1 below. As a
preclinical model of the treatment of ocular hyperproliferative
disease, three human ocular fibroblast (HOF) cell lines obtained
from different human donors were prepared and infected with the
recombinant adenoviral constructs in substantial accordance with
the teaching of Examples 2, 3 and 4 herein. The percentage of
cellular proliferation was determined by FACS analysis. As can be
seen from the data presented in Table 1 below, in each case the
vector containing the p21 or p16 transgene (TOCC or XTCC
respectively) was more effective than control vector (ZZCC) or the
vector containing the p56 RB transgene (QLCC) as measured by BrdU
labeling. A dose dependent inhibition of BrdU incorporation was
detected in each case. This was not limited to the kinase inhibitor
rAd-constructs, as the null control and RB56 rAd-constructs also
inhibited BrdU incorporation in a dose dependent manner.
TABLE-US-00001 TABLE 1 Inhibition of Human Ocular Fibroblasts Cell
Line Vector dose G1 (%) S (%) G2 (%) HOF-GON ZZCC 5 .times.
10.sup.8 49 48 3 HOF-GON QLCC 5 .times. 10.sup.8 52 43 5 HOF-GON
TOCC 5 .times. 10.sup.8 81 7 12 HOF-GON XTCC 5 .times. 10.sup.8 88
4 7 HOF-GON ZZCC 5 .times. 10.sup.9 67 3 0 HOF-GON QLCC 5 .times.
10.sup.9 79 13 7 HOF-GON TOCC 5 .times. 10.sup.9 91 0 9 HOF-GON
XTCC 5 .times. 10.sup.9 90 0 10 HOF-NEP ZZCC 5 .times. 10.sup.8 19
76 5 HOF-NEP QLCC 5 .times. 10.sup.8 21 74 5 HOF-NEP TOCC 5 .times.
10.sup.8 47 37 16 HOF-NEP ZZCC 5 .times. 10.sup.9 52 36 12 HOF-NEP
QLCC 5 .times. 10.sup.9 61 26 13 HOF-NEP TOCC 5 .times. 10.sup.9 81
0 19 HOF-SCH ZZCC 5 .times. 10.sup.8 40 53 6 HOF-SCH QLCC 5 .times.
10.sup.8 42 51 8 HOF-SCH XTCC 5 .times. 10.sup.8 83 5 12 HOF-SCH
ZZCC 5 .times. 10.sup.9 78 8 15 HOF-SCH QLCC 5 .times. 10.sup.9 83
6 11 HOF-SCH XTCC 5 .times. 10.sup.9 88 0 11
[0013] These data were confirmed by a second method,
.sup.3H-thymidine incorporation to assess cell proliferation. In
this assay a dose response was measured and compared to untreated
control cells. These data supported the BrdU incorporation assay in
that the p21 and p16 rAd constructs were able to inhibit
.sup.3H-thymidine incorporation at lower doses than the no
transgene or RB56 rAd constructs. The estimated ED.sub.50 was about
10 fold lower with the p16 and p21 vector constructs than rAd-null
or rAd-RB56. Also similar to the observation in the BrdU
incorporation assay, there was a dose-dependent inhibition of
3H-thymidine incorporation with the control rAd-null and rAd-RB56
constructs.
[0014] Additional experiments were performed comparing the activity
of rAd-p56RB, rAd-p21, rAd-p16 and rAd-p53 in additional human
ocular fibroblast cells. Treatment of fibroblasts with rAd
expressing p21, p16, or p53 resulted in dramatically reduced
percentage of cells incorporating BrdU and conversely retained a
higher percentage of cells in G0/G1 phase. Based on the GFP
analysis it can be estimated that at a dose of 1.times.10.sup.9
PN/ml all of the cells were expressing the respective transgene.
This was the second lot of TOCC to be tested and cells responded to
this lot similarly as to the first lot. Cells treated with QLCC did
not respond differently than cells treated with ZZCC. Cells derived
from two different human donors gave comparable responses to each
of the constructs. It is therefore expected that TOCB and other
vector constructs expressing p21 will be effective at inhibition of
S phase entry as demonstrated with TOCC. TABLE-US-00002 TABLE 2
Cell cycle analysis of HOF cells treated with rAd and released from
serum starvation. Vector % g0/g1 % s % g2/m HOFtol ut 37 60 4
HOFtol gfcb 1e8 39 57 4 HOFtol ftcb 1e8 47 49 4 HOFtol gfcb 1e9 50
45 5 HOFtol ftcb 1e9 78 14 8 HOFtol qlcc 1e8 38 57 4 HOFtol tocc
1e8 65 27 8 HOFtol xtcc 1e8 69 23 8 HOFtol zzcc 1e8 43 52 5 HOFtol
qlcc 1e9 44 50 7 HOFtol tocc 1e9 89 0 11 HOFtol xtcc 1e9 91 2 8
HOFtol zzcc 1e9 47 50 3 HOFcar ut 50 41 9 HOFcar gfcb 1e8 52 41 7
HOFcar ftcb 1e8 62 30 8 HOFcar gfcb 1e9 68 25 7 HOFcar ftcb 1e9 88
2 9 HOFcar qlcc 1e8 50 41 8 HOFcar tocc 1e8 82 9 10 HOFcar xtcc 1e8
80 11 9 HOFcar zzcc 1e8 62 31 6 HOFcar qlcc 1e9 69 23 8 HOFcar tocc
1e9 91 1 9 HOFcar xtcc 1e9 89 1 10 HOFcar zzcc 1e9 71 22 7
[0015] In summary, the treatment of these cells with QLCC
(rAD-RB56) did not demonstrate an effect greater than the control
vector, ZZCC. However, these data demonstrate that treatment of
human ocular cells with genes expressing cyclin dependent kinase
inhibitors such as p16 or p21 is an effective therapy for treatment
of ocular diseases.
B. Cyclin Dependent Kinase Inhibitors
[0016] The term cyclin dependent kinase inhibitors includes the
human, p27kip, p57kip2, p15ink4b, p18ink4c, p19ink4d, p16ink4a and
p21sdi wild-type proteins, homologous protein sequences from other
organisms, as well as any mutations or truncations thereof which
display essentially the same function as the wild-type
polynucleotide or protein sequence as well as polynucleotide
sequences encoding same.
[0017] 1. p16
[0018] The term "p16" is meant to refer to a 156 amino acid
polypeptide having the amino acid sequence provided below:
(Sequence. ID NO: 2): TABLE-US-00003
MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQ
VMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRA
GARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEG PSDIPD
The p16 molecule has been referred to in the literature by the
following names: CDKN2A, CDK4I, Hs.1174, MLM, p16, INK4, MTS1,
CMM2, CDKN2 and cyclin-dependent kinase inhibitor 2A. The human p16
genomic coding sequence is arranged in three exons on human
chromosome 9p. The human cDNA coding sequence is well known in the
literature (See Okamoto, A. et al., (1994) Proc. Natl. Acad. Sci.
U.S.A. 91 (23), 11045-11049) and is available as GenBank Accession
Number L27211
[0019] 2. p21
[0020] The wild type p21 protein is a 164 amino acid protein having
cell regulatory functions. The cDNA and protein sequence are
desribed in Smith, et al., U.S. Pat. No. 5,302,706 issued Apr. 12,
1994, the entire teaching of which is herein incorporated by
reference. p21 is also known in the scientific literature as
p21sdi, p21waf1, p21cip1 and p21pic1. The term p21 also includes
polynucleotide sequence encoding the human wild-type protein and
homologous sequences from other organisms, as well as any
mutations, truncations, or anti-sense nucleic acid which displays
essentially the same function as the wild-type polynucleotide or
protein sequence.
C. Delivery Systems
[0021] When the method of treatment to be employed is to introduce
a nucleotide sequence encoding p16 or p21, it is possible to
incorporate the naked plasmid into a cell However, when delivering
the p16 or p21 coding sequence to the target cell, it is preferred
that the plasmid be incorporated into a viral or non-viral delivery
system.
1. Non-Viral Delivery Systems
[0022] Examples of non viral delivery systems used to introduce the
p16 or p21 gene to the target cell include expression plasmids
capable of directing the expression of the therapeutic gene of
interest in the target cell. Expression plasmids are autonomously
replicating, extrachromosomal circular DNA molecules, distinct from
the normal genome and nonessential for cell survival under
nonselective conditions capable of effecting the expression of a
DNA sequence in the target cell. Plasmids autonomously replicate in
bacteria to facilitate bacterial production, but it is not
necessary that such plasmids containing the cyclin dependent kinase
gene replicate in the target cell in order to achieve the
therapeutic effect. The transgene may also be under control of a
tissue specific promoter region allowing expression of the
transgene only in particular cell types. Those of skill in the art
will readily appreciate the variety of expression plasmids which
may be useful in the practice of the present invention.
[0023] The expression plasmid may also contain promoter, enhancer
or other sequences aiding expression of the therapeutic gene and/or
secretion can also be included in the expression vector. Additional
genes, such as those encoding drug resistance, can be included to
allow selection or screening for the presence of the recombinant
vector. Such additional genes can include, for example, genes
encoding neomycin resistance, multi-drug resistance, thymidine
kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and
chloramphenicol acetyl transferase.
[0024] The expression plasmid containing the therapeutic gene may
be encapsulated in liposomes. Liposomes include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
dispersions, lamellar layers and the like. The delivery of DNA
sequences to target cells using liposome carriers is well known in
the art. A variety of methods are available for preparing
liposomes, as described in, e.g., Szoka et al. Ann. Rev. Biophys.
Bioeng. 9:467 (1980), Szoka, et al. U.S. Pat. No. 4,394,448 issued
Jul. 19, 1983, as well as U.S. Pat. Nos. 4,235,871, 4,501,728,
4,837,028, and 5,019,369, incorporated herein by reference.
Liposomes useful in the practice of the present invention may be
formed from one or more standard vesicle-forming lipids, which
generally include neutral and negatively charged phospholipids and
a sterol, such as cholesterol. Examples of such vesicle forming
lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC,
DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol carbamate and
other cationic lipids as disclosed in U.S. Pat. No. 5,650,096. The
selection of lipids is generally guided by consideration of, e.g.,
liposome size, acid lability and stability of the liposomes in the
blood stream. Additional components may be added to the liposome
formulation to increase serum half-life such as polyethylene glycol
coating (so called "PEG-ylation") as described in U.S. Pat. Nos.
5,013,556 issued May 7, 1991 and 5,213,804 issued May 25, 1993.
[0025] In order to insure efficient delivery of the therapeutic
gene to a particular tissue or organ, it may be advantageous to
incorporate elements into the non-viral delivery system which
facilitate cellular targeting. For example, a lipid encapsulated
expression plasmid may incorporate modified surface cell receptor
ligands to facilitate targeting. Although a simple liposome
formulation may be administered, the liposomes either filled or
decorated with a desired composition of the invention of the
invention can delivered systemically, or can be directed to a
tissue of interest, where the liposomes then deliver the selected
therapeutic/immunogenic peptide compositions. Examples of such
ligands includes antibodies, monoclonal antibodies, humanized
antibodies, single chain antibodies, chimeric antibodies or
functional fragments (Fv, Fab, Fab') thereof.
[0026] Alternatively, the DNA constructs of the invention can be
linked through a polylysine moiety to a targeting moiety as
described in Wu, et al. U.S. Pat. No. 5,166,320 issued Nov. 24,
1992 and Wu, et al, U.S. Pat. No. 5,635,383 issued Jun. 3, 1997,
the entire teachings of which are herein incorporated by
reference.
2. Viral Delivery Systems
[0027] In other instances, the DNA sequence is delivered by a viral
delivery system wherein the therapeutic gene of interest is
incorporated into a viral genome capable of infecting the target
cell and the gene is operably linked to expression and control
sequences such that the gene of interest is expressed under
appropriate conditions in the target cell. The vectors useful in
the practice of the present invention may also be derived from the
viral genomes. Vectors which may be employed include recombinantly
modified enveloped or non-enveloped DNA and RNA viruses, preferably
selected from baculoviridiae, parvoviridiae, picornaviridiae,
herpesveridiae, poxviridae or adenoviridiae. Chimeric vectors may
also be employed which exploit advantageous elements of each of the
parent vector properties (See e.g., Feng, et al. (1997) Nature
Biotechnology 15:866-870.) Such viral genomes may be modified by
recombinant DNA techniques to include the cyclin dependent kinase
inhibitor gene and may be engineered to be replication deficient,
conditionally replicating or replication competent. In the
preferred practice of the invention, the vectors are replication
deficient or conditionally replicating. Preferred vectors are
derived from the adenoviral, adeno-associated viral and retroviral
genomes. In the most preferred practice of the invention, the
vectors are replication incompetent vectors derived from the human
adenovirus genome. The transgene may also be under control of a
tissue specific promoter region allowing expression of the
transgene only in particular cell types.
[0028] It may be valuable in some instances to utilize or design
vectors to achieve introduction of the exogenous transgene in a
particular cell type. Certain vectors exhibit a natural tropism for
certain tissue types. For example, vectors derived from the genus
herpesviridiae have been shown to have preferential infection of
neuronal cells. Examples of recombinantly modified herpesviridiae
vectors are disclosed in U.S. Pat. No. 5,328,688 issued Jul. 12,
1994.
[0029] In other instances, to insure efficient delivery of the
therapeutic gene to a particular tissue or organ, it may be
advantageous to incorporate elements into the viral delivery system
which facilitate cellular targeting. Viral envelopes used for
packaging the constructs of the invention can be modified by the
addition of receptor ligands or antibodies specific for a receptor
to permit receptor-mediated endocytosis into specific cells (e.g.,
WO 93/20221, WO 93/14188; WO 94/06923). In some embodiments of the
invention, the DNA constructs of the invention are linked to viral
proteins, such as adenovirus particles, to facilitate endocytosis.
(See, e.g. Curiel, et al. (1991) Proc. Natl. Acad. Sci. U.S.A.
88:8850-8854). Cell type specificity or cell type targeting may
also be achieved in vectors derived from viruses having
characteristically broad infectivities by the modification of the
viral envelope proteins. For example, cell targeting has been
achieved with adenovirus vectors by selective modification of the
viral genome knob and fiber coding sequences to achieve expression
of modified knob and fiber domains having specific interaction with
unique cell surface receptors. Examples of such modifications are
described in Wickham, et al. (1997) J. Virol. 71(11):8221-8229
(incorporation of RGD peptides into adenoviral fiber proteins);
Arnberg, et al. (1997) Virology 227:239-244 (modification of
adenoviral fiber genes to achieve tropism to the eye and genital
tract); Harris and Lemoine (1996) TIG 12(10):400-405; Stevenson, et
al. (1997) J. Virol. 71(6):4782-4790; Michael, et al. (1995) Gene
Therapy 2:660-668 (incorporation of gastrin releasing peptide
fragment into adenovirus fiber protein); and Ohno, et al.(1997)
Nature Biotechnology 15:763-767 (incorporation of Protein A-IgG
binding domain into Sindbis virus). Also see U.S. Pat. Nos.
5,721,126 and 5,559,099, herein incorporated by reference. Other
methods of cell specific targeting have been achieved by the
conjugation of antibodies or antibody fragments to the envelope
proteins (see, e.g., Michael, et al. (1993) J. Biol. Chem.
268:6866-6869, Watkins, et al. (1997) Gene Therapy 4:1004-1012;
Douglas, et al.(1996) Nature Biotechnology 14: 1574-1578.
Alternatively, particular moieties may be conjugated to the viral
surface to achieve targeting (See, e.g. Nilson, et al. (1996) Gene
Therapy 3:280-286 (conjugation of EGF to retroviral proteins)).
[0030] Conditionally replicating viral vectors are used to achieve
selective expression in particular cell types while avoiding
untoward broad spectrum infection. Examples of conditionally
replicating vectors are described in Bischoff, et al.(1996) Science
274:373-376; Pennisi, E. (1996) Science 274:342-343; Russell, S. J.
(1994) Eur. J. of Cancer 30A(8):1165-1171. Additionally, the viral
genome may be modified to include inducible promoters which achieve
replication or expression of the transgene only under certain
conditions. Examples of inducible promoters are known in the
scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem.
Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J. Virol.
70(9):6054-6059; Hwang, et al.(1997) J. Virol. 71(9):7128-7131;
Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et
al.(1997) J. Biol. Chem. 272(46); 29364-29371). The transgene may
also be under control of a tissue specific promoter region allowing
expression of the transgene only in particular cell types.
[0031] In some instances, particularly when employing a
conditionally replicating or replication competent vector, it may
be desirable to include a suicide gene in the viral vector in
addition to the therapeutic gene. A suicide gene is a nucleic acid
sequence, the expression of which renders the cell susceptible to
killing by external factors or causes a toxic condition in the
cell. A well known example of a suicide gene is the thymidine
kinase (TK) gene (see e.g. Woo, et al. U.S. Pat. No. 5,631,236
issued May 20, 1997 and Freeman, et al. U.S. Pat. No. 5,601,818
issued Feb. 11, 1997) in which the cells expressing the TK gene
product are susceptible to selective killing by the administration
of gancyclovir. This provides a "safety valve" to the viral vector
delivery system to prevent widespread infection due to the
spontaneous generation of fully replication competent viral vectors
of broad range infectivity.
[0032] In the preferred practice of the invention, the vector is
derived from genus adenoviridiae. Particularly preferred vectors
are derived from the human adenovirus type 2 or type 5. Such
vectors are preferably are replication deficient by modifications
or deletions in the E1a and/or E1b coding regions. Other
modifications to the viral genome to achieve particular expression
characteristics or permit repeat administration or lower immune
response are preferred. More preferred are recombinant adenoviral
vectors having complete or partial deletions of the E4 coding
region, optionally retaining (or deleting) E4 ORF6 and ORF 6/7. The
E3 coding sequence has been demonstrated to be nonessential and may
be deleted from adenoviral vectors but is preferably retained. In
particular, it is preferred that the promoter operator region of E3
be modified to increase expression of E3 to achieve a more
favorable immunological profile for the therapeutic vectors. Most
preferred are human adenoviral type 5 vectors containing a DNA
sequence encoding a cyclin dependent kinase inhibitor under control
of the cytomegalovirus promoter region and the tripartite leader
sequence having E3 under control of the CMV promoter and deletion
of E4 coding regions while retaining E4 ORF6 and ORF 6/7. In the
most preferred practice of the invention as exemplified herein, the
cyclin dependent kinase inhibitor is p16 or p21.
3. Protein Delivery Systems
[0033] Alternatively to viral or non-viral delivery of p16 or p21
coding sequences, the p16 or p21 protein may also be administered
directly. When the protein is to be administered directly, it is
preferred that the protein be incorporated into a formulation which
facilitates or enhances the uptake of the protein into the target
ocular cell.
[0034] Formulations may include excipients which stabilize
polypeptides, such as methionine (U.S. Pat. No. 5,358,708);
osmolytes, lyotropic salts, water-soluble synthetic and natural
polymers, surfactants, sulfated polysaccharides, proteins, and
buffers (U.S. Pat. No. 5,580,856); fatty acids, amino acids,
vitamins (U.S. Pat. No. 5,078,997), and so on.
D. Pharmaceutical Formulation
[0035] The invention further provides pharmaceutical formulations
comprising the therapeutic gene in a viral or non-viral delivery
system for administration. The compositions of the invention will
be formulated for administration by manners known in the art
acceptable for administration to a mammalian subject, preferably a
human. In particular delivery systems may be formulated for
intramuscular, intravenous, injectable depot type devices or
topical administration.
[0036] The compositions of the invention can also be administered
in topical formulations or polymer matrices, hydrogel matrices,
polymer implants, or encapsulated formulations to allow slow or
sustained release of the compositions. A particularly preferred
formulation is a suspension or solution of the delivery system in a
topical ocular formulation, such as eye drops.
1. Carriers
[0037] When the delivery system is formulated as a solution or
suspension, the delivery system is in an acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers may be
used, e.g., water, buffered water, 0.8% saline, 0.3% glycine,
hyaluronic acid and the like. These compositions may be sterilized
by conventional, well known sterilization techniques, or may be
sterile filtered. The resulting aqueous solutions may be packaged
for use as is, or lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration.
[0038] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH adjusting and buffering agents, tonicity
adjusting agents, wetting agents and the like, for example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, sorbitan monolaurate, triethanolamine oleate,
etc.
[0039] The concentration of the compositions of the invention in
the pharmaceutical formulations can vary widely, i.e., from less
than about 0.1%, usually at or at least about 2% to as much as 20%
to 50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
2. Delivery Enhancers
[0040] The pharmaceutical formulations of the invention may
optionally include one or more delivery-enhancing agents, The term
"delivery enhancing agents" includes agents which facilitate the
transfer of the nucleic acid or protein molecule to the target
cell. Examples of such delivery enhancing agents detergents,
alcohols, glycols, surfactants, bile salts, heparin antagonists,
cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
Alcohols include for example the aliphatic alcohols such as
ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol.
Glycols include glycerine, propyleneglycol, polyethyleneglycol and
other low molecular weight glycols such as glycerol and
thioglycerol. Acetates such as acetic acid, gluconic acid, and
sodium acetate are further examples of delivery-enhancing agents.
Hypertonic salt solutions like 1M NaCl are also examples of
delivery-enhancing agents. Examples of surfactants are sodium
dodecyl sulfate (SDS) and lysolecithin, polysorbate 80,
nonylphenoxypolyoxyethylene, lysophosphatidylcholine,
polyethylenglycol 400, polysorbate 80, polyoxyethylene ethers,
polyglycol ether surfactants and DMSO. Bile salts such as
taurocholate, sodium tauro-deoxycholate, deoxycholate,
chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid
and other astringents like silver nitrate may be used.
Heparin-antagonists like quaternary amines such as protamine
sulfate may also be used. Cyclooxygenase inhibitors such as sodium
salicylate, salicylic acid, and non-steroidal antiinflammatory drug
(NSAIDS) like indomethacin, naproxen, diclofenac may be used.
[0041] Detergents include anionic, cationic, zwitterionic, and
nonionic detergents. Exemplary detergents include but are not
limited to taurocholate, deoxycholate, taurodeoxycholate,
cetylpyridium, benalkonium chloride, ZWITTERGENT.RTM. 3-14
detergent, CHAPS
(3-{(3-Cholamidopropyl)dimethylammoniol}-1-propanesulfonate
hydrate, Aldrich), Big CHAP, Deoxy Big CHAP, TRITON.RTM.-X-100
detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC.RTM.-F68
detergent, TWEEN.RTM. 20 detergent, and TWEEN.RTM. 80 detergent
(CALBIOCHEM.RTM. Biochemicals).
[0042] The concentration of the delivery-enhancing agent will
depend on a number of factors known to one of ordinary skill in the
art such as the particular delivery-enhancing agent being used, the
buffer, pH, target tissue or organ and mode of administration. The
concentration of the delivery-enhancing agent will be in the range
of 1% to 50% (v/v), preferably 10% to 40% (v/v) and most preferably
15% to 30% (v/v). Preferably, the detergent concentration in the
final formulation administered to the patient is about 0.5-2.times.
the critical micellization concentration (CMC).
[0043] In order to facilitate the improved gene transfer for
nucleic acid formulations comprising commercial Big-CHAP
preparations, the concentration of Big CHAP will vary based on its
commercial source. When the Big CHAP is sourced from
CALBIOCHEM.RTM., it is preferred that the concentration be in a
range of 2 to 10 millimolar. More preferred is 4 to 8 millimolar.
Most preferred is approximately 7 millimolar.
[0044] When the Big CHAP is sourced from Sigma, it is preferred
that the concentration of Big CHAP be in a range of 15 to 35
millimolar. More preferred is 20 to 30 millimolar. Most preferred
is approximately 25 millimolar.
[0045] In a further embodiment of the invention, delivery-enhancing
agents having Formula I are provided: ##STR1## wherein n is an
integer from 2-8, X1 is a cholic acid group or deoxycholic acid
group, and X2 and X3 are each independently selected from the group
consisting of a cholic acid group, a deoxycholic acid group, and a
saccharide group. At least one of X2 and X3 is a saccharide group.
The saccharide group may be selected from the group consisting of
pentose monosaccharide groups, hexose monosaccharide groups,
pentose-pentose disaccharide groups, hexose-hexose disaccharide
groups, pentose-hexose disaccharide groups, and hexose-pentose
disaccharide groups. In one preferred embodiment, the compounds of
the present invention have the Formula II: ##STR2## wherein X1 and
X2 are selected from the group consisting of a cholic acid group
and a deoxycholic acid group and X3 is a saccharide group.
[0046] These compounds are preferably used in the range of about
0.002 to 2 mg/ml, more preferably about 0.02 to 2 mg/ml, most
preferably about 0.2 to 2 mg/ml in the formulations of the
invention. Most preferred is approximately 2 mg/ml.
[0047] Phosphate buffered saline (PBS) is the preferred
solubilizing agent for these compounds. However, one of ordinary
skill in the art will recognize that certain additional excipients
and additives may be desirable to achieve solubility
characteristics of these agents for various pharmaceutical
formulations. For example, the addition of well known solubilizing
agents such as detergents, fatty acid esters, surfactants may be
added in appropriate concentrations so as to facilitate the
solubilization of the compounds in the various solvents to be
employed. When the solvent is PBS, a preferred solubilizing agent
is Tween 80 at a concentration of approximately 0.15%.
[0048] These delivery-enhancing compounds may be used alone, in
combination with each other, or in combination with another
delivery-enhancing agent.
E. Diseases Amenable to Treatment
[0049] The formulations of the present invention are useful in the
treatment of ocular diseases. The term ocular diseases includes but
is not limited to diseases of the eye associated with the
hyperproliferation cells in the eye. Examples of hyperproliferative
disorders include glaucoma surgery failure and proliferative
vitreoretinopathy. Other ocular diseases associated with excessive
angiogenesis such as age related macular degeneration, retinopathy
of prematurity, and diabetic retinopathy may also be treated in
accordance with the practice of the present invention.
[0050] Proliferative vitreoretinopathy describes a condition
whereby the retina of eye is pulled away from the wall of the eye
caused by a hyperproliferation of cells of the retinal pigmented
epithelium in response to the injury which caused the retinal
detachment. Subsequent contraction of the cellular membrane in the
time course of proliferative vitreoretinopathy is a primary cause
of failure of retinal reattachment surgery.
[0051] Glaucoma results from an abnormally high pressure in the
eye. The commonly accepted surgical treatment for glaucoma is to
provide a drain for excess vitreous humor from the eye to relieve
the excess pressure. A significant complication of this surgery
results from the reocclusion of the drain site from the
hyperproliferation of the fibroblasts resulting in repeated
surgical intervention.
[0052] The present invention provides methods and compositions for
the treatment of "glaucoma surgery failure" by administration of
p21 or p16 prevent hyperproliferation in these tissues surrounding
the drainage site to prevent the occlusion of the drainage
duct.
F. Methods of Administration
[0053] The therapeutic agents of the invention can be introduced
into the tissue of interest in vivo or ex vivo by a variety of
methods. In some embodiments of the invention, the therapeutic
agent is introduced to cells by such methods as liposome fusion,
injection, topically or biolistics. In some embodiments of the
invention, the compositions of the invention can be administered
directly into the eye, such as to the intraophthalmic artery,
subretinal, intravitreal or subconjunctival space. The formulations
of the present invention may be administered to subconjunctival and
scleral tissues at the time of surgery, preferably in the form of
topical drop formulations or with depot devices such as a gel foam
(Weck cell) sponge. Additionally, the formulations of the present
invention may be administered following surgery, by subconjuctival
injection. The formulations of the present invention may be
administered in a single dose or in multiple doses. Preferably,
when doses are administered, the factor determining the frequency
of repeat administration is the duration of transgene
expression.
[0054] In the case of glaucoma surgery failure and proliferative
vitreoretinopathy, the formulations of the present invention are
preferably administered at the time of surgery. In the case of
angiogenic diseases such as age related macular degeneration and
diabetic retinopathy, the formulations of the present invention are
administered over a course of treatment ranging from weeks to
years. The preferred routes of administration for the treatment of
such diseases include ophthalmic artery administration, subretinal
injection, intravitreal injection. Sustained release formulations
such as implants would also be appropriate for the treatment of
such long term disease indications. These formulations may also be
administered in combination with other anti-angiogenic agents.
[0055] Subretinal injections for the treatment of retinal
proliferative disease, especially proliferative vitreoretinopathy.
This procedure may be performed before, during or after surgery,
preferably during the surgical procedure.
[0056] Alternatively, angiogenic or proliferative diseases of the
eye (especially the cells of the retinal pigment epithelium) may be
treated by the intra-arterial, or subretinal, administration of
viral or non-viral formulations of the present invention. In
particular, viral or non-viral formulations may be delivered via
the opthalmic artery to enhance delivery to the target tissue.
1. Therapy
[0057] In one embodiment of the invention, a DNA sequence encoding
a cyclin dependent kinase inhibitor such as p27kip, p57kip2,
p15ink4b, p18ink4c, p19ink4d p16ink4a or p21sdi is administered to
the eye. In some embodiments the cyclin dependent kinase inhibitor
gene or polypeptide is provided in combination with each other
and/or one or more of the following genes or proteins: p53, RB, a
suicide gene or gene product.
[0058] In some embodiments of the invention, the formulations of
the present invention may also be administered in combination with
other chemotherapeutic agents. Examples of chemotherapeutic agents
useful in the practice of the present invention includes
fluorouracil and mitomycin-C. Additional agents, such
corticosteroids, may also be administered in the course
therapy.
[0059] In some embodiments of the invention, therapeutic
polypeptides of the invention are administered directly to a
patient in need of treatment. A "therapeutically effective" dose is
an amount of therapeutic gene or polypeptide sufficient to prevent
or reduce severity of the pathogeneis of the disease.
[0060] In some embodiments of the invention, the compositions of
the invention are administered ex vivo to cells or tissues
explanted from a patient, then returned to the patient. Examples of
ex vivo administration of therapeutic gene constructs include
Arteaga et al. (1996) Cancer Research 56(5):1098-1103; Nolta et al.
(1996) Proc. Natl. Acad. Sci. U.S.A. 93(6):2414-9; Koc et al.
(1996) Seminars in Oncology 23(1):46-65; Raper et al. (1996) Annals
of Surgery 223(2):116-26; Dalesandro et al. (1996) J. Thorac.
Cardi. Surg. 11(2):416-22; and Makarov et al. (1996) Proc. Natl.
Acad. Sci. U.S.A. 93(1):402-6.
G. Dosage Ranges
[0061] Therapeutically effective amounts of the pharmaceutical
composition comprising a therapeutic gene, such as p16 or p21 in a
recombinant viral vector delivery system formulated in a buffer
comprising a delivery-enhancing agent will be administered in
accord with the teaching of this invention. For example,
therapeutically effective amounts of the p16 or p21 cyclin
dependent kinase inhibitor gene in a recombinant adenoviral vector
formulated in a buffer optionally containing a delivery-enhancing
agent are in the range of about 1.times.10.sup.8 particles/ml to
1.times.10.sup.12 particles/ml, more typically about
1.times.10.sup.8 particles/ml to 5.times.10.sup.11 particles/ml,
most typically 1.times.10.sup.9 particles/ml to 1.times.10.sup.11
particles/ml (PN/ml).
EXAMPLES
[0062] The following examples provide the methodology and results
of experiments demonstrating the recombinant adenoviruses (rAd)
that express the p16 and/or p21 cell cycle control genes to inhibit
proliferation of target cells. As will be apparent to those skilled
in the art to which the invention pertains, the present invention
may be embodied in forms other than those specifically disclosed
above, without departing from the spirit or essential
characteristics of the invention. The particular embodiments of the
invention described below, are therefore to be considered as
illustrative and not restrictive.
[0063] The following examples are intended to illustrate, not limit
the scope of this invention. In the following examples, "g" means
grams, "ml" means milliliters, "mol" means moles, "EC" means
degrees Centigrade, "min." means minutes, "FBS" means fetal bovine
serum, and "PN" specifies particle number. All temperatures are in
degrees Centigrade unless otherwise specified.
Example 1
Construction of Recombinant Adenoviral Vectors
[0064] Recombinant adenoviruses were constructed to express the p16
and p21 coding sequences following infection of primary ocular
fibroblasts. The DNA sequence encoding p16 employed in the
construction of the present recombinant adenoviral vectors was
(Sequence ID No: 1): TABLE-US-00004 ATG GAG CCT TCG GCT GAC TGG CTG
GCC ACG GCC GCG GCC CGG GGT CGG GTA GAG GAG GTG CGG GCG CTG CTG GAG
GCG GGG GCG CTG CCC AAC GCA CCG AAT AGT TAC GGT CGG AGG CCG ATC CAG
GTC ATG ATG ATG GGC AGC GCC CGA GTG GCG GAG CTG CTG CTG CTC CAC GGC
GCG GAG CCC AAC TGC GCC GAC CCC GCC ACT CTC ACC CGA CCC GTG CAC GAC
GCT GCC CGG GAG GGC TTC CTG GAC ACG CTG GTG GTG CTG CAC CGG GCC GGG
GCG CGG CTG GAC GTG CGC GAT GCC TGG GGC CGT CTG CCC GTG GAC CTG GCT
GAG GAG CTG GGC CAT CGC GAT GTC GCA CGG TAC CTG CGC GCG GCT GCG GGG
GGC ACC AGA GGC AGT AAC CAT GCC CGC ATA GAT GCC GCG GAA GGT CCC TCA
GAC ATC CCC GAT TGA
[0065] The DNA sequence encoding p21 employed in the construction
of the present recombinant adenoviral vectors was as follows
(Sequence ID No: 3): TABLE-US-00005 ATG TCA GAA CCG GCT GGG GAT GTC
CGT CAG AAC CCA TGC GGC AGC AAG GCC TGC CGC CGC CTC TTC GGC CCA GTG
GAC AGC GAG CAG CTG AGC CGC GAC TGT GAT GCG CTA ATG GCG GGC TGC ATC
CAG GAG GCC CGT GAG CGA TGG AAC TTC GAC TTT GTC ACC GAG ACA CCA CTG
GAG GGT GAC TTC GCC TGG GAG CGT GTG CGG GGC CTT GGC CTG CCC AAG CTC
TAC CTT CCC ACG GGG CCC CGG CGA GGC CGG GAT GAG TTG GGA GGA GGC AGG
CGG CCT GGC ACC TCA CCT GCT CTG CTG CAG GGG ACA GCA GAG GAA GAC CAT
GTG GAC CTG TCA CTG TCT TGT ACC CTT GTG CCT CGC TCA GGG GAG CAG GCT
GAA GGG TCC CCA GGT GGA CCT GGA GAC TCT CAG GGT CGA AAA CGG CGG CAG
ACC AGC ATG ACA GAT TTC TAC CAC TCC AAA CGC CGG CTG ATC TTC TCC AAG
AGG AAG CCC TAA
[0066] The DNA sequence encoding p56-Rb employed in the
construction of the present recombinant adenoviral vectors was as
follows (Sequence ID No: 5): TABLE-US-00006 ATG AAC ACT ATC CAA CAA
TTA ATG ATG ATT TTA AAT TCT GCA AGT GAT CAA CCT TCA GAA AAT CTG ATT
TCC TAT TTT AAC AAC TGC ACA GTG AAT CCA AAA GAA AGT ATA CTG AAA AGA
GTG AAG GAT ATA GGA TAC ATC TTT AAA GAG AAA TTT GCT AAA GCT GTG GGA
CAG GGT TGT GTC GAA ATT GGA TCA CAG CGA TAC AAA CTT GGA GTT CGC TTG
TAT TAC CGA GTA ATG GAA TCC ATG CTT AAA TCA GAA GAA GAA CGA TTA TCC
ATT CAA AAT TTT AGC AAA CTT CTG AAT GAC AAC ATT TTT CAT ATG TCT TTA
TTG GCG TGC GCT CTT GAG GTT GTA ATG GCC ACA TAT AGC AGA AGT ACA TCT
CAG AAT CTT GAT TCT GGA ACA GAT TTG TCT TTC CCA TGG ATT CTG AAT GTG
CTT AAT TTA AAA GCC TTT GAT TTT TAC AAA GTG ATC GAA AGT TTT ATC AAA
GCA GAA GGC AAC TTG ACA AGA GAA ATG ATA AAA CAT TTA GAA CGA TGT GAA
CAT CGA ATC ATG GAA TCC CTT GCA TGG CTC TCA GAT TCA CCT TTA TTT GAT
CTT ATT AAA CAA TCA AAG GAC CGA GAA GGA CCA ACT GAT CAC CTT GAA TCT
GCT TGT CCT CTT AAT CTT CCT CTC CAG AAT AAT CAC ACT GCA GCA GAT ATG
TAT CTT TCT CCT GTA AGA TCT CCA AAG AAA AAA GGT TCA ACT ACG CGT GTA
AAT TCT ACT GCA AAT GCA GAG ACA CAA GCA ACC TCA GCC TTC CAG ACC CAG
AAG CCA TTG AAA TCT ACC TCT CTT TCA CTG TTT TAT AAA AAA GTG TAT CGG
CTA GCC TAT CTC CGG CTA AAT ACA CTT TGT GAA CGC CTT CTG TCT GAG CAC
CCA GAA TTA GAA CAT ATC ATC TGG ACC CTT TTC CAG CAC ACC CTG CAG AAT
GAG TAT GAA CTC ATG AGA GAC AGG CAT TTG GAC CAA ATT ATG ATG TGT TCC
ATG TAT GGC ATA TGC AAA GTG AAG AAT ATA GAC CTT AAA TTC AAA ATC ATT
GTA ACA GCA TAC AAG GAT CTT CCT CAT GCT GTT CAG GAG ACA TTC AAA CGT
GTT TTG ATC AAA GAA GAG GAG TAT GAT TCT ATT ATA GTA TTC TAT AAC TCG
GTC TTC ATG CAG AGA CTG AAA ACA AAT ATT TTG CAG TAT GCT TCC ACC AGG
CCC CCT ACC TTG TCA CCA ATA CCT CAC ATT CCT CGA AGC CCT TAC AAG TTT
CCT AGT TCA CCC TTA CGG ATT CCT GGA GGG AAC ATC TAT ATT TCA CCC CTG
AAG AGT CCA TAT AAA ATT TCA GAA GGT CTG CCA ACA CCA ACA AAA ATG ACT
CCA AGA TCA AGA ATC TTA GTA TCA ATT GGT GAA TCA TTC GGG ACT TCT GAG
AAG TTC CAG AAA ATA AAT CAG ATG GTA TGT AAC AGC GAC CGT GTG CTC AAA
AGA AGT GCT GAA GGA AGC AAC CCT CCT AAA CCA CTG AAA AAA CTA CGC TTT
GAT ATT GAA GGA TCA GAT GAA GCA GAT GGA AGT AAA CAT CTC CCA GGA GAG
TCC AAA TTT CAG CAG AAA CTG GCA GAA ATG ACT TCT ACT CGA ACA CGA ATG
CAA AAG CAG AAA ATG AAT GAT AGC ATG GAT ACC TCA AAC AAG GAA GAG AAA
TGA
[0067] A viral vector backbone was created based on a human
adenovirus type 5 genome comprising deletions of the E1a and E1b
and protein IX gene functions and partial deletion of the E4 coding
region (retaining the function of the E4 orf 6 and E4 orf 6/7
genes). The recombinant viral vectors for expression of p16 and p21
were constructed as described in Wang, et al. (1997) Cancer
Research 57:351-354. The recombinant viral vectors for expression
of p56 was constructed as described in Smith, et al. (1997)
Circulation 96(6):1899-1905. This sequence was inserted into the
viral vector backbone so as to be under control of the CMV promoter
element. The resulting vector was designated QLCC.
Example 2
Preparation of Target Cells
[0068] Human ocular fibroblast were used in in vitro assays as they
are the cellular component causing pathology in glaucoma surgery
failure. Primary ocular fibroblast cell lines from three different
human sources (HOF-gon, HOF-nep and HOF-sch) were obtained from by
Drs. S. Schwartz, D. Farber, and S. Ogueta, Jules Stein Eye
Institute, University of California Los Angeles. Cells were
synchronized in G1 by incubation in low serum containing HAMs
F12/DME media (commercially available from Irvine Scientific,
Irvine Calif.) containing 0.5% FBS for a period of 3 days.
Example 3
Evaluation of Activity in a Human Ocular Fibroblasts Model
[0069] Human ocular fibroblast cells (prepared in substantial
accordance with the teaching of Example 2 above) were infected with
rAd constructs (prepared in substantial accordance with the
teaching of Example 1 above) in low serum media (F12/DME media
containing 0.5% FBS) for 20-24% with a dose of either
5.times.10.sup.8 and 5.times.10.sup.9 adenoviral particles.
[0070] The cells were stimulated to enter the cell cycle by
incubation in complete media (10% FBS) and assayed 18-24 hours
after stimulation. Response to rAd mediated gene expression was
measured by 3H-thymidine incorporation or by BrdU incorporation.
Plates were rinsed and re-fed with complete media (10% FBS). 16 h
after release BrdU was added for 5 h and cells were harvested and
analyzed by FACS for BrdU incorporation (DNA synthesis) and PI
staining (DNA content). Cells that had incorporated BrdU were
stained using a FITC conjugated mAb to BrdU (commercially available
from Becton-Dickinson) and detected by FACS analysis.
[0071] Bivariate analysis on DNA content and BrdU positive cells
was used to analyze the data. The response to treatment comparing
Ad-null (ZZCC) with rAd-p21 (TOCC) are represented in the
two-dimensional plot is shown in FIG. 1. The X-axis (FL2-A)
corresponds to propidium iodide (PI) staining or DNA content. The
Y-axis (FL1-H) corresponds to FITC staining or BrdU incorporation.
Therefore, in the lower right quadrant are cells that did not exit
gl phase during labeling with BrdU. The cells in the arc from the
lower left to the upper right quadrants represent cells in the
process of BrdU incorporation at the time of harvest. Cells in the
upper left quadrant had incorporated BrdU during the labeling
period and then divided.
[0072] The percent of cells remaining in G1 was determined from the
lower left quadrant. The percent of cells in S phase was determined
from the both upper quadrants and therefore represent the percent
cells in S-phase during labeling. The cells in the lower right
quadrant are labeled G2. Two doses of virus were tested,
5.times.10.sup.8 and 5.times.10.sup.9 particles per ml of media.
The from the analysis of human ocular fibroblasts (HOF-) from 3
human donors (GON, NEP and SCH) and data is summarized in Table 1
above. Dose response to rAd treatment measured by tritiated
thymidine incorporation shown in FIG. 2.
Example 4
Construction of Additional Viral Vectors
[0073] A viral vector backbone was created based on a human
adenovirus type 5 genome comprising deletions of the E1a and E1b
and protein IX gene functions and partial deletion of the E3 coding
region. Specifically, the deletions of base pairs 355 to 3325 was
used to eliminate E1a and E1b functions, deletion of base pairs
3325 to 4021 was used to eliminate protein IX function and
deletions of 28592 to 30470 were used to eliminate E3 functions.
See Wills, et al. (1994) Human Gene Therapy 5:1079-1088. The DNA
sequence encoding the cytomegalovirus immediate early promoter
without the presence of the CMV promoter intron was inserted into
the rAd viral genome. This vector without an exogenous transgene
was used as control vector and was designated ZZCB.
[0074] The green fluorescent protein (GFP) coding sequence was
obtained as a NheI to BclI restriction endonuclease cleavage
fragment from the vector pEGFP-C1 (commercially available from
CloneTech). This sequence was inserted into the XbaI to BamHI site
of the resulting vector was designated GFCB.
[0075] The recombinant viral vectors for expression of p53 were
constructed as described in Wills, et al., supra.
Example 5
Comparison of p53, p56RB and p21 Activity
[0076] Human Ocular Fibroblast (HOF) cells obtained from 2 human
donors (HOFtol and HOFcar) were prepared in substantial accordance
with Example 2 above. The GFCB and FTCB virus constructions
prepared in accordance with the teaching of Example 4 were tested.
in comparison with the previously constructed p16 and p56RB vectors
prepared in accordance with Example 1. Two doses of virus were
tested, 1.times.10.sup.8 and 1.times.10.sup.9 particles per ml of
media 24 h. Plates were rinsed and re-fed with complete media (10%
FBS). 16 h after release BrdU was added for 5 h and cells were
harvested and analyzed by FACS for BrdU incorporation (DNA
synthesis) and PI staining (DNA content). Controls included
untreated cells released (ut 10). In addition to cell cycle
analysis the percent of gfcb treated cells expressing GFP (green
fluorescent protein) transgene was determined by FACS immediately
after cells were harvested. The percent of cells that incorporated
BrdU during the labeling period (16-21 h post-release) is reported
as % s.
Results
[0077] Serum starved human ocular fibroblast were treated with rAd
for 24 h then rAd was washed. Cells were released from quiescence
by feeding cells with complete media and cells were harvested 21 h
later for analysis of transgene expression by FACS. Treatment with
1.times.10.sup.8 pn/ml resulted in 62% and 79% positive cells in
HOF-TOL and HOF-CAR respectively. A dose of 1.times.10.sup.9
resulted in 100% of the cells expressing GFP transgene for both
HOF-TOL and HOF-CAR. Cell cycle analysis of cells is tabulated in
Table 2 above.
[0078] As will be apparent to those skilled in the art to which the
invention pertains, the present invention may be embodied in forms
other than those specifically disclosed above, without departing
from the spirit or essential characteristics of the invention. The
particular embodiments of the invention described above, are,
therefore to be considered as illustrative and not restrictive. The
scope of the present invention is as set forth in the appended
claims rather than being limited to the examples contained in the
foregoing description.
Sequence CWU 1
1
* * * * *