U.S. patent application number 11/088219 was filed with the patent office on 2007-02-22 for enzymatic nucleic acid-mediated treatment of ocular diseases or conditions related to levels of vascular endothelial growth factor receptor (vegf-r).
This patent application is currently assigned to Sirna Therapeutics, Inc.. Invention is credited to James McSwiggen, Pamela Pavco, Daniel Stinchcomb.
Application Number | 20070042029 11/088219 |
Document ID | / |
Family ID | 34084793 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042029 |
Kind Code |
A1 |
Pavco; Pamela ; et
al. |
February 22, 2007 |
Enzymatic nucleic acid-mediated treatment of ocular diseases or
conditions related to levels of vascular endothelial growth factor
receptor (VEGF-R)
Abstract
The present invention relates to nucleic acid molecules which
modulate the synthesis, expression and/or stability of an mRNA
encoding one or more receptors of vascular endothelial growth
factor, such as flt-1 and KDR. Nucleic acid molecules and methods
for the inhibition of angiogenesis and treatment of cancer and
ocular diseases are provided, optionally in conjunction with other
therapeutic agents.
Inventors: |
Pavco; Pamela; (Lafayette,
CO) ; McSwiggen; James; (Boulder, CO) ;
Stinchcomb; Daniel; (Ft. Collins, CO) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Sirna Therapeutics, Inc.
Boulder
CO
|
Family ID: |
34084793 |
Appl. No.: |
11/088219 |
Filed: |
March 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10138674 |
May 3, 2002 |
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11088219 |
Mar 23, 2005 |
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09870161 |
May 29, 2001 |
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10138674 |
May 3, 2002 |
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09708690 |
Nov 7, 2000 |
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09870161 |
May 29, 2001 |
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09371722 |
Aug 10, 1999 |
6534872 |
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09708690 |
Nov 7, 2000 |
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08584040 |
Jan 11, 1996 |
6346398 |
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09371722 |
Aug 10, 1999 |
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60005974 |
Oct 26, 1995 |
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Current U.S.
Class: |
424/450 ;
435/458; 514/44A |
Current CPC
Class: |
C12N 2310/121 20130101;
C12N 15/1136 20130101; C12N 2310/111 20130101; C12N 2310/322
20130101; C12N 2310/122 20130101; A61K 38/00 20130101; C12N
2310/317 20130101; C12N 2310/321 20130101; C12N 15/113 20130101;
C12N 2310/332 20130101; C12N 2310/3521 20130101; C12N 2310/315
20130101; C12N 2310/12 20130101; C12N 2310/321 20130101; C12N
2310/318 20130101; C12N 2310/53 20130101; C12N 15/1138
20130101 |
Class at
Publication: |
424/450 ;
514/044; 435/458 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/127 20060101 A61K009/127; C12N 15/88 20060101
C12N015/88 |
Claims
1: A method of locally or intraconjunctivally administering to a
cell a nucleic acid molecule that down regulates the expression of
a flt-1 gene, wherein said nucleic acid molecule comprises sequence
that is complementary to RNA encoded by said flt-1 gene and is
17-30 nucleotides in length, comprising contacting said cell with
said nucleic acid molecule under conditions suitable for said
administration.
2: The method of claim 1, wherein said cell is a mammalian
cell.
3: The method of claim 1, wherein said cell is a human cell.
4: The method of claim 1, wherein said administration is in the
presence of a delivery reagent.
5: The method of claim 4, wherein said delivery reagent is a
lipid.
6: The method of claim 5, wherein said lipid is a cationic
lipid.
7: The method of claim 5, wherein said lipid is a phospholipid.
8: The method of claim 4, wherein said delivery reagent is a
liposome.
9: A method of inhibiting diabetic retinopathy in a patient
comprising the step of locally or intraconjunctivally administering
a nucleic acid molecule that down regulates the expression of a
flt-1 gene to a patient under conditions suitable for said
inhibition, wherein said nucleic acid molecule comprises sequence
that is complementary to RNA encoded by said flt-1 gene and is
17-30 nucleotides in length.
10: A method of inhibiting age related macular degeneration in a
patient comprising the step of locally or intraconjunctivally
administering a nucleic acid molecule that down regulates the
expression of a flt-1 gene to a patient under conditions suitable
for said inhibition, wherein said nucleic acid molecule comprises
sequence that is complementary to RNA encoded by said flt-1 gene
and is 17-30 nucleotides in length.
11: A method of inhibiting neovascular glaucoma in a patient
comprising the step of locally or intraconjunctivally administering
a nucleic acid molecule that down regulates the expression of a
flt-1 gene to a patient under conditions suitable for said
inhibition, wherein said nucleic acid molecule comprises sequence
that is complementary to RNA encoded by said flt-1 gene and is
17-30 nucleotides in length.
12: A method of locally or intraconjunctivally administering to a
mammal a nucleic acid molecule that down regulates the expression
of a flt-1 gene, wherein said nucleic acid molecule comprises
sequence that is complementary to RNA encoded by said fit-1 gene
and is 17-30 nucleotides in length, comprising contacting the
mammal with said nucleic acid molecule under conditions suitable
for said administration.
13: The method of claim 12, wherein said mammal is a human.
14: The method of claim 12, wherein said administration is in the
presence of a delivery reagent.
15: The method of claim 14, wherein said delivery reagent is a
lipid.
16: The method of claim 15, wherein said lipid is a cationic
lipid.
17: The method of claim 15, wherein said lipid is a
phospholipid.
18: The method of claim 14, wherein said delivery reagent is a
liposome.
Description
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/138,674 filed May 3, 2002, which is a
continuation-in-part of U.S. patent application Ser. No. 09/870,161
filed May 29, 2001, now abandoned, which is a continuation-in-part
of U.S. patent application Ser. No. 09/708,690, now abandoned,
filed Nov. 7, 2000, which is a continuation-in-part of U.S. patent
application Ser. No. 09/371,722, now U.S. Pat. No. 6,566,127, which
is a continuation-in-part of U.S. patent application Ser. No.
08/584,040, now U.S. Pat. No. 6,346,398, which claims the benefit
of U.S. Provisional Patent Application No. 60/005,974 filed on Oct.
26, 1995. Each of these applications is hereby incorporated by
reference herein in its entirety including the drawings and
tables.
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods and reagents for the
treatment of diseases or conditions relating to the levels of
expression of vascular endothelial growth factor (VEGF)
receptor(s).
[0003] The following is a discussion of relevant art, none of which
is admitted to be prior art to the present invention.
[0004] VEGF, also referred to as vascular permeability factor (VPF)
and vasculotropin, is a potent and highly specific mitogen of
vascular endothelial cells (for a review see Ferrara, 1993 Trends
Cardiovas. Med. 3, 244; Neufeld et al., 1994 Prog. Growth Factor
Res. 5, 89). VEGF induced neovascularization is implicated in
various pathological conditions such as tumor angiogenesis,
proliferative diabetic retinopathy, hypoxia-induced angiogenesis,
rheumatoid arthritis, psoriasis, wound healing and others.
[0005] VEGF, an endothelial cell-specific mitogen, is a 34-45 kDa
glycoprotein with a wide range of activities that include promotion
of angiogenesis, enhancement of vascular-permeability and others.
VEGF belongs to the platelet-derived growth factor (PDGF) family of
growth factors with approximately 18% homology with the A and B
chain of PDGF at the amino acid level. Additionally, VEGF contains
the eight conserved cysteine residues common to all growth factors
belonging to the PDGF family (Neufeld et al., supra). VEGF protein
is believed to exist predominantly as disulfide-linked homodimers;
monomers of VEGF have been shown to be inactive (Plouet et al.,
1989 EMBO J. 8, 3801).
[0006] VEGF exerts its influence on vascular endothelial cells by
binding to specific high-affinity cell surface receptors. Covalent
cross-linking experiments with .sup.125I-labeled VEGF protein have
led to the identification of three high molecular weight complexes
of 225, 195 and 175 kDa presumed to be VEGF and VEGF receptor
complexes (Vaisman et al., 1990 J. Biol. Chem. 265, 19461). Based
on these studies VEGF-specific receptors of 180, 150 and 130 kDa
molecular mass were predicted. In endothelial cells, receptors of
150 and the 130 kDa have been identified. The VEGF receptors belong
to the superfamily of receptor tyrosine kinases (RTKs)
characterized by a conserved cytoplasmic catalytic kinase domain
and a hydrophylic kinase sequence. The extracellular domains of the
VEGF receptors consist of seven immunoglobulin-like domains that
are thought to be involved in VEGF binding functions.
[0007] The two most abundant and high-affinity receptors of VEGF
are flt-1 (fms-like tyrosine kinase) cloned by Shibuya et al., 1990
Oncogene 5, 519 and KDR (kinase-insert-domain-containing receptor)
cloned by Terman et al., 1991 Oncogene 6, 1677. The murine homolog
of KDR, cloned by Mathews et al., 1991, Proc. Natl. Acad. Sci.,
USA, 88, 9026, shares 85% amino acid homology with KDR and is
termed as flk-1 (fetal liver kinase-1). Recently it has been shown
that the high-affinity binding of VEGF to its receptors is
modulated by cell surface-associated heparin and heparin-like
molecules (Gitay-Goren et al., 1992 J. Biol. Chem. 267, 6093).
[0008] VEGF expression has been associated with several
pathological states such as tumor angiogenesis, several forms of
blindness, rheumatoid arthritis, psoriasis and others. Following is
a brief summary of evidence supporting the involvement of VEGF in
various diseases:
[0009] 1) Tumor angiogenesis: Increased levels of VEGF gene
expression have been reported in vascularized and edema-associated
brain tumors (Berkman et al., 1993 J Clini. Invest. 91, 153). A
more direct demostration of the role of VEGF in tumor angiogenesis
was demonstrated by Jim Kim et al., 1993 Nature 362, 841 wherein,
monoclonal antibodies against VEGF were successfully used to
inhibit the growth of rhabdomyosarcoma, glioblastoma multiforme
cells in nude mice. Similarly, expression of a dominant negative
mutated form of the flt-1 VEGF receptor inhibits vascularization
induced by human glioblastoma cells in nude mice (Millauer et al.,
1994, Nature 367, 576).
[0010] 2) Ocular diseases: Aiello et al., 1994 New Engl. J. Med.
331, 1480, showed that the ocular fluid of a majority of patients
suffering from diabetic retinopathy and other retinal disorders,
contains a high concentration of VEGF. Miller et al., 1994 Am. J.
Pathol. 145, 574, reported elevated levels of VEGF mRNA in patients
suffering from retinal ischemia. These observations support a
direct role for VEGF in ocular diseases.
[0011] 3) Psoriasis: Detmar et al., 1994 J. Exp. Med. 180, 1141
reported that VEGF and its receptors were over-expressed in
psoriatic skin and psoriatic dermal microvessels, suggesting that
VEGF plays a significant role in psoriasis.
[0012] 4) Rheumatoid arthritis: Immunohistochemistry and in situ
hybridization studies on tissues from the joints of patients
suffering from rheumatoid arthritis show an increased level of VEGF
and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341).
Additionally, Koch et al., 1994 J. Immunol. 152, 4149, found that
VEGF-specific antibodies were able to significantly reduce the
mitogenic activity of synovial tissues from patients suffering from
rheumatoid arthritis. These observations support a direct role for
VEGF in rheumatoid arthritis.
[0013] In addition to the above data on pathological conditions
involving excessive angiogenesis, a number of studies have
demonstrated that VEGF is both necessary and sufficient for
neovascularization. Takashita et al., 1995 J. Clin. Invest. 93,
662, demonstrated that a single injection of VEGF augmented
collateral vessel development in a rabbit model of ischemia. VEGF
also can induce neovascularization when injected into the cornea.
Expression of the VEGF gene in CHO cells is sufficient to confer
tumorigenic potential to the cells. Kim et al., supra and Millauer
et al., supra used monoclonal antibodies against VEGF or a dominant
negative form of flk-1 receptor to inhibit tumor-induced
neovascularization.
[0014] During development, VEGF and its receptors are associated
with regions of new vascular growth (Millauer et al., 1993 Cell 72,
835; Shalaby et al., 1993 J. Clin. Invest. 91, 2235). Furthermore,
transgenic mice lacking either of the VEGF receptors are defective
in blood vessel formation, in fact these mice do not survive; flk-1
appears to be required for differentiation of endothelial cells,
while flt-1 appears to be required at later stages of vessel
formation (Shalaby et al., 1995 Nature 376, 62; Fung et al., 1995
Nature 376, 66). Thus, these receptors must be present to properly
signal endothelial cells or their precursors to respond to
vascularization-promoting stimuli.
[0015] All of the conditions listed above, involve extensive
vascularization. This hyper-stimulation of endothelial cells may be
alleviated by VEGF antagonists. Thus most of the therapeutic
efforts for the above conditions have concentrated on finding
inhibitors of the VEGF protein.
[0016] Kim et al., 1993 Nature 362, 841 have been successful in
inhibiting VEGF-induced tumor growth and angiogenesis in nude mice
by treating the mice with VEGF-specific monoclonal antibody.
[0017] Koch et al., 1994 J. Immunol. 152, 4149 showed that the
mitogenic activity of microvascular endothelial cells found in
rheumatoid arthritis (RA) synovial tissue explants and the
chemotactic property of endothelial cells from RA synovial fluid
can be neutralized significantly by treatment with VEGF-specific
antibodies.
[0018] Ullrich et al., International PCT Publication No. WO
94/11499 and Millauer et al., 1994 Nature 367, 576 used a soluble
form of flk-1 receptor (dominant-negative mutant) to prevent
VEGF-mediated tumor angiogenesis in immunodeficient mice.
[0019] Kendall and Thomas, International PCT Publication No. WO
94/21679 describe the use of naturally occuring or
recombinantly-engineered soluble forms of VEGF receptors to inhibit
VEGF activity.
[0020] Robinson, International PCT Publication No. WO 95/04142
describes the use of antisense oligonucleotides targeted against
VEGF RNA to inhibit VEGF expression.
[0021] Jellinek et al., 1994 Biochemistry 33, 10450 describe the
use of VEGF-specific high-affinity RNA aptamers to inhibit the
binding of VEGF to its receptors.
[0022] Rockwell and Goldstein, International PCT Publication No. WO
95/21868, describe the use of anti-VEGF receptor monoclonal
antibodies to neutralize the effect of VEGF on endothelial
cells.
SUMMARY OF THE INVENTION
[0023] The invention features novel nucleic acid-based compounds
[e.g., enzymatic nucleic acid molecules (ribozymes such as Inozyme,
G-cleaver, amberzyme, zinzyme), DNAzyme, antisense nucleic acids,
2-5A antisense chimeras, triplex forming nucleic acid, decoy
nucleic acids, aptamers, allozymes, antisense nucleic acids
containing RNA cleaving chemical groups (Cook et al., U.S. Pat. No.
5,359,051)] and methods for their use to down regulate or inhibit
the expression of receptors of VEGF (VEGF-R).
[0024] In one embodiment, the invention features the use of one or
more of the nucleic acid-based compounds to inhibit the expression
of flt-1 and/or flk-1/KDR receptors.
[0025] In another embodiment, the present invention features a
compound having Formula I: (SEQ ID NO: 20818). TABLE-US-00001 5'
g.sub.sa.sub.sg.sub.su.sub.sugcUGAuGagg ccgaaa ggccGaaAgucugB
3'
[0026] wherein each a is 2'-O-methyl adenosine nucleotide, each g
is a 2'-O-methyl guanosine nucleotide, each c is a 2'-O-methyl
cytidine nucleotide, each u is a 2'-O-methyl uridine nucleotide,
each A is adenosine, each G is guanosine, each s individually
represents a phosphorothioate internucleotide linkage, U is
2'-deoxy-2'-C-allyl uridine, and B is an inverted deoxyabasic
moiety.
[0027] In another embodiment, the present invention features a
compound having Formula II: (SEQ ID NO: 13488). TABLE-US-00002
5'-u.sub.sa.sub.sc.sub.s a.sub.sau ucU GAu Gag gcg aaa gcc Gaa Aag
aca aB-3'
[0028] wherein each a is 2'-O-methyl adenosine nucleotide, each g
is a 2'-O-methyl guanosine nucleotide, each c is a 2'-O-methyl
cytidine nucleotide, each u is a 2'-O-methyl uridine nucleotide,
each A is adenosine, each G is guanosine, each s individually
represents a phosphorothioate internucleotide linkage, U is
2'-deoxy-2'-C-allyl uridine, and B is an inverted deoxyabasic
moiety.
[0029] In one embodiment, the invention features a composition
comprising a compound of Formula I and/or II in a pharmaceutically
acceptable carrier or diluent.
[0030] In another embodiment, the invention features a method of
administering to a cell, for example a mammalian cell or human
cell, the compound of Formula I and/or II, comprising contacting
the cell with the compound under conditions suitable for
administration, for example in the presence of a delivery reagent.
Examples of suitable delivery reagents include a lipid, cationic
lipid, phospholipid, or liposome as described herein and known in
the art.
[0031] In one embodiment, the invention features a method of
administering to a cell the compound of Formula I or II in
conjunction with a chemotherapeutic agent comprising contacting the
cell with the compound and the chemotherapeutic agent under
conditions suitable for administration.
[0032] Examples of chemotherapeutic agents that can be combined
with the compound of Formula I and/or II include but are not
limited to 5-fluoro uridine, Leucovorin, Irinotecan (CAMPTOSAR.RTM.
or CPT-11 or Camptothecin-11 or Campto), Paclitaxel, or Carboplatin
or a combination thereof.
[0033] In another embodiment, the present invention also features a
cell comprising the compound of Formula I and/or II, wherein the
cell is a mammalian cell. For example, in one embodiment the
mammalian cell is a human cell.
[0034] In one embodiment, the invention features a method of
inhibiting angiogenesis, for example tumor angiogenesis, in a
patient comprising the step of contacting the patient with the
compound of Formula I and/or II under conditions suitable for said
inhibition. In one embodiment, the patient is a mammal, for
example, a human.
[0035] In another embodiment, the invention features a method of
treatment of a patient having a condition associated with an
increased level of VEGF receptor, for example, cancers such as
breast cancer, lung cancer, colorectal cancer, renal cancer,
pancreatic cancer, or melanoma, or ocular indications such as
diabetic retinopathy, or age related macular degeneration,
comprising contacting one or more cells of the patient with the
compound of Formula I and/or II, under conditions suitable for the
treatment. In one embodiment, the patient is a human.
[0036] In another embodiment, the invention features a method of
treatment of a patient having an ocular condition associated with
an increased level of a VEGF receptor, for example, diabetic
retinopathy, or age related macular degeneration, comprising
contacting one or more cells of the patient with a nucleic acid
molecule, such as an enzymatic nucleic acid molecule, targeted
against a VEGF receptor RNA, e.g., a molecule according to Formula
I and/or II, under conditions suitable for the treatment. In one
embodiment, the patient is a human.
[0037] In yet another embodiment, a method of treatment of the
invention further comprises the use of one or more drug therapies
under conditions suitable for the treatment.
[0038] In one embodiment, the present invention also features a
method of cleaving RNA comprising a sequence of flt-1 comprising
contacting the compound of Formula I with the RNA under conditions
suitable for the cleavage of the RNA, for example, where the
cleavage is carried out in the presence of a divalent cation such
as Mg2+.
[0039] In another embodiment, the invention features a method of
administering to a mammal, for example a human, the compound of
Formula I and/or II comprising contacting the mammal with the
compound under conditions suitable for the administration, for
example, in the presence of a delivery reagent such as a lipid,
cationic lipid, phospholipid, or liposome.
[0040] In yet another embodiment, the invention features a method
of administering to a mammal the compound of Formula I and/or II in
conjunction with a chemotherapeutic agent comprising contacting the
mammal, for example a human, with the compound and the
chemotherapeutic agent under conditions suitable for the
administration.
[0041] In another embodiment, the invention features a composition
comprising the nucleic acid molecule of the instant invention and a
pharmaceutically acceptable carrier or diluent.
[0042] By "inhibit" it is meant that the activity of VEGF-R or
level of VEGF-R mRNAs or equivalent RNAs encoding VEGF-R is reduced
below that observed in the absence of a nucleic acid molecule of
the instant invention. In one embodiment, inhibition with enzymatic
nucleic acid preferably is below that level observed in the
presence of an enzymatically inactive or attenuated molecule that
is able to bind to the same site on the mRNA, but is unable to
cleave that RNA. In another embodiment, inhibition with antisense
oligonucleotides is preferably below that level observed in the
presence of, for example, an oligonucleotide with scrambled
sequence or with mismatches. In another embodiment, inhibition of a
VEGF-R gene with the nucleic acid molecule of the instant invention
is greater in the presence of the nucleic acid molecule than in its
absence.
[0043] By "enzymatic nucleic acid molecule" it is meant an RNA
molecule which has complementarity in a substrate binding region to
a specified gene target, and also has an enzymatic activity which
is active to specifically cleave target RNA. That is, the enzymatic
RNA molecule is able to intermolecularly cleave RNA and thereby
inactivate a target RNA molecule. The complementary region(s)
allows sufficient hybridization of the enzymatic RNA molecule to
the target RNA and thus permit cleavage. One hundred percent
complementarity is preferred, but complementarity as low as 50-75%
can also be useful in this invention. The nucleic acids can be
modified at the base, sugar, and/or phosphate groups. The term
enzymatic nucleic acid is used interchangeably with phrases such as
ribozymes, catalytic RNA, enzymatic RNA, enzymatic DNA, catalytic
DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, Zinzyme, RNA
enzyme, endoribonuclease, endonuclease, minizyme, leadzyme,
oligozyme or DNA enzyme. All of these terminologies describe
nucleic acid molecules with enzymatic activity. The specific
enzymatic nucleic acid molecules described in the instant
application are not meant to be limiting and those skilled in the
art will recognize that all that is important in an enzymatic
nucleic acid molecule of this invention is that it have a specific
substrate binding site which is complementary to one or more of the
target nucleic acid regions, and that it have nucleotide sequences
within or surrounding that substrate binding site which impart a
nucleic acid cleaving activity to the molecule (Cech et al., U.S.
Pat. No. 4,987,071; Cech et al., 1988, JAMA).
[0044] By "enzymatic portion" or "catalytic domain" is meant that
portion/region of the enzymatic nucleic acid essential for cleavage
of a nucleic acid substrate (for example see FIG. 1).
[0045] By "substrate binding arm" or "substrate binding domain" is
meant that portion/region of a enzymatic nucleic acid which is
complementary to (i.e., able to base-pair with) a portion of its
substrate. Generally, such complementarity is 100%, but can be less
if desired. For example, as few as 10 bases out of 14 can be
base-paired. Such arms are shown generally in FIG. 1. That is,
these arms contain sequences within an enzymatic nucleic acid are
intended to bring enzymatic nucleic acid and target RNA together
through complementary base-pairing interactions. The enzymatic
nucleic acid of the invention can have binding arms that are
contiguous or non-contiguous and can be of varying lengths. The
length of the binding arm(s) are preferably greater than or equal
to four nucleotides, and are of sufficient length to stably
interact with the target RNA; specifically 12-100 nucleotides; more
specifically 14-24 nucleotides long. If two binding arms are
chosen, the design is such that the length of the binding arms are
symmetrical (i.e., each of the binding arms is of the same length;
e.g., five and five nucleotides, six and six nucleotides or seven
and seven nucleotides long) or asymmetrical (i.e., the binding arms
are of different length; e.g., six and three nucleotides; three and
six nucleotides long; four and five nucleotides long; four and six
nucleotides long; four and seven nucleotides long; and the
like).
[0046] By "DNAzyme" is meant an enzymatic nucleic acid molecule
lacking a 2'-OH group. In particular embodiments, the enzymatic
nucleic acid molecule can have an attached linker(s) or other
attached or associated groups, moieties, or chains containing one
or more nucleotides with 2'-OH groups.
[0047] By "zinzyme" motif or configuration is meant a class II
enzymatic nucleic acid molecule comprising a motif as generally
described in FIG. 32 and in Beigelman et al., International PCT
publication No. WO 99/55857 and U.S. patent application Ser. No.
09/918,728, which is herein incorporated by reference in its
entirety, including the drawings. Zinzymes possess endonuclease
activity to cleave RNA substrates having a cleavage triplet
including but not limited to, YG/Y, where Y is uridine or cytidine,
and G is guanosine and/represents the cleavage site. Zinzymes can
be chemically modified to increase nuclease stability through
various substitutions, including substituting 2'-O-methyl guanosine
nucleotides for guanosine nucleotides. In addition, differing
nucleotide and/or non-nucleotide linkers can be used to substitute
the 5'-gaaa-2' loop of the motif. Zinzymes represent a non-limiting
example of an enzymatic nucleic acid molecule that does not require
a ribonucleotide (2'-OH) group within its own nucleic acid sequence
for activity.
[0048] By "sufficient length" is meant a nucleic acid molecule long
enough to provide the intended function under the expected
condition. For example, a nucleic acid molecule of the invention
needs to be of "sufficient length" to provide stable binding to a
target site under the expected binding conditions and environment.
In another non-limiting example, for the binding arms of an
enzymatic nucleic acid, "sufficient length" means that the binding
arm sequence is long enough to provide stable binding to a target
site under the expected reaction conditions and environment. The
binding arms are not so long as to prevent useful turnover of the
nucleic acid molecule.
[0049] By "stably interact" is meant interaction of the
oligonucleotides with target, such as a target protein or target
nucleic acid (e.g., by forming hydrogen bonds with complementary
amino acids or nucleotides in the target under physiological
conditions) that is sufficient for the intended purpose (e.g.,
specific binding to a protein target to disrupt the function of
that protein or cleavage of target RNA/DNA by an enzyme).
[0050] By "equivalent" RNA to VEGF-R is meant to include those
naturally occurring RNA molecules having homology (partial or
complete) to VEGF-R, or encoding for proteins with similar function
as VEGF-R in various animals, including human, rodent, primate,
rabbit and pig. The equivalent RNA sequence also includes in
addition to the coding region, regions such as 5'-untranslated
region, 3'-untranslated region, introns, intron-exon junction and
the like.
[0051] By "homology" is meant the nucleotide sequence of two or
more nucleic acid molecules is partially or completely
identical.
[0052] By "antisense nucleic acid" it is meant a non-enzymatic
nucleic acid molecule that binds to target RNA by means of RNA-RNA
or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993
Nature 365, 566) interactions and alters the activity of the target
RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).
Typically, antisense molecules will be complementary to a target
sequence along a single contiguous sequence of the antisense
molecule. However, in certain embodiments, an antisense molecule
may bind to substrate such that the substrate molecule forms a
loop, and/or an antisense molecule may bind such that the antisense
molecule forms a loop. Thus, the antisense molecule may be
complementary to two (or even more) non-contiguous substrate
sequences or two (or even more) non-contiguous sequence portions of
an antisense molecule may be complementary to a target sequence or
both.
[0053] By "2-5A antisense chimera" it is meant, an antisense
oligonucleotide-containing a 5' phosphorylated 2'-5'-linked
adenylate residues. These chimeras bind to target RNA in a
sequence-specific manner and activate a cellular 2-5A-dependent
ribonuclease which, in turn, cleaves the target RNA (Torrence et
al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
[0054] By "triplex DNA" it is meant an oligonucleotide that can
bind to a double-stranded DNA in a sequence-specific manner to form
a triple-strand helix. Formation of such triple helix structure has
been shown to inhibit transcription of the targeted gene
(Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89,
504).
[0055] By "gene" it is meant a nucleic acid that encodes an RNA,
for example, nucleic acid sequences including, but not limited to,
structural genes encoding a polypeptide.
[0056] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. In
reference to the nucleic molecules of the present invention, the
binding free energy for a nucleic acid molecule with its target or
complementary sequence is sufficient to allow the relevant function
of the nucleic acid to proceed, e.g., ribozyme cleavage, antisense
or triple helix inhibition. Determination of binding free energies
for nucleic acid molecules is well known in the art (see, e.g.,
Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier
et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et
al., 1987, J. Am. Chem. Soc. 109:3783-3785. A percent
complementarity indicates the percentage of contiguous residues in
a nucleic acid molecule which can form hydrogen bonds (e.g.,
Watson-Crick base pairing) with a second nucleic acid sequence
(e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%,
and 100% complementary). "Perfectly complementary" means that all
the contiguous residues of a nucleic acid sequence will hydrogen
bond with the same number of contiguous residues in a second
nucleic acid sequence.
[0057] Seven basic varieties of naturally-occurring enzymatic RNAs
are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans (and thus can cleave other RNA
molecules) under physiological conditions. Table I summarizes some
of the characteristics of these ribozymes. In general, enzymatic
nucleic acids act by first binding to a target RNA. Such binding
occurs through the target binding portion of a enzymatic nucleic
acid which is held in close proximity to an enzymatic portion of
the molecule that acts to cleave the target RNA. Thus, the
enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct
site, acts enzymatically to cut the target RNA. Strategic cleavage
of such a target RNA destroys its ability to direct synthesis of an
encoded protein. After an enzymatic nucleic acid has bound and
cleaved its RNA target, it is released from that RNA to search for
another target and can repeatedly bind and cleave new targets.
Thus, a single enzymatic nucleic acid molecule is able to cleave
many molecules of target RNA. In addition, the enzymatic nucleic
acid is a highly specific inhibitor of gene expression, with the
specificity of inhibition depending not only on the base-pairing
mechanism of binding to the target RNA, but also on the mechanism
of target RNA cleavage. Single mismatches, or base-substitutions,
near the site of cleavage can completely eliminate catalytic
activity of a ribozyme.
[0058] Enzymatic nucleic acids that cleave the specified sites in
VEGF-R mRNAs represent a novel therapeutic approach to treat tumor
angiogenesis, and cancers including, but not limited to, tumor and
cancer types shown under Diagnosis in Table XX, ocular diseases,
rhuematoid arthritis, psoriasis and others. The enzymatic nucleic
acid molecules of the instant invention are able to inhibit the
activity of VEGF-R (specifically fit-1 and flk-1/KDR) and the
catalytic activity of the enzymatic nucleic acid molecules is
required for their inhibitory effect. Those of ordinary skill in
the art will find it clear from the exemplary nucleic acid
molecules described that other enzymatic nucleic acid molecules
that cleave VEGF-R mRNAs can be readily designed and are within the
scope of the invention.
[0059] In one of the embodiments of the inventions described
herein, the enzymatic nucleic acid molecule is formed in a
hammerhead or hairpin motif, but can also be formed in the motif of
a hepatitis delta virus, group I intron, group II intron or RNase P
RNA (in association with an RNA guide sequence), Neurospora VS RNA,
DNAzymes, Zinzymes, NCH cleaving motifs, or G-cleavers. Examples of
such hammerhead motifs are described by Dreyfus, supra, Rossi et
al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin
motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989
Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53,
Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990
Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, U.S. Pat. No.
5,631,359; of the hepatitis delta virus motif is described by
Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif
by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman,
1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24,
835; Neurospora VS RNA ribozyme motif is described by Collins
(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins,
1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive,
1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J 14,
363); Group II introns are described by Griffin et al., 1995, Chem.
Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle
et al., International PCT Publication No. WO 96/22689; of the Group
I intron by Cech et al., U.S. Pat. No. 4,987,071; of Zinzymes as is
generally described by Beigelman et al., International PCT
publication No. WO 99/55857 (see for example FIG. 32); and of
DNAzymes as is generally described by Usman et al., International
PCT Publication No. WO 95/11304; Chartrand et al., 1995, NAR 23,
4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al.,
1997, PNAS 94, 4262 (see for example FIG. 33). NCH cleaving motifs
are described in Ludwig & Sproat, International PCT Publication
No. WO 98/58058; and G-cleavers are described in Kore et al., 1998,
Nucleic Acids Research 26, 4116-4120 and Eckstein et al.,
International PCT Publication No. WO 99/16871. These specific
motifs are not limiting in the invention and those skilled in the
art will recognize that all that is important in an enzymatic
nucleic acid molecule of this invention is that it has a specific
substrate binding site which is complementary to one or more of the
target gene RNA regions, and that it have nucleotide sequences
within or surrounding that substrate binding site which impart an
RNA cleaving activity to the molecule (Cech et al., U.S. Pat. No.
4,987,071).
[0060] Enzymatic nucleic acid molecules of the invention that are
allosterically regulated ("allozymes") can be used to modulate VEGR
receptor expression. These allosteric enzymatic nucleic acids or
allozymes (see for example George et al., U.S. Pat. Nos. 5,834,186
and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al.,
U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT
publication No. WO 00/24931, Breaker et al., International PCT
Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al.,
International PCT publication No. WO 99/29842) are designed to
respond to a signaling agent. For example, allozymes can be
designed to respond to signaling agents, such as flt-1 or kdr
protein, flt-1 or kdr RNA, other proteins and/or RNAs involved in
VEGF activity, and also, for example, compounds, metals, polymers,
molecules and/or drugs that are targeted to VEGF or VEGF receptor,
such as flt-1 or kdr expressing cells etc., which in turn modulate
the activity of the enzymatic nucleic acid molecule. In response to
interaction with a predetermined signaling agent, the activity of
the allosteric enzymatic nucleic acid molecule is activated or
inhibited such that the expression of a particular target is
selectively down-regulated. The target can comprise flt-1 or kdr
and/or a predetermined cellular component or receptor that
modulates VEGF activity.
[0061] In a specific example, allosteric enzymatic nucleic acid
molecules that are activated by interaction with a RNA encoding a
flt-1 protein are used as therapeutic agents in vivo. The presence
of RNA encoding the flt-1 protein activates the allosteric
enzymatic nucleic acid molecule that subsequently cleaves the RNA
encoding the flt-1 protein, resulting in the inhibition of flt-1
protein expression. In this manner, cells that express the flt-1
protein are selectively targeted.
[0062] In another non-limiting example, an allozyme can be
activated by an flt-1 protein or peptide that caused the allozyme
to inhibit the expression of flt-1 gene by, for example, cleaving
RNA encoded by flt-1 gene. In this non-limiting example, the
allozyme acts as a decoy to inhibit the function of flt-1 and also
inhibit the expression of flt-1 once activated by the fit-1
protein.
[0063] In one embodiment, the nucleic acid molecule of the
invention, e.g., antisense molecule, triplex DNA, or ribozyme, is
13 to 100 nucleotides in length, e.g., in specific embodiments 35,
36, 37, or 38 nucleotides in length (e.g., for particular
ribozymes). In particular embodiments, the nucleic acid molecule is
15-100, 17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100,
32-100, 35-100, 40-100, 50-100, 60-100, 70-100, or 80-100
nucleotides in length. Instead of 100 nucleotides being the upper
limit on the length ranges specified above, the upper limit of the
length range can be, for example, 30, 40, 50, 60, 70, or 80
nucleotides. Thus, for any of the length ranges, the length range
for particular embodiments has a lower limit as specified, with an
upper limit as specified which is greater than the lower limit. For
example, in a particular embodiment, the length range can be 35-50
nucleotides in length. All such ranges are expressly included. Also
in particular embodiments, a nucleic acid molecule can have a
length which is any of the lengths specified above, for example, 21
nucleotides in length.
[0064] In one embodiment, the invention provides a method for
producing a class of enzymatic cleaving agents which exhibit a high
degree of specificity for the RNA of a desired target. The
enzymatic nucleic acid molecule is preferably targeted to a highly
conserved sequence region of target mRNAs encoding VEGF-R proteins
(specifically flt-1 and flk-1/KDR) such that specific treatment of
a disease or condition can be provided with either one or several
enzymatic nucleic acids. Such enzymatic nucleic acid molecules can
be delivered exogenously to specific tissue or cellular targets as
required. Alternatively, the enzymatic nucleic acid molecules can
be expressed from DNA and/or RNA vectors that are delivered to
specific cells.
[0065] By "highly conserved sequence region" is meant a nucleotide
sequence of one or more regions in a nucleic acid molecule that
does not vary significantly from one generation to the other or
from one biological system to the other.
[0066] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
nucleic acid motifs (e.g., antisense oligonucleotides, hammerhead
or the hairpin ribozymes) are used for exogenous delivery. The
simple structure of these molecules increases the ability of the
nucleic acid to invade targeted regions of the mRNA structure.
However, these nucleic acid molecules can also be expressed within
cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985
Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad.
Sci. USA 83, 399; SullengerScanlon et al., 1991, Proc. Natl. Acad.
Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res.
Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41;
Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992
Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic
Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247, 1222-1225;
Thompson et al., 1995 Nucleic Acids Res. 23, 2259). Those skilled
in the art realize that any nucleic acid can be expressed in
eukaryotic cells from the appropriate DNA/RNA vector. The activity
of such nucleic acids can be augmented by their release from the
primary transcript by a ribozyme (Draper et al., PCT WO93/23569,
and Sullivan et al., PCT WO94/02595, both hereby incorporated in
their totality by reference herein; Ohkawa et al., 1992 Nucleic
Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res.,
19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55;
Chowrira et al., 1994 J. Biol. Chem. 269, 25856).
[0067] The nucleic acid molecules of the invention are useful for
the prevention of diseases and conditions related to the level of
VEGF-R, including cancer (including but not limited to tumor and
cancer types shown under Diagnosis in Table XX), diabetic
retinopathy, macular degeneration, neovascular glaucoma, myopic
degeneration, arthritis, psoriasis, verruca vulgaris, angiofibroma
of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome,
Kippel-Trenaunay-Weber syndrome, Osler-Weber-Rendu syndrome and any
other diseases or conditions that are related to the levels of
VEGF-R (specifically flt-1 and flk-1/KDR) in a cell or tissue.
[0068] By "diseases or conditions related to the level of VEGF-R"
is meant that the reduction of VEGF-R (specifically flt-1 and
flk-1/KDR) RNA levels and thus reduction in the level of the
respective protein will relieve, to some extent, the symptoms of
the disease or condition.
[0069] Nucleic acid molecules of the invention are added directly,
or can be complexed with cationic lipids, packaged within
liposomes, or otherwise delivered to target cells or tissues. The
nucleic acid or nucleic acid complexes can be locally administered
to relevant tissues ex vivo, or in vivo through injection, infusion
pump or stent, with or without their incorporation in
biopolymers.
[0070] In one embodiment, the enzymatic nucleic acid molecule of
the invention has one or more binding arms which are complementary
to the substrate sequences in Tables II to IX, XIV-XIX, XXII, and
XXIII. Examples of such enzymatic nucleic acid molecules also are
shown in Tables II to IX, XIV-XIX, XXII, and XXIII. Examples of
such enzymatic nucleic acid molecules consist essentially of
sequences defined in these Tables.
[0071] In yet another embodiment, the invention features antisense
nucleic acid molecules and 2-5A chimera including sequences
complementary to the target sequences shown in Tables II to IX,
XIV-XIX, XXII, and XXIII. Such nucleic acid molecules can include
sequences as shown for the binding arms of the enzymatic nucleic
acid molecules in Tables II to IX, XIV-XIX, XXII, and XXIII.
Similarly, triplex molecules can be provided targeted to the
corresponding DNA target regions, and containing the DNA equivalent
of a target sequence or a sequence complementary to the specified
target (substrate) sequence. Typically, antisense molecules are
complementary to a target sequence along a single contiguous
sequence of the antisense molecule. However, in certain
embodiments, an antisense molecule can bind to substrate such that
the substrate molecule forms a loop, and/or an antisense molecule
can bind such that the antisense molecule forms a loop. Thus, the
antisense molecule can be complementary to two (or even more)
non-contiguous substrate sequences or two (or even more)
non-contiguous sequence portions of an antisense molecule can be
complementary to a target sequence or both.
[0072] By "consists essentially of" is meant that the active
nucleic acid molecule of the invention, for example, an enzymatic
nucleic acid molecule, contains an enzymatic center or core
equivalent to those in the examples, and binding arms able to bind
RNA such that cleavage at the target site occurs. Other sequences
can be present that do not interfere with such cleavage. Thus, a
core region can, for example, include one or more loop, stem-loop
structure, or linker that does not prevent enzymatic activity.
Thus, the underlined regions in the sequences in Tables II, IV, VI,
VIII, XIV and XVI can be such a loop, stem-loop, nucleotide linker,
and/or non-nucleotide linker and can be represented generally as
sequence "X". For example, a core sequence for a hammerhead
enzymatic nucleic acid can comprise a conserved sequence, such as
5'-CUGAUGAG-3' and 5'-CGAA-3' connected by "X", where X is
5'-GCCGUUAGGC-3' (SEQ ID NO: 20822), or any other Stem II region
known in the art, or a nucleotide and/or non-nucleotide linker.
Similarly, for other nucleic acid molecules of the instant
invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme,
antisense, 2-5A antisense, triplex forming nucleic acid, and decoy
nucleic acids, other sequences or non-nucleotide linkers can be
present that do not interfere with the function of the nucleic acid
molecule.
[0073] X can be a linker of .gtoreq.2 nucleotides in length,
preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the
nucleotides can preferably be internally base-paired to form a stem
of preferably .gtoreq.2 base pairs. Alternatively or in addition, X
may be a non-nucleotide linker. In yet another embodiment, the
nucleotide linker (X) can be a nucleic acid aptamer, such as an ATP
aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others
(for a review see Gold et al., 1995, Annu. Rev. Biochem., 64, 763;
and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland
and Atkins, pp. 511, CSH Laboratory Press).
[0074] A "nucleic acid aptamer" as used herein is meant to indicate
a nucleic acid sequence capable of interacting with a ligand. The
ligand can be any natural or a synthetic molecule, including but
not limited to a resin, metabolites, nucleosides, nucleotides,
drugs, toxins, transition state analogs, peptides, lipids,
proteins, amino acids, nucleic acid molecules, hormones,
carbohydrates, receptors, cells, viruses, bacteria and others.
[0075] In yet another embodiment, the non-nucleotide linker (X) is
as defined herein. The term "non-nucleotide" as used herein include
either abasic nucleotide, polyether, polyamine, polyamide, peptide,
carbohydrate, lipid, or polyhydrocarbon compounds. Specific
examples include those described by Seela and Kaiser, Nucleic Acids
Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and
Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and
Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic
Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et
al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides
& Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.
1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et
al., International Publication No. WO 89/02439; Usman et al.,
International Publication No. WO 95/06731; Dudycz et al.,
International Publication No. WO 95/11910 and Ferentz and Verdine,
J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein. A "non-nucleotide" further means any group or
compound which can be incorporated into a nucleic acid chain in the
place of one or more nucleotide units, including either sugar
and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic activity. The group or compound can be
abasic in that it does not contain a commonly recognized nucleotide
base, such as adenosine, guanine, cytosine, uracil or thymine.
Thus, in one embodiment, the invention features an enzymatic
nucleic acid molecule having one or more non-nucleotide moieties,
and having enzymatic activity to cleave an RNA or DNA molecule.
[0076] In another aspect of the invention, enzymatic nucleic acid
molecules that cleave target RNA molecules and inhibit VEGF-R
(specifically flt-1 and flk-1/KDR) activity are expressed from
transcription units inserted into DNA or RNA vectors. The
recombinant vectors are preferably DNA plasmids or viral vectors.
Enzymatic nucleic acid expressing viral vectors can be constructed
based on, but not limited to, adeno-associated virus, retrovirus,
adenovirus, or alphavirus vectors. Preferably, the recombinant
vectors capable of expressing the enzymatic nucleic acid molecules
are delivered as described above, and persist in target cells.
Alternatively, viral vectors can be used that provide for transient
expression of enzymatic nucleic acids. Such vectors can be
repeatedly administered as necessary. Once expressed, the enzymatic
nucleic acids cleave the target mRNA. Delivery of enzymatic nucleic
acids expressing vectors can be systemic, such as by intravenous or
intramuscular administration, by administration to target cells
ex-planted from the patient followed by reintroduction into the
patient, or by any other means that would allow for introduction
into the desired target cell.
[0077] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0078] By "patient" is meant an organism which is a donor or
recipient of explanted cells or the cells themselves. "Patient"
also refers to an organism to which the nucleic acid molecules of
the invention can be administered. In one embodiment, the patient
is a mammal or mammalian cells. Preferably, the patient is a human
or human cells.
[0079] The nucleic acid molecules of the instant invention,
individually, or in combination or in conjunction with other drugs,
can be used to treat diseases or conditions discussed above. For
example, to treat a disease or condition associated with VEGF-R,
the patient can be treated, or other appropriate cells can be
treated, as is evident to those skilled in the art, individually
with a nucleic acid molecule of the invention or in combination
with one or more drugs under conditions suitable for the
treatment.
[0080] For example, to treat a disease or condition associated with
VEGF-R levels, such as cancer (e.g., colorectal cancer, breast
cancer) or ocular diseases (e.g., diabetic retinopathy or age
related macular degeneration) a patient may be treated, or other
appropriate cells may be treated, as is evident to those skilled in
the art, individually or in combination with one or more drugs
under conditions suitable for the treatment.
[0081] In a further embodiment, the described molecules, such as
antisense or enzymatic nucleic acid molecules can be used in
combination with other known treatments to treat conditions or
diseases discussed above. For example, the described molecules can
be used in combination with one or more known therapeutic agents to
treat cancer.
[0082] In another embodiment, the invention features nucleic
acid-based techniques (e.g., enzymatic nucleic acid molecules
(ribozymes such as Inozyme, G-cleaver, amberzyme, zinzyme),
DNAzyme, antisense nucleic acids, 2-5A antisense chimeras, triplex
forming nucleic acid, decoy nucleic acids, aptamers, allozymes,
antisense nucleic acids containing RNA cleaving chemical groups
(Cook et al., U.S. Pat. No. 5,359,051)] and methods for their use
to down regulate or inhibit the expression of genes capable of
inducing angiogenesis (e.g., flt-1 and kdr).
[0083] In another embodiment, the invention features nucleic
acid-based techniques (e.g., enzymatic nucleic acid molecules
(ribozymes such as Inozyme, G-cleaver, amberzyme, zinzyme),
DNAzyme, antisense nucleic acids, 2-5A antisense chimeras, triplex
forming nucleic acid, decoy nucleic acids, aptamers, allozymes,
antisense nucleic acids containing RNA cleaving chemical groups
(Cook et al., U.S. Pat. No. 5,359,051)] and methods for their use
to down regulate or inhibit the expression of VEGF receptor.
[0084] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1 is a diagrammatic representation of the hammerhead
ribozyme domain known in the art. Stem II can be .gtoreq.2
base-pair long.
[0086] FIGS. 2a-d show hammerhead ribozyme substrate motifs. FIG.
2a is a diagrammatic representation of the hammerhead ribozyme
domain known in the art; FIG. 2b is a diagrammatic representation
of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature,
327, 596-600) into a substrate and enzyme portion; FIG. 2c is a
similar diagram showing the hammerhead divided by Haseloff and
Gerlach (1988, Nature, 334, 585-591) into two portions; and FIG. 2d
is a similar diagram showing the hammerhead divided by Jeffries and
Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two
portions.
[0087] FIG. 3 is a diagrammatic representation of the general
structure of a hairpin ribozyme. Helix 2 (H2) is provided with at
least 4 base pairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be
optionally provided of length 2 or more bases (preferably 3-20
bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 can be
covalently linked by one or more bases (i.e., r is .gtoreq.1 base).
Helix 1, 4 or 5 can also be extended by 2 or more base pairs (e.g.,
4-20 base pairs) to stabilize the ribozyme structure, and
preferably is a protein binding site. In each instance, each N and
N' independently is any normal or modified base and each dash
represents a potential base-pairing interaction. These nucleotides
can be modified at the sugar, base or phosphate. Complete
base-pairing is not required in the helices, but is preferred.
Helix 1 and 4 can be of any size (i.e., o and p is each
independently from 0 to any number, e.g., 20) as long as some
base-pairing is maintained. Essential bases are shown as specific
bases in the structure, but those in the art will recognize that
one or more can be modified chemically (abasic, base, sugar and/or
phosphate modifications) or replaced with another base without
significant effect. Helix 4 can be formed from two separate
molecules, i.e., without a connecting loop. The connecting loop
when present can be a ribonucleotide with or without modifications
to its base, sugar or phosphate. "q" is .gtoreq.2 bases. The
connecting loop can also be replaced with a non-nucleotide linker
molecule. H refers to bases A, U, or C. Y refers to pyrimidine
bases. ".sub.------------" refers to a covalent bond.
[0088] FIG. 4 is a representation of the general structure of the
hepatitis delta virus ribozyme domain known in the art.
[0089] FIG. 5 is a representation of the general structure of the
VS RNA ribozyme domain.
[0090] FIG. 6 is a schematic representation of an RNAseH
accessibility assay. Specifically, the left side of FIG. 6 is a
diagram of complementary DNA oligonucleotides bound to accessible
sites on the target RNA. Complementary DNA oligonucleotides are
represented by broad lines labeled A, B, and C. Target RNA is
represented by the thin, twisted line. The right side of FIG. 6 is
a schematic of a gel separation of uncut target RNA from a cleaved
target RNA. Detection of target RNA is by autoradiography of
body-labeled, T7 transcript. The bands common to each lane
represent uncleaved target RNA; the bands unique to each lane
represent the cleaved products.
[0091] FIG. 7 shows the effect of hammerhead ribozymes targeted
against flt-1 receptor on the binding of VEGF to the surface of
human microvascular endothelial cells. Sequences of the ribozymes
used are shown in Table II; the length of stem II region is 3 bp.
The hammerhead ribozymes were chemically modified such that the
ribozyme consists of ribose residues at five positions (see FIG.
11); U4 and U7 positions contain 2'-NH.sub.2 modifications, the
remaining nucleotide positions contain 2'-O-methyl substitutions;
four nucleotides at the 5' terminus contains phosphorothioate
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose. The results of two
separate experiments are shown as separate bars for each set. Each
bar represents the average of triplicate samples. The standard
deviation is shown with error bars. For the flt-1 data, 500 nM
ribozyme (3:1 charge ratio with LipofectAMINE.RTM.) was used.
Control 1-10 is the control for ribozymes 307-2797, control 11-20
is the control for ribozymes 3008-5585. The Control 1-10 and
Control 11-20 represent the treatment of cells with
LipofectAMINE.RTM. alone without any ribozymes.
[0092] FIG. 8 shows the effect of hammerhead ribozymes targeted
against KDR receptor on the binding of VEGF to KDR on the surface
of human microvascular endothelial cells. Sequences of the
ribozymes used are shown in Table IV; the length of stem II region
is 3 bp. The hammerhead ribozymes were chemically modified such
that the ribozyme consists of ribose residues at five positions
(see FIG. 11); U4 and U7 positions contain 2'-NH.sub.2
modifications, the remaining nucleotide positions contain
2'-O-methyl substitutions; four nucleotides at the 5' terminus
contains phosphorothioate substitutions. Additionally, the 3' end
of the ribozyme contains a 3'-3' linked inverted abasic
deoxyribose. The Control 1-10 and Control 11-20 represent the
treatment of cells with LipofectAMINE.RTM. alone without any
ribozymes. Irrel. RZ, is a control experiment wherein the cells are
treated with a non-KDR-targeted ribozyme complexed with
Lipofectamine.RTM.. 200 nM ribozyme (3:1 charge ratio with
LipofectAMINE.RTM.) was used. In addition to the KDR-targeted
ribozymes, the effect on VEGF binding of a ribozyme targeted to an
irrelevant mRNA (irrel. RZ) is also shown. Because the affinity of
KDR for VEGF is about 10-fold lower than the affinity of flt-1 for
VEGF, a higher concentration of VEGF was used in the binding
assay.
[0093] FIG. 9 shows the specificity of hammerhead ribozymes
targeted against flt-1 receptor. Inhibition of the binding of VEGF,
urokinase plasminogen activator (UPA) and fibroblast growth factor
(FGF) to their corresponding receptors as a function of anti-FLT
ribozymes is shown. The sequence and description of the ribozymes
used are as described in FIG. 7 above. The average of triplicate
samples is given; percent inhibition as calculated below.
[0094] FIG. 10 shows the inhibition of the proliferation of Human
aortic endothelial cells (HAEC) mediated by phosphorothioate
antisense oligodeoxynucleotides targeted against human KDR receptor
RNA. Cell proliferation (O.D. 490) as a function of antisense
oligodeoxynucleotide concentration is shown. KDR 21AS represents a
21 nt phosphorothioate antisense oligodeoxynucleotide targeted
against KDR RNA. KDR 21 Scram represents a 21 nt phosphorothioate
oligodeoxynucleotide having a scrambled sequence. LF represents the
lipid carrier Lipofectin.
[0095] FIGS. 11A and B show a diagrammatic representation of
hammerhead ribozymes targeted against flt-1 RNA and in vitro
cleavage of flt-1 RNA by hammerhead ribozymes. The hammerhead (HH)
ribozymes were chemically modified such that the ribozyme consists
of ribose residues at five positions; U4 and U7 positions contain
2'-NH.sub.2 modifications, the remaining nucleotide positions
contain 2'-O-methyl substitutions; four nucleotides at the 5'
terminus contains phosphorothioate substitutions. Additionally, the
3' end of the ribozyme contains a 3'-3' linked inverted abasic
deoxyribose (designated as 3'-1H). FIG. 11A shows hammerhead
ribozymes 1358 HH-A and 4229 HH-A, which contain a 3 base-paired
stem II region. FIG. 11B shows hammerhead ribozymes 1358 HH-B and
4229 HH-B, which contain a 4 base-paired stem II region. FIGS. 11C
and 1 ID show in vitro cleavage kinetics of hammerhead ribozymes
targeted against sites 1358 and 4229 within the flt-1 RNA.
[0096] FIG. 12A shows a diagrammatic representation of hammerhead
(HH) ribozymes targeted against sites 1358 and 4229 within the
flt-1 RNA. The hammerhead (HH) ribozymes were chemically modified
such that the ribozyme consists of ribose residues at five
positions; U4 position contains 2'-C-allyl modification, the
remaining nucleotide positions contain 2'-O-methyl substitutions;
four nucleotides at the 5' terminus contains phosphorothioate
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose (designated as 3'-1H).
FIG. 12B shows a graphical representation of the inhibition of cell
proliferation mediated by 1358HH and 4229HH ribozymes.
[0097] FIG. 13 shows inhibition of human microvascular endothelial
cell proliferation mediated by anti-KDR hammerhead ribozymes. The
figure is a graphical representation of the inhibition of cell
proliferation mediated by hammerhead ribozymes targeted against
sites 527, 730, 3702 and 3950 within the KDR RNA. Irrelevant HH RZ
is a hammerhead ribozyme targeted to an irrelevant target. All of
these ribozymes, including the Irrelevant HH RZ, were chemically
modified such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions; four nucleotides at the 5' termini contain
phosphorothioate substitutions. Additionally, the 3' end of the
ribozyme contains a 3'-3' linked inverted abasic deoxyribose
(3'-1H).
[0098] FIG. 14 shows in vitro cleavage of KDR RNA by hammerhead
ribozymes. The hammerhead (HH) ribozymes were chemically modified
such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose (designated as 3'-1H). 726
HH and 527 HH contain 4 base-paired stem II region. Percent in
vitro cleavage kinetics as a function of time of HH ribozymes
targeted against sites 527 and 726 within the KDR RNA is shown.
[0099] FIG. 15 shows in vitro cleavage of KDR RNA by hammerhead
ribozymes. The hammerhead (HH) ribozymes were chemically modified
such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose (designated as 3'-1H).
3702 HH and 3950 HH contain 4 base-paired stem II region. Percent
in vitro cleavage kinetics as a function of time of HH ribozymes
targeted against sites 3702 and 3950 within the KDR RNA is
shown.
[0100] FIG. 16 shows in vitro cleavage of RNA by hammerhead
ribozymes that are targeted to sites that are conserved between
flt-1 and KDR RNA. The hammerhead (HH) ribozymes were chemically
modified such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose (designated as 3'-1H).
FLT/KDR-1HH ribozyme was synthesized with either a 4 base-paired or
a 3 base-paired stem II region. FLT/KDR-1HH can cleave site 3388
within flt-1 RNA and site 3151 within KDR RNA. Percent in vitro
cleavage kinetics as a function of time of HH ribozymes targeted
against sites 3702 and 3950 within the KDR RNA is shown.
[0101] FIG. 17 shows inhibition of human microvascular endothelial
cell proliferation mediated by anti-KDR and anti-flt-1 hammerhead
ribozymes. The figure is a graphical representation of the
inhibition of cell proliferation mediated by hammerhead ribozymes
targeted against sites KDR sites-527, 726 or 3950 or flt-1 site
4229. The figure also shows enhanced inhibition of cell
proliferation by a combination of flt-1 and KDR hammerhead
ribozymes. 4229+527, indicates the treatment of cells with both the
flt 4229 and the KDR 527 ribozymes. 4229+726, indicates the
treatment of cells with both the flt 4229 and the KDR 726
ribozymes. 4229+3950, indicates the treatment of cells with both
the flt 4229 and the KDR 3950 ribozymes. VEGF--, indicates the
basal level of cell proliferation in the absence of VEGF. A,
indicates catalytically active ribozyme; I, indicates catalytically
inactive ribozyme. All of these ribozymes were chemically modified
such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions; four nucleotides at the 5' termini contain
phosphorothioate substitutions. Additionally, the 3' end of the
ribozyme contains a 3'-3' linked inverted abasic deoxyribose
(3'-1H).
[0102] FIG. 18 shows the inhibition of VEGF-induced angiogenesis in
rat cornea mediated by anti-flt-1 hammerhead ribozyme. All of these
ribozymes were chemically modified such that the ribozyme consists
of ribose residues at five positions; U4 position contains
2'-C-allyl modifications, the remaining nucleotide positions
contain 2'-O-methyl substitutions; four nucleotides at the 5'
termini contain phosphorothioate substitutions. Additionally, the
3' end of the ribozyme contains a 3'-3' linked inverted abasic
deoxyribose (3'-iH). A decrease in the Surface Area corresponds to
a reduction in angiogenesis. VEGF alone corresponds to treatment of
the cornea with VEGF and no ribozymes. Vehicle alone corresponds to
the treatment of the cornea with the carrier alone and no VEGF.
This control gives a basal level of Surface Area. Active 4229 HH,
corresponds to the treatment of cornea with the flt-1 4229 HH
ribozyme in the absence of any VEGF. This control also gives a
basal level of Surface Area. Active 4229 HH+VEGF, corresponds to
the co-treatment of cornea with the flt-1 4229 HH ribozyme and
VEGF. Inactive 4229 HH+VEGF, corresponds to the co-treatment of
cornea with a catalytically inactive version of 4229 HH ribozyme
and VEGF.
[0103] FIG. 19 shows ribozyme-mediated inhibition of cell
proliferation. Cultured HMVEC-d were treated with ribozyme or
attenuated controls as LIPOFECTAMINE.TM. complexes. After
treatment, cells were stimulated with VEGF.sub.165 or bFGF and
allowed to grow for 48 h prior to determining the cell number. Each
ribozyme was tested in triplicate at three concentrations and data
are presented as mean cell number per well +SD. The data obtained
following ribozyme treatment and VEGF stimulation are presented in
panels A & B for anti-Flt-1 ribozymes and panels D & E for
anti-KDR ribozymes. Representative data obtained following ribozyme
treatment and bFGF stimulation are shown in panel C for one
anti-Flt-1 ribozyme and in panel F for one anti-KDR ribozyme. In
all panels, active ribozymes are represented with filled symbols;
attenuated controls with open symbols. In addition to the ribozymes
and attenuated controls listed in Table XII, a second set having
the same sequences but with an additional basepair in the "stem II"
region of the ribozyme are also shown for VEGF-induced
proliferation studies. These 4 bp stem II ribozymes and attenuated
controls have one additional base pair such that the stem II/loop
sequence is ggccgaaaggcc (SEQ ID NO: 20823). Therefore, ribozymes
and controls with 3 or 4 basepair stem IIs are denoted with circles
and squares, respectively. The data for one irrelevant ribozyme
(filled triangle, panel B) are also shown. This irrelevant ribozyme
contains an active core sequence but has no binding site in either
Flt-1 or KDR mRNA. Its sequence is
5'-g.sub.sa.sub.sa.sub.sg.sub.sgaacUGAuGaggccgaaaggccGaaAgauggcT-3'
(SEQ ID NO: 20824) with modifications as in Table XII except that T
indicates a 3'-3' inverted deoxythymidine. For reference, the
average number of cells in control wells after 48 h in the absence
of VEGF or bFGF for each of the panels are as follows: A, B, C,
12477.+-.617; D, E, F, 17182.+-.1053.
[0104] FIG. 20 shows target specificity of anti-Flt-1 and KDR
ribozymes. Cultured HMVEC-d were treated with LIPOFECTAMINE.TM.
complexes containing 200 nM active ribozyme (A) or attenuated
control (C) and analyzed by RNAse protection following 24 h of
VEGF-stimulated growth. Data obtained for ribozymes and attenuated
controls that target Flt-1 site 4229 or KDR site 726 are shown.
Data were normalized to the level of an internal mRNA control
(cyclophilin) and are presented as percent decrease in Flt-1 (left
panel) or KDR mRNA (right panel) relative to an untreated control.
Error bars indicate the range of duplicate samples.
[0105] FIG. 21 shows antiangiogenic efficacy of ribozyme in the rat
corneal model of VEGF-induced angiogenesis. The percent inhibition
of VEGF-induced angiogenesis for locally administered anti-Flt-1
(site 4229) ribozyme (filled circles) and their attenuated controls
(open circles) are plotted over the dose range tested. Pixels
associated with background structures including the iris were
subtracted from all treatment groups. Data are expressed as mean
percent reduction in VEGF-induced angiogenesis .+-.SEM. *p<0.05
relative to VEGF/vehicle treated controls by Dunnett's, **p<0.05
relative to attenuated dose-matched controls by Tukey-Kramer.
[0106] FIG. 22 shows antiangiogenic efficacy of ribozyme in the rat
corneal model of VEGF-induced angiogenesis. The percent inhibition
of VEGF-induced angiogenesis for locally administered anti-KDR
(site 726) ribozyme (filled circles) and their attenuated controls
(open circles) are plotted over the dose range tested. Pixels
associated with background structures including the iris were
subtracted from all treatment groups. Data are expressed as mean
percent reduction in VEGF-induced angiogenesis .+-.SEM. *p<0.05
relative to VEGF/vehicle treated controls by Dunnett's, **p<0.05
relative to attenuated dose-matched controls by Tukey-Kramer.
[0107] FIG. 23 shows the effect of subcutaneous bolus
administration of ANGIOZYME.TM. in a mouse Lewis Lung Carcinoma
(LLC) model.
[0108] FIG. 24 shows the effect of ANGIOZYME.TM. in combination
with gemcitabine or cyclophosphamide on primary tumor growth in the
mouse LLC model.
[0109] FIG. 25 shows the effect of ANGIOZYME.TM. in combination
with gemcitabine or cyclophosphamide on tumor metastases in the
mouse LLC model.
[0110] FIG. 26 shows a secondary structure model of ANGIOZYME.TM.
ribozyme bound to its RNA target.
[0111] FIG. 27 shows a time course of inhibition of primary tumor
growth following systemic administration of ANGIOZYME.TM. in the
LLC mouse model.
[0112] FIG. 28 shows inhibition of primary tumor growth following
systemic administration of ANGIOZYME.TM. according to a certain
dosing regimen in the LLC mouse model.
[0113] FIG. 29 shows a dose-dependent inhibition of tumor
metastases following systemic administration of ANGIOZYME.TM. in a
mouse colorectal model.
[0114] FIG. 30 shows inhibition of liver metastases following
systemic administration of ANGIOZYME.TM. in a mouse colorectal
model.
[0115] FIG. 31 is a graph showing the plasma concentration profile
of ANGIOZYME.TM. after a single subcutaneous (SC) dose of 10, 30,
100 or 300 mg/m.sup.2.
[0116] FIG. 32 shows an example of the Zinzyme enzymatic nucleic
acid motif that is chemically stabilized (see for example Beigelman
et al., International PCT publication No. WO 99/55857, incorporated
by reference herein; also referred to as Class A or Class II
Motif). The Zinzyme motif is a class of enzymatic nucleic molecules
that do not require the presence of a ribonucleotide (2'-OH) group
for its activity.
[0117] FIG. 33 shows an example of a DNAzyme motif described
generally, for example in Santoro et al., 1997, PNAS, 94, 4262.
[0118] FIGS. 34 A and B show a mouse model protocol and results of
proliferative retinopathy. FIG. 34A shows anoutline for the mouse
model of proliferative retinopathy showing the points of ribozyme
administration. FIG. 34B shows a graph demonstrating the efficacy
of a VEGF-receptor-targeted enzymatic nucleic acid molecule in a
mouse model of proliferative retinopathy.
DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
[0119] Antisense: Antisense molecules can be modified or unmodified
RNA, DNA, or mixed polymer oligonucleotides and primarily function
by specifically binding to matching sequences resulting in
inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm,
20-33). The antisense oligonucleotide binds to target RNA by Watson
Crick base-pairing and blocks gene expression by preventing
ribosomal translation of the bound sequences either by steric
blocking or by activating RNase H enzyme. Antisense molecules can
also alter protein synthesis by interfering with RNA processing or
transport from the nucleus into the cytoplasm (Mukhopadhyay &
Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
[0120] In addition, binding of single stranded DNA to RNA can
result in nuclease degradation of the heteroduplex (Wu-Pong, supra;
Crooke, supra). To date, the only backbone modified DNA chemistry
which acts as substrates for RNase H are phosphorothioates and
phosphorodithioates. Recently it has been reported that 2'-arabino
and 2'-fluoro arabino-containing oligos can also activate RNase H
activity.
[0121] A number of antisense molecules have been described that
utilize novel configurations of chemically modified nucleotides,
secondary structure, and/or RNase H substrate domains (Woolf et
al., International PCT Publication No. WO 98/13526; Thompson et
al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998;
Hartmann et al., U.S. Ser. No. 60/101,174 which was filed on Sep.
21, 1998) all of these are incorporated by reference herein in
their entirety.
[0122] Triplex Forming Oligonucleotides (TFO): Single stranded DNA
can be designed to bind to genomic DNA in a sequence specific
manner. TFOs are comprised of pyrimidine-rich oligonucleotides
which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong,
supra). The resulting triple helix composed of the DNA sense, DNA
antisense, and TFO disrupts RNA synthesis by RNA polymerase. The
TFO mechanism can result in gene expression or cell death since
binding may be irreversible (Mukhopadhyay & Roth, supra)
[0123] 2-5A Antisense Chimera: The 2-5A system is an interferon
mediated mechanism for RNA degradation found in higher vertebrates
(Mitra et al., 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two
types of enzymes, 2-5A synthetase and RNase L, are required for RNA
cleavage. The 2-5A synthetases require double stranded RNA to form
2'-5' oligoadenylates (2-5A). 2-5A then acts as an allosteric
effector for utilizing RNase L which has the ability to cleave
single stranded RNA. The ability to form 2-5A structures with
double stranded RNA makes this system particularly useful for
inhibition of viral replication.
[0124] (2'-5') oligoadenylate structures can be covalently linked
to antisense molecules to form chimeric oligonucleotides capable of
RNA cleavage (Torrence, supra). These molecules putatively bind and
activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex
then binds to a target RNA molecule which can then be cleaved by
the RNase enzyme.
[0125] Enzymatic Nucleic Acid: Seven basic varieties of
naturally-occurring enzymatic RNAs are presently known. In
addition, several in vitro selection (evolution) strategies (Orgel,
1979, Proc. R. Soc. London, B 205, 435) have been used to evolve
new nucleic acid catalysts capable of catalyzing cleavage and
ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87;
Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific
American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel
et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93;
Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.
Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,
4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994,
supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995,
supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are
incorporated by reference herein). Each can catalyze a series of
reactions including the hydrolysis of phosphodiester bonds in trans
(and thus can cleave other RNA molecules) under physiological
conditions.
[0126] Enzymatic nucleic acid molecules of this invention block to
some extent VEGF-R (specifically flt-1 and flk-1/KDR) production
and can be used to treat disease or diagnose such disease.
Enzymatic nucleic acid molecules are delivered to cells in culture,
to cells or tissues in animal models of angiogenesis and/or RA and
to human cells or tissues ex vivo or in vivo. Enzymatic nucleic
acid molecule cleavage of VEGF-R RNAs (specifically RNAs that
encode flt-1 and flk-1/KDR) in these systems can alleviate disease
symptoms.
[0127] The enzymatic nature of enzymatic nucleic acid molecules,
such as ribozymes, has significant advantages, such as the
concentration of enzymatic nucleic acid necessary to affect a
therapeutic treatment is lower. This advantage reflects the ability
of the enucleic acid molecule to act enzymatically. Thus, a single
enzymatic nucleic acid molecule is able to cleave many molecules of
target RNA. In addition, the enzymatic nucleic acid molecule is a
highly specific inhibitor, with the specificity of inhibition
depending not only on the base-pairing mechanism of binding to the
target RNA, but also on the mechanism of target RNA cleavage.
Single mismatches, or base-substitutions, near the site of cleavage
can be chosen to completely eliminate catalytic activity of an
enzymatic nucleic acid molecule.
[0128] Nucleic acid molecules having an endonuclease enzymatic
activity are able to repeatedly cleave other separate RNA molecules
in a nucleotide base sequence-specific manner. Such enzymatic
nucleic acid molecules can be targeted to virtually any RNA
transcript, and achieved efficient cleavage in vitro (Zaug et al.,
324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al.,
84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein
Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585,
1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic
Acids Research 1371, 1989; Santoro et al., 1997 supra).
[0129] Because of their sequence specificity, enzymatic nucleic
acids, such as trans-cleaving ribozymes can be used as therapeutic
agents for human disease (Usman & McSwiggen, 1995 Ann. Rep.
Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem.
38, 2023-2037). Enzymatic nucleic acid molecules can be designed to
cleave specific RNA targets within the background of cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates
protein expression from that RNA. In this manner, synthesis of a
protein associated with a disease state can be selectively
inhibited.
Target Sites
[0130] Targets for useful enzymatic nucleic acid molecules and
antisense nucleic acids can be determined as disclosed in Draper et
al., WO 93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO
94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat.
No. 5,525,468, and hereby incorporated by reference herein in
totality. Other examples include the following PCT applications
which concern inactivation of expression of disease-related genes:
WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference
herein. Rather than repeat the guidance provided in those documents
here, below are provided specific examples of such methods, not
limiting to those with skill in the art. Enzymatic nucleic acid
molecules to such targets are designed as described in those
applications and synthesized to be tested in vitro and in vivo, as
also described. The sequence of human and mouse flt-1, KDR and/or
flk-1 mRNAs were screened for optimal enzymatic nucleic acid target
sites using a computer folding algorithm. Hammerhead, hairpin, NCH,
or G-Cleaver ribozyme cleavage sites were identified. These sites
are shown in Tables II to IX, XIV-XIX, XXII, and XXIII (all
sequences are 5' to 3' in the tables; X can be any base-paired
sequence, the actual sequence is not relevant here). The nucleotide
base position is noted in the Tables as that site to be cleaved by
the designated type of ribozyme. While mouse and human sequences
can be screened and enzymatic nucleic acid molecules thereafter
designed, the human targeted sequences are of most utility.
However, as discussed in Stinchcomb et al., WO 95/23225, mouse
targeted enzymatic nucleic acid can be useful to test efficacy of
action of the enzymatic nucleic acid molecule prior to testing in
humans. The nucleotide base position is noted in the Tables as that
site to be cleaved by the designated type of enzymatic nucleic
acid.
[0131] For example, hammerhead or hairpin ribozymes were designed
that could bind and cleave target RNA in a sequence-specific
manner. The ribozymes were individually analyzed by computer
folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706)
to assess whether the ribozyme sequences fold into the appropriate
secondary structure. Those ribozymes with unfavorable
intramolecular interactions between the binding arms and the
catalytic core were eliminated from consideration. Varying binding
arm lengths can be chosen to optimize activity.
[0132] Referring to FIG. 6, mRNA was screened for accessible
cleavage sites by the method described generally in Draper et al.,
PCT WO93/23569, hereby incorporated by reference herein. Briefly,
DNA oligonucleotides complementary to potential hammerhead or
hairpin ribozyme cleavage sites were synthesized. A polymerase
chain reaction was used to generate substrates for T7 RNA
polymerase transcription from human and mouse flt-1, KDR and/or
flk-1 cDNA clones. Labeled RNA transcripts were synthesized in
vitro from the templates. The oligonucleotides and the labeled
transcripts were annealed, RNAseH was added and the mixtures were
incubated for the designated times at 37.degree. C. Reactions were
stopped and RNA separated on sequencing polyacrylamide gels. The
percentage of the substrate cleaved was determined by
autoradiographic quantitation using a PhosphorImaging system. From
these data, antisense oligonucleotides, and ribozymes, such as
hammerhead or hairpin ribozyme sites are chosen as the most
accessible.
[0133] Ribozymes of the hammerhead or hairpin motif were designed
to anneal to various sites in the mRNA message. The binding arms
are complementary to the target site sequences described above. The
ribozymes were chemically synthesized. The method of synthesis used
follows the procedure for normal RNA synthesis as described below
and in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et
al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995
Nucleic Acids Res. 23, 2677-2684.
Synthesis of Nucleic Acid Molecules
[0134] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods, and the therapeutic
cost of such molecules is prohibitive. In this invention, small
nucleic acid motifs ("small refers to nucleic acid motifs no more
than 100 nucleotides in length, preferably no more than 80
nucleotides in length, and most preferably no more than 50
nucleotides in length; e.g., antisense oligonucleotides, hammerhead
or the hairpin ribozymes) are preferably used for exogenous
delivery. The simple structure of these molecules increases the
ability of the nucleic acid to invade targeted regions of RNA
structure. Exemplary molecules of the instant invention were
chemically synthesized, and others can similarly be synthesized.
Oligodeoxyribonucleotides were synthesized using standard protocols
as described in Caruthers et al., 1992, Methods in Enzymology 211,
3-19, and is incorporated herein by reference.
[0135] The method of synthesis used for normal RNA including
certain enzymatic nucleic acid molecules follows the procedure as
described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845;
Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et
al., 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common
nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
In a non-limiting example, small scale syntheses were conducted on
a 394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.mol
scale protocol with a 7.75 min coupling step for alkylsilyl
protected nucleotides and a 2.5 min coupling step for
2'-O-methylated nucleotides. Table XI outlines the amounts and the
contact times of the reagents used in the synthesis cycle.
Alternatively, syntheses at the 0.2 .mu.mol scale can be done on a
96-well plate synthesizer, such as the instrument produced by
Protogene (Palo Alto, Calif.) with minimal modification to the
cycle. A 15-fold excess (31 .mu.L of 0.1 M=3.1 .mu.mol) of
phosphoramidite and a 38.7-fold excess of S-ethyl tetrazole (31
.mu.L of 0.25 M=7.75 .mu.mol) relative to polymer-bound 5'-hydroxyl
was used in each coupling cycle. Average coupling yields on the 394
Applied Biosystems, Inc. synthesizer, determined by calorimetric
quantitation of the trityl fractions, were 97.5-99%. Other
oligonucleotide synthesis reagents for the 394 Applied Biosystems,
Inc. synthesizer; detritylation solution was 3% TCA in methylene
chloride (ABI); capping was performed with 16% N-methyl imidazole
in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF
(ABI); oxidation solution was 16.9 mM I.sub.2, 49 mM pyridine, 9%
water in THF (PERSEPTIVE.TM.). Burdick & Jackson Synthesis
Grade acetonitrile was used directly from the reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) was made up from
the solid obtained from American International Chemical, Inc.
[0136] Deprotection of the RNA was performed using either a two-pot
or one-pot protocol. For the two-pot protocol, the polymer-bound
trityl-on oligoribonucleotide was transferred to a 4 mL glass screw
top vial and suspended in a solution of 40% aq. methylamine (1 mL)
at 65.degree. C. for 10 min. After cooling to -20.degree. C., the
supernatant was removed from the polymer support. The support was
washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and
the supernatant was then added to the first supernatant. The
combined supernatants, containing the oligoribonucleotide, were
dried to a white powder. The base deprotected oligoribonucleotide
was resuspended in anhydrous TEA/HF/NMP solution (300 .mu.L of a
solution of 1.5 mL N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL
TEA.3HF to provide a 1.4 M HF concentration) and heated to
65.degree. C. After 1.5 h, the oligomer was quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0137] Alternatively, for the one-pot protocol, the polymer-bound
trityl-on oligoribonucleotide was transferred to a 4 mL glass screw
top vial and suspended in a solution of 33% ethanolic
methylamine/DMSO:1/1 (0.8 mL) at 65.degree. C. for 15 min. The vial
was brought to r.t. TEA.3HF (0.1 mL) was added and the vial was
heated at 65.degree. C. for 15 min. The sample was cooled at
-20.degree. C. and then quenched with 1.5 M NH.sub.4HCO.sub.3.
[0138] For purification of the trityl-on oligomers, the quenched
NH.sub.4HCO.sub.3 solution was loaded onto a C-18 containing
cartridge that had been prewashed with acetonitrile followed by 50
mM TEAA. After washing the loaded cartridge with water, the RNA was
detritylated with 0.5% TFA for 13 min. The cartridge was then
washed again with water, salt exchanged with 1 M NaCl and washed
with water again. The oligonucleotide was then eluted with 30%
acetonitrile.
[0139] Inactive hammerhead ribozymes or binding attenuated control
(BAC) oligonucleotides) were synthesized by substituting a U for
G.sub.5 and a U for A.sub.14 (numbering from Hertel, K. J., et al.,
1992, Nucleic Acids Res., 20, 3252).
[0140] The average stepwise coupling yields were >98% (Wincott
et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary
skill in the art will recognize that the scale of synthesis can be
adapted to be larger or smaller than the example described above
including but not limited to 96 well format, all that is important
is the ratio of chemicals used in the reaction.
[0141] Alternatively, the nucleic acid molecules of the present
invention can be synthesized separately and joined together by
ligation (Moore et al., 1992, Science 256, 9923; Draper et al.,
International PCT publication No. WO 93/23569; Shabarova et al.,
1991, Nucleic Acids Research 19, 4247)
[0142] Enzymatic nucleic acid molecules are modified extensively to
enhance stability by modification with nuclease resistant groups,
for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H
(for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et
al., 1994 Nucleic Acids Symp. Ser. 31, 163). Enzymatic nucleic acid
molecules are purified by gel electrophoresis using general methods
or are purified by high pressure liquid chromatography (HPLC; See
Wincott et al., Supra, the totality of which is hereby incorporated
herein by reference) and are resuspended in water.
[0143] For example, the sequences of the ribozymes that are
chemically synthesized, useful in this study, are shown in Tables
II to IX, XIV-XIX, XXII, and XXIII. Those in the art will recognize
that these sequences are representative only of many more such
sequences where the enzymatic portion of the ribozyme (all but the
binding arms) is altered to affect activity. Stem-loop IV sequence
of hairpin ribozymes listed in, for example, Table III
(5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or
insertion) to contain any sequence, provided a minimum of two
base-paired stem structure can form. Preferably, no more than 200
bases are inserted at these locations. The sequences listed in
Tables II to X, XII-XIX, XXII, and XXIII may be formed of
ribonucleotides or other nucleotides or non-nucleotides. Such
ribozymes with enzymatic activity are equivalent to the ribozymes
described specifically in the Tables.
Optimizing Activity of the Nucleic Acid Molecule of the
Invention.
[0144] Enzymatic nucleic acid activity can be optimized as
described by Stinchcomb et al., supra. The details will not be
repeated here, but include altering the length of the enzymatic
nucleic acid binding arms (stems I and III, see FIG. 2c), or
chemically synthesizing enzymatic nucleic acid with modifications
that prevent their degradation by serum ribonucleases (see e.g.,
Eckstein et al., International Publication No. WO 92/07065;
Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science
253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17,
334; Usman et al., International Publication No. WO 93/15187; Rossi
et al., International Publication No. WO 91/03162; Beigelman et
al., 1995 J. Biol. Chem. in press; as well as Sproat, U.S. Pat. No.
5,334,711 which describe various chemical modifications that can be
made to the sugar moieties of enzymatic RNA molecules).
Modifications which enhance their efficacy in cells, and removal of
stem II bases to shorten RNA synthesis times and reduce chemical
requirements are desired. (All these publications are hereby
incorporated by reference herein).
[0145] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into enzymatic
nucleic acid molecules without significantly effecting catalysis
and with significant enhancement in their nuclease stability and
efficacy. For example, enzymatic nucleic acid molecules are
modified to enhance stability and/or enhance catalytic activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H, nucleotide base
modifications (for a review see Usman and Cedergren, 1992 TIBS 17,
34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et
al., 1996 Biochemistry 35, 14090). Sugar modification of enzymatic
nucleic acid molecules have been extensively described in the art
(see Eckstein et al., International Publication PCT No. WO
92/07065; Perrault et al. Nature 1990, 344, 565-568; Pieken et al.
Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.
Sci. 1992, 17, 334-339; Usman et al. International Publication PCT
No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et
al., 1995 J. Biol. Chem. 270, 25702; all of the references are
hereby incorporated in their totality by reference herein). Such
publications describe general methods and strategies to determine
the location of incorporation of sugar, base and/or phosphate
modifications and the like into enzymatic nucleic acid without
inhibiting catalysis, and are incorporated by reference herein. In
view of such teachings, similar modifications can be used as
described herein to modify the nucleic acid catalysts of the
instant invention.
[0146] Nucleic acid catalysts having chemical modifications which
maintain or enhance enzymatic activity are provided. Such nucleic
acid is also generally more resistant to nucleases than unmodified
nucleic acid. Thus, in a cell and/or in vivo the activity may not
be significantly lowered. As exemplified herein such enzymatic
nucleic acid molecules are useful in a cell and/or in vivo even if
activity over all is reduced 10 fold (Burgin et al., 1996,
Biochemistry, 35, 14090). Such enzymatic nucleic acid molecules
herein are said to "maintain" the enzymatic activity of an all RNA
enzymatic nucleic acid molecules.
[0147] Therapeutic nucleic acid molecules (e.g., enzymatic nucleic
acid molecules and antisense nucleic acid molecules) delivered
exogenously must optimally be stable within cells until translation
of the target RNA has been inhibited long enough to reduce the
levels of the undesirable protein. This period of time varies
between hours to days depending upon the disease state. Clearly,
these nucleic acid molecules must be resistant to nucleases in
order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of nucleic acid molecules
described in the instant invention and in the art have expanded the
ability to modify nucleic acid molecules by introducing nucleotide
modifications to enhance their nuclease stability as described
above.
[0148] By "enhanced enzymatic activity" is meant to include
activity measured in cells and/or in vivo where the activity is a
reflection of both catalytic activity and enzymatic nucleic acid
stability. In this invention, the product of these properties is
increased or not significantly (less that 10 fold) decreased in
vivo compared to an all RNA enzymatic nucleic acid molecule.
[0149] In one embodiment, the nucleic acid molecules comprise a 5'
and/or a 3'-cap structure.
[0150] By "cap structure" is meant chemical modifications, which
have been incorporated at the terminus of the oligonucleotide (see
for example Wincott et al., WO 97/26270, incorporated by reference
herein). These terminal modifications protect the nucleic acid
molecule from exonuclease degradation, and may help in delivery
and/or localization within a cell. The cap may be present at the
5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or may be
present on both terminus. In non-limiting examples: the 5'-cap is
selected from the group comprising inverted abasic residue
(moiety), 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl)
nucleotide, 4'-thio nucleotide, carbocyclic nucleotide;
1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides;
modified base nucleotide; phosphorodithioate linkage;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl
nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic
moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic
moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate;
3'-phosphorothioate; phosphorodithioate; or bridging or
non-bridging methylphosphonate moiety (for more details see
Beigelman et al., International PCT publication No. WO 97/26270,
incorporated by reference herein).
[0151] In yet another embodiment, the 3'-cap is selected from a
group comprising, 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moeity; 5'-5'-inverted abasic moeity;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moeities (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0152] By the term "non-nucleotide" is meant any group or compound
which can be incorporated into a nucleic acid chain in the place of
one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their enzymatic activity. The group or compound is abasic in that
it does not contain a commonly recognized nucleotide base, such as
adenosine, guanine, cytosine, uracil or thymine.
[0153] By "nucleotide" as used herein is as recognized in the art
to include natural bases (standard), and modified bases well known
in the art. Such bases are generally located at the 1' position of
a sugar moiety. A nucleotide generally comprises a base, sugar and
a phosphate group. The nucleotide may also be abasic, i.e., lacking
a base. The nucleotides can be unmodified or modified at the sugar,
phosphate and/or base moiety, (also referred to interchangeably as
nucleotide analogs, modified nucleotides, non-natural nucleotides,
non-standard nucleotides and other; see for example, Usman and
McSwiggen, supra; Eckstein et al., International PCT Publication
No. WO 92/07065; Usman et al., International PCT Publication No. WO
93/15187; all hereby incorporated by reference herein). Several
examples of modified nucleic acid bases are known in the art and
has recently been summarized by Limbach et al., 1994, Nucleic Acids
Res. 22, 2183. Some of the non-limiting examples of base
modifications that can be introduced into enzymatic nucleic acids
without significantly effecting their catalytic activity include,
inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil,
dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,
5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or
6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et
al., 1996, Biochemistry, 35, 14090).
[0154] By "modified bases" in this aspect is meant nucleotide bases
other than adenine, guanine, cytosine and uracil at 1' position or
their equivalents; such bases may be used within the catalytic core
of the enzyme and/or in the substrate-binding regions.
[0155] By "abasic" is meant sugar moieties lacking a base or having
other chemical groups in place of a base at the 1' position.
[0156] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, uracil joined to the 1' carbon of
.beta.-D-ribo-furanose.
[0157] By "modified nucleoside" is meant any nucleotide base that
contains a modification in the chemical structure of an unmodified
nucleotide base, sugar and/or phosphate.
[0158] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH.sub.2 or
2'-O--NH.sub.2, which can be modified or unmodified. Such modified
groups are described, for example, in Eckstein et al., U.S. Pat.
No. 5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively,
which are both incorporated by reference in their entireties.
[0159] Various modifications to nucleic acid (e.g., antisense and
ribozyme) structure can be made to enhance the utility of these
molecules. Such modifications enhance shelf-life, half-life in
vitro, stability, and ease of introduction of such oligonucleotides
to the target site, e.g., to enhance penetration of cellular
membranes, and confer the ability to recognize and bind to targeted
cells.
[0160] Use of these molecules can lead to better treatment of the
disease progression by affording the possibility of combination
therapies (e.g., multiple enzymatic nucleic acid molecules targeted
to different genes, enzymatic nucleic acid molecules coupled with
known small molecule inhibitors, or intermittent treatment with
combinations of enzymatic nucleic acid molecules (including
different ribozyme motifs) and/or other chemical or biological
molecules). The treatment of patients with nucleic acid molecules
can also include combinations of different types of nucleic acid
molecules. Therapies can be devised which include a mixture of
enzymatic nucleic acid molecules (including different ribozyme
motifs), antisense and/or 2-5A chimera molecules to one or more
targets to alleviate symptoms of a disease.
Administration of Nucleic Acid Molecules
[0161] Sullivan, et al., supra, describes the general methods for
delivery of enzymatic RNA molecules. Enzymatic nucleic acid
molecules can be administered to cells by a variety of methods
known to those familiar to the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres. For some indications, enzymatic nucleic acid
molecules can be directly delivered ex vivo to cells or tissues
with or without the aforementioned vehicles. Alternatively, the
nucleic acid/vehicle combination is locally delivered by direct
injection or by use of a catheter, infusion pump or stent. Other
routes of delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of nucleic acid delivery and administration are
provided in Sullivan et al., supra and Draper et al., supra which
have been incorporated by reference herein.
[0162] Methods for the delivery of nucleic acid molecules is
described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and
Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.
Akhtar, 1995 which are both incorporated herein by reference.
Sullivan et al., PCT WO 94/02595, further describes the general
methods for delivery of enzymatic RNA molecules. These protocols
can be utilized for the delivery of virtually any nucleic acid
molecule. Nucleic acid molecules can be administered to cells by a
variety of methods known to those familiar to the art, including,
but not restricted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres. For some indications, nucleic acid
molecules can be directly delivered ex vivo to cells or tissues
with or without the aforementioned vehicles. Alternatively, the
nucleic acid/vehicle combination is locally delivered by direct
injection or by use of a catheter, infusion pump or stent. Other
routes of delivery include, but are not limited to, intravascular,
intramuscular, subcutaneous or joint injection, aerosol inhalation,
oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of nucleic acid delivery and administration are
provided in Sullivan et al., supra and Draper et al., PCT
WO93/23569 which have been incorporated by reference herein.
[0163] The molecules of the instant invention can be used as
pharmaceutical agents. Pharmaceutical agents prevent, inhibit the
occurrence, or treat (alleviate a symptom to some extent,
preferably all of the symptoms) of a disease state in a
patient.
[0164] The negatively charged polynucleotides of the invention can
be administered (e.g., RNA, DNA or protein) and introduced into a
patient by any standard means, with or without stabilizers,
buffers, and the like, to form a pharmaceutical composition. When
it is desired to use a liposome delivery mechanism, standard
protocols for formation of liposomes can be followed. The
compositions of the present invention may also be formulated and
used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions;
suspensions for injectable administration; and the like.
[0165] The present invention also includes pharmaceutically
acceptable formulations of the compounds described. These
formulations include salts of the above compounds, e.g., acid
addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and benzene sulfonic acid.
[0166] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic administration, into a cell or patient, preferably a
human. Suitable forms, in part, depend upon the use or the route of
entry, for example oral, transdermal, or by injection. Such forms
should not prevent the composition or formulation to reach a target
cell (i.e., a cell to which the negatively charged polymer is
desired to be delivered to). For example, pharmacological
compositions injected into the blood stream should be soluble.
Other factors are known in the art, and include considerations such
as toxicity and forms which prevent the composition or formulation
from exerting its effect.
[0167] By "systemic administration" is meant in vivo systemic
absorption or accumulation of drugs in the blood stream followed by
distribution throughout the entire body. Administration routes
which lead to systemic absorption include, without limitations:
intravenous, subcutaneous, intraperitoneal, inhalation, oral,
intrapulmonary and intramuscular. Each of these administration
routes expose the desired negatively charged polymers, e.g.,
nucleic acids, to an accessible diseased tissue. The rate of entry
of a drug into the circulation has been shown to be a function of
molecular weight or size. The use of a liposome or other drug
carrier comprising the compounds of the instant invention can
potentially localize the drug, for example, in certain tissue
types, such as the tissues of the reticular endothelial system
(RES). A liposome formulation which can facilitate the association
of drug with the surface of cells, such as, lymphocytes and
macrophages is also useful. This approach may provide enhanced
delivery of the drug to target cells by taking advantage of the
specificity of macrophage and lymphocyte immune recognition of
abnormal cells, such as the cancer cells.
[0168] The invention also features the use of a composition
comprising surface-modified liposomes containing poly(ethylene
glycol) lipids (PEG-modified, or long-circulating liposomes or
stealth liposomes). These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;
Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such
liposomes have been shown to accumulate selectively in tumors,
presumably by extravasation and capture in the neovascularized
target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et
al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating
liposomes enhance the pharmacokinetics and pharmacodynamics of DNA
and RNA, particularly compared to conventional cationic liposomes
which are known to accumulate in tissues of the MPS (Liu et al., J.
Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT
Publication No. WO 96/10391; Ansell et al., International PCT
Publication No. WO 96/10390; Holland et al., International PCT
Publication No. WO 96/10392; all of these are incorporated by
reference herein). Long-circulating liposomes are also likely to
protect drugs from nuclease degradation to a greater extent
compared to cationic liposomes, based on their ability to avoid
accumulation in metabolically aggressive MPS tissues such as the
liver and spleen. All of these references are incorporated by
reference herein.
[0169] The present invention also includes compositions prepared
for storage or administration which include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated
by reference herein. For example, preservatives, stabilizers, dyes
and flavoring agents may be provided. Id. at 1449. These include
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
In addition, antioxidants and suspending agents can be used.
Id.
[0170] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors which those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the negatively charged polymer.
[0171] The nucleic acid molecules of the invention and formulations
thereof can be administered orally, topically, parenterally, by
inhalation or spray or rectally in dosage unit formulations
containing conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and vehicles. The term parenteral as used
herein includes percutaneous, subcutaneous, intravascular (e.g.,
intravenous), intramuscular, or intrathecal injection or infusion
techniques and the like. In addition, there is provided a
pharmaceutical formulation comprising a nucleic acid molecule of
the invention and a pharmaceutically acceptable carrier. One or
more nucleic acid molecules of the invention can be present in
association with one or more non-toxic pharmaceutically acceptable
carriers and/or diluents and/or adjuvants, and if desired other
active ingredients. The pharmaceutical compositions containing
nucleic acid molecules of the invention can be in a form suitable
for oral use, for example, as tablets, troches, lozenges, aqueous
or oily suspensions, dispersible powders or granules, emulsion,
hard or soft capsules, or syrups or elixirs.
[0172] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate can be
employed.
[0173] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0174] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0175] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0176] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0177] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0178] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0179] The nucleic acid molecules of the invention can also be
administered in the form of suppositories, e.g., for rectal
administration of the drug. These compositions can be prepared by
mixing the drug with a suitable non-irritating excipient that is
solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Such
materials include cocoa butter and polyethylene glycols.
[0180] Nucleic acid molecules of the invention can be administered
parenterally in a sterile medium. The drug, depending on the
vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anesthetics, preservatives and buffering agents can be dissolved in
the vehicle.
[0181] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
patient per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depending upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 500 mg of an active ingredient.
[0182] It is understood that the specific dose level for any
particular patient depends upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0183] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0184] The nucleic acid molecules of the present invention can also
be administered to a patient in combination with other therapeutic
compounds to increase the overall therapeutic effect. The use of
multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.
[0185] Another means of accumulating high concentrations of a
nucleic acid molecule of the invention (e.g., ribozyme or
antisense) within cells is to incorporate the nucleic acid-encoding
sequences into a DNA or RNA expression vector. Transcription of the
nucleic acid sequences are driven from a promoter for eukaryotic
RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase III (pol III). Transcripts from pol II or pol III
promoters will be expressed at high levels in all cells; the levels
of a given pol II promoter in a given cell type will depend on the
nature of the gene regulatory sequences (enhancers, silencers,
etc.) present nearby. Prokaryotic RNA polymerase promoters are also
used, providing that the prokaryotic RNA polymerase enzyme is
expressed in the appropriate cells (Elroy-Stein and Moss, 1990
Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic
Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzymol., 217,
47-66; Zhou et al., 1990 Mol. Cell. Biol., 10, 4529-37; Thompson et
al., 1995 supra). Several investigators have demonstrated that
enzymatic nucleic acid or antisese expressed from such promoters
can function in mammalian cells (e.g. Izant and Weintraub, 1985
Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad.
Sci. USA 83, 399; Kashani-Sabet et al., 1992 Antisense Res. Dev.,
2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89,
10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et
al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et
al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl.
Acad. Sci. U.S.A., 90, 8000-4; Thompson et al., 1995 Nucleic Acids
Res. 23, 2259). The above nucleic acid transcription units can be
incorporated into a variety of vectors for introduction into
mammalian cells, including but not restricted to, plasmid DNA
vectors, viral DNA vectors (such as adenovirus or adeno-associated
virus vectors), or viral RNA vectors (such as retroviral or
alphavirus vectors).
[0186] In one embodiment of the invention, a transcription unit
expressing an enzymatic nucleic acid that cleaves RNAs that encode
flt-1, KDR and/or flk-1 are inserted into a plasmid DNA vector or
an adenovirus or adeno-associated virus DNA viral vector or a
retroviral RNA vector. Viral vectors have been used to transfer
genes and lead to either transient or long term gene expression
(Zabner et al., 1993 Cell 75, 207; Carter, 1992 Curr. Opi. Biotech.
3, 533). The adenovirus, AAV or retroviral vector is delivered as
recombinant viral particles. The DNA may be delivered alone or
complexed with vehicles (as described for RNA above). The
recombinant adenovirus or AAV or retroviral particles are locally
administered to the site of treatment, e.g., through incubation or
inhalation in vivo or by direct application to cells or tissues ex
vivo. Retroviral vectors have also been used to express enzymatic
nucleic acid in mammalian cells (Ojwang et al., 1992 supra;
Thompson et al., 1995 supra).
[0187] Flt-1, KDR and/or flk-1 are attractive nucleic acid-based
therapeutic targets by several criteria. The interaction between
VEGF and VEGF-R is well-established. Efficacy can be tested in
well-defined and predictive animal models. Finally, the disease
conditions are serious and current therapies are inadequate.
Whereas protein-based therapies would inhibit VEGF activity nucleic
acid-based therapy provides a direct and elegant approach to
directly modulate flt-1, KDR and/or flk-1 expression.
[0188] Because flt-1 and KDR mRNAs are highly homologous in certain
regions, some enzymatic nucleic acid target sites are also
homologous (see Table X). In this case, a single enzymatic nucleic
acid can target both flt-1 and KDR mRNAs. At partially homologous
sites, a single enzymatic nucleic acid can sometimes be designed to
accommodate a site on both mRNAs by including G/U base pairing. For
example, if there is a G present in an enzymatic nucleic acid
target site in KDR mRNA at the same position there is an A in the
flt-1 ribozyme target site, the enzymatic nucleic acid can be
synthesized with a U at the complementary position and it will bind
both to sites. The advantage of one enzymatic nucleic acid that
targets both VEGF-R mRNAs is clear, especially in cases where both
VEGF receptors may contribute to the progression of angiogenesis in
the disease state.
[0189] "Angiogenesis" refers to formation of new blood vessels,
which is an essential process in reproduction, development and
wound repair. "Tumor angiogenesis" refers to the induction of the
growth of blood vessels from surrounding tissue into a solid tumor.
Tumor growth and tumor metastasis are dependent on angiogenesis
(for a review see Folkman, 1985 supra; Folkman 1990 J. Natl. Cancer
Inst., 82, 4; Folkman and Shing, 1992 J. Biol. Chem. 267,
10931).
[0190] Angiogenesis plays an important role in other diseases such
as arthritis wherein new blood vessels have been shown to invade
the joints and degrade cartilage (Folkman and Shing, supra).
[0191] "Retinopathy" refers to inflammation of the retina and/or
degenerative condition of the retina which may lead to occlusion of
the retina and eventual blindness. In "diabetic retinopathy"
angiogenesis causes the capillaries in the retina to invade the
vitreous resulting in bleeding and blindness which is also seen in
neonatal retinopathy (for a review see Folkman, 1985 supra; Folkman
1990 supra; Folkman and Shing, 1992 supra).
[0192] The following examples further illustrate the present
invention but should not be construed to limit the present
invention in any way.
EXAMPLE 1
flt-1, KDR and/or flk-1 Ribozymes
[0193] By engineering ribozyme motifs, Applicant has designed
several ribozymes directed against flt-1, KDR and/or flk-1 encoded
mRNA sequences. These ribozymes were synthesized with modifications
that improve their nuclease resistance (Beigelman et al., 1995 J.
Biol. Chem. 270, 25702) and enhance their activity in cells. The
ability of ribozymes to cleave target sequences in vitro was
evaluated essentially as described in Thompson et al., PCT
Publication No. WO 93/23057; Draper et al., PCT Publication No. WO
95/04818.
EXAMPLE 2
Effect of Ribozymes on the Binding of VEGF to flt-1, KDR and/or
flk-1 Receptors
[0194] Several common human cell lines are available that express
endogenous flt-1, KDR and/or flk-1. flt-1, KDR and/or flk-1 which
can be detected easily with monoclonal antibodies. Use of
appropriate fluorescent reagents and fluorescence-activated
cell-sorting (FACS) permit direct quantitation of surface flt-1,
KDR and/or flk-1 on a cell-by-cell basis. Active ribozymes are
expected to directly reduce flt-1, KDR and/or flk-1 expression and
thereby reduce VEGF binding to the cells. In this example, human
umbelical cord microvascular endothelial cells were used.
Cell Preparation:
[0195] Plates were coated with 1.5% gelatin and allowed to stand
for one hour. Cells (e.g., microvascular endothelial cells derived
from human umbilical cord vein) were plated at 20,000 cells/well
(24 well plate) in 200 .mu.l growth media and incubated overnight
(.about.1 doubling) to yield .about.40,000 cells (75-80%
confluent).
Ribozyme Treatment:
[0196] Media was removed from cells and the cells were washed two
times with 300 .mu.l 1.times.PBS: Ca.sup.2+: Mg.sup.2+ mixture. A
complex of 200-500 nM ribozyme and LipofectAMINE.RTM. (3:1
lipid:phosphate ratio) in 200 .mu.l OptiMEM.RTM. (5% FBS) was added
to the cells. The cells were incubated for 6 hr (equivalent to 2-3
VEGF-R turnovers).
.sup.125I VEGF Binding Assay:
[0197] The assay was carried out on ice to inhibit internalization
of VEGF during the experiment. The media containing the ribozyme
was removed from the cells and the cells were washed twice with 300
.mu.l 1.times.PBS:Ca.sup.2+:Mg.sup.2+ mixture containing 1% BSA.
Appropriate 1251 VEGF solution (100,000 cpm/well, +/-10.times. cold
1.times.PBS, 1% BSA) was applied to the cells. The cells were
incubated on ice for 1 hour. .sup.125I VEGF-containing solution was
removed and the cells were washed three times with 300 .mu.l
1.times.PBS:Ca.sup.2+:Mg.sup.2+ mixture containing 1% BSA. To each
well 300 .mu.l of 100 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 was
added and the mixture was incubated for 2 minutes. The .sup.125I
VEGF-binding was quantitated using standard scintillation counting
techniques. Percent inhibition was calculated as follows: Percent
.times. .times. Inhibition = cpm 125 .times. IVEGF .times. .times.
.times. bound .times. .times. by .times. .times. the .times.
.times. ribozyme - treated .times. .times. samples cpm 125 .times.
IVEGF .times. .times. .times. bound .times. .times. by .times.
.times. the .times. .times. .times. Control .times. .times. sample
.times. 100 ##EQU1##
EXAMPLE 3
Effect of Hammerhead Ribozymes Targeted Against flt-1 Receptor on
the Binding of VEGF
[0198] Hammerhead ribozymes targeted to twenty sites within flt-1
RNA were synthesized as described above. The sequences of the
ribozymes used are shown in Table II; the length of the stem II
region is 3 bp. The hammerhead ribozymes were chemically modified
such that the ribozyme consists of ribose residues at five
positions; U4 and U7 positions contain 2'-NH.sub.2 modifications,
the remaining nucleotide positions contain 2'-O-methyl
substitutions; four nucleotides at the 5' terminus contains
phosphorothioate substitutions. Additionally, 3' end of the
ribozyme contains a 3'-3' linked inverted abasic ribose.
[0199] Referring to FIG. 7, the effect of hammerhead ribozymes
targeted against flt-1 receptor on the binding of VEGF to flt-1 on
the surface of human microvascular endothelial cells is shown. The
majority of the ribozymes tested were able to inhibit the
expression of flt-1 and thereby were able to inhibit the binding of
VEGF.
[0200] In order to determine the specificity of ribozymes targeted
against flt-1 RNA, the effect of five anti-flt-1 ribozymes on the
binding of VEGF, UPA (urokinase plasminogen activator) and FGF
(fibroblast growth factor) to their corresponding receptors were
assayed. As shown in FIG. 9, there was significant inhibition of
VEGF binding to its receptors on cells treated with anti-flt-1
ribozymes. There was no specific inhibition of the binding of UPA
and FGF to their corresponding receptors. These data strongly
suggest that anti-flt-1 ribozymes specifically cleave flt-1 RNA and
not RNAs encoding the receptors for UPA and FGF, resulting in the
inhibition of flt-1 receptor expression on the surface of the
cells. Thus the ribozymes are responsible for the inhibition of
VEGF binding but not the binding of UPA and FGF.
EXAMPLE 4
Effect of Hammerhead Ribozymes Targeted Against KDR Receptor on the
Binding of VEGF
[0201] Hammerhead ribozymes targeted to twenty-one sites within KDR
RNA were synthesized as described above. The sequences of the
ribozymes used are shown in Table IV; the length of stem II region
is 3 bp. The hammerhead ribozymes were chemically modified such
that the ribozyme consists of ribose residues at five positions; U4
and U7 positions contain 2'-NH.sub.2 modifications, the remaining
nucleotide positions contain 2'-O-methyl substitutions; four
nucleotides at the 5' terminus contains phosphorothioate
substitutions. Additionally, the 3' end of the ribozyme contains a
3'-3' linked inverted abasic deoxyribose.
[0202] Referring to FIG. 8, the effect of hammerhead ribozymes
targeted against KDR receptor on the binding of VEGF to KDR on the
surface of human microvascular endothelial cells is shown. A
majority of the ribozymes tested were able to inhibit the
expression of KDR and thereby were able to inhibit the binding of
VEGF. As a control, the cells were treated with a ribozyme that is
not targeted towards KDR RNA (irrel. RZ); there was no specific
inhibition of VEGF binding. The results from this control
experiment strongly suggest that the inhibition of VEGF binding
observed with anti-KDR ribozymes is a ribozyme-mediated
inhibition.
EXAMPLE 5
Effect of Ribozymes Targeted Against VEGF Receptors on Cell
Proliferation
Cell Preparation:
[0203] 24-well plates were coated with 1.5% gelatin (porcine skin
300 bloom). After 1 hour, excess gelatin is washed off of the
plate. Microvascular endothelial cells were plated at 5,000
cells/well (24 well plate) in 200 .mu.l growth media. The cells
were allowed to grow for 18 hours (.about.1 doubling) to yield
.about.10,000 cells (25-30% confluent).
Ribozyme Treatment:
[0204] Media was removed from the cells, and the cells were washed
two times with 300 .mu.l 1.times.PBS:Ca.sup.2+:Mg.sup.2+
mixture.
[0205] For anti-flt-1HH ribozyme experiment (FIG. 12) a complex of
500 nM ribozyme; 15 .mu.M LFA (3:1 lipid:phosphate ratio) in 200
.mu.l OptiMEM (5% FCS) media was added to the cells. Incubation of
cells was carried out for 6 hours (equivalent to 2-3 VEGF receptor
turnovers).
[0206] For anti-KDR HH ribozyme experiment (FIG. 13) a complex of
200 nM ribozyme; 5.25 .mu.M LFA (3:1 lipid:phosphate ratio) in 200
.mu.l OptiMEM (5% FCS) media was added to the cells. Incubation of
cells was carried out for 3 hours.
Proliferation:
[0207] After three or six hours, the media was removed from the
cells and the cells were washed with 300 .mu.l 1.times.PBS:
Ca.sup.2+: Mg.sup.2+ mixture. Maintenance media (contains dialyzed
10% FBS)+/-VEGF or basic FGF at 10 ng/ml was added to the cells.
The cells were incubated for 48 or 72 hours. The cells were
trypsinized and counted (Coulter counter). Trypan blue was added on
one well of each treatment as a control.
[0208] As shown in FIG. 12B, VEGF and basic FGF stimulate human
microvascular endothelial cell proliferation. However, treatment of
cells with 1358 HH or 4229 HH ribozymes, targeted against flt-1
mRNA, results in a significant decrease in the ability of VEGF to
stimulate endothelial cell proliferation. These ribozymes do not
inhibit the FGF-mediated stimulation of endothelial cell
proliferation.
[0209] Human microvascular endothelial cells were also treated with
hammerhead ribozymes targeted against sites 527, 730, 3702 or 3950
within the KDR mRNA. As shown in FIG. 13, all four ribozymes caused
significant inhibition of VEGF-mediated induction of cell
proliferation. No significant inhibition of cell proliferation was
observed when the cells were treated with a hammerhead ribozyme
targeted to an irrelevant RNA. Additionally, none of the ribozymes
inhibited FGF-mediated stimulation of cell proliferation.
[0210] These results strongly suggest that hammerhead ribozymes
targeted against either flt-1 or KDR mRNA specifically inhibit
VEGF-mediated induction of endothelial cell proliferation.
EXAMPLE 6
Effect of Antisense Oligonucleotides Targeted Against VEGF
Receptors on Cell Proliferation (Colorimetric Assay)
[0211] The following are some of the reagents used in the
proliferation assay:
[0212] Cells: Human aortic endothelial cells (HAEC) from
Clonetics.RTM.. Cells at early passage are preferably used.
[0213] Uptake Medium: EBM (from Clonetics.RTM.); 1% L-Glutamine; 20
mM Hepes; No serum; No antibiotics.
[0214] Growth Medium: EGM (from Clonetics.RTM.); FBS to 20%; 1%
L-Glutamine; 20 mM Hepes.
[0215] Cell Plating: 96-well tissue culture plates were coated with
0.2% gelatin (50 .mu.l/well). The gelatin was incubated in the
wells at room temperature for 15-30 minutes. The gelatin was
removed by aspiration and the wells were washed with
PBS:Ca.sup.2+:Mg.sup.2+ mixture. PBS mixture was left in the wells
until cells were ready to be added. HAEC cells were detached by
trypsin treatment and resuspended at 1.25.times.10.sup.4/ml in
growth medium. PBS was removed from plates and 200 .mu.l of cells
(i.e. 2.5.times.10.sup.3 cells/well) were added to each well. The
cells were allowed to grow for 48 hours before the proliferation
assay.
[0216] Assay: Growth medium was removed from the wells. The cells
were washed twice with PBS:Ca.sup.2+:Mg.sup.2+ mixture without
antibiotics. A formulation of lipid/antisense oligonucleotide
(antisense oligonucleotide is used here as a non-limiting example)
complex was added to each well (100 .mu.l/well) in uptake medium.
The cells were incubated for 2-3 hours at 37.degree. C. in a
CO.sub.2 incubator. After uptake, 100 .mu.l/well of growth medium
was added (gives final FBS concentration of 10%). After
approximately 72 hours, 40 .mu.l MTS.RTM. stock solution (made as
described by manufacturer) was added to each well and incubated at
37.degree. C. for 1-3 hours, depending on the color development.
(For this assay, 2 hours was sufficient). The intensity of color
formation was determined on a plate reader at 490 nM.
[0217] Phosphorothioate-substituted antisense oligodeoxynucleotides
were custom synthesized by The Midland Certified Reagent
Company.RTM., Midland, Tex. Following non-limiting antisense
oligodeoxynucleotides targeted against KDR RNA were used in the
proliferation assay: TABLE-US-00003 KDR 21 AS: (SEQ ID NO: 20825)
5'-GCA GCA CCT TGC TCT CCA TCC-3' SCRAMBLED CONTROL: (SEQ ID NO:
20826) 5'-CTG CCA ACT TCC CAT GCC TGC-3'
[0218] As shown in FIG. 10, proliferation of HAEC cells is
specifically inhibited by increasing concentrations of the
phosphorothioate anti-KDR-antisense oligodeoxynucleotide. The
scrambled antisense oligonucleotide is not expected to bind the KDR
RNA and therefore is not expected to inhibit KDR expression. As
expected, there is no detectable inhibition of proliferation of
HAEC cells treated with a phosphorothioate antisense
oligonucleotide with scrambled sequence.
EXAMPLE 7
In Vitro Cleavage of flt-1 RNA by Hammerhead Ribozymes
[0219] Referring to FIG. 11A, hammerhead ribozymes (HH) targeted
against sites 1358 and 4229 within the flt-1 RNA were synthesized
as described above.
RNA Cleavage Assay In Vitro:
[0220] Substrate RNA was 5' end-labeled using [.gamma.-.sup.32P]
ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage
reactions were carried out under ribozyme "excess" conditions.
Trace amount (<1 nM) of 5' end-labeled substrate and 40 nM
unlabeled ribozyme were denatured and renatured separately by
heating to 90.degree. C. for 2 minutes and snap-cooling on ice for
10-15 minutes. The ribozyme and substrate were incubated,
separately, at 37.degree. C. for 10 minutes in a buffer containing
50 mM Tris-HCl and 10 mM MgCl.sub.2. The reaction was initiated by
mixing the ribozyme and substrate solutions and incubating at
37.degree. C. Aliquots of 5 .mu.l were taken at regular intervals
of time and the reaction was quenched by mixing with equal volume
of 2.times. formamide stop mix. The samples were resolved on 20%
denaturing polyacrylamide gels. The results were quantified and
percentage of target RNA cleaved is plotted as a function of
time.
[0221] Referring to FIGS. 11B and 11C, hammerhead ribozymes
targeted against sites 1358 and 4229 within the flt-1 RNA are
capable of cleaving target RNA efficiently in vitro.
EXAMPLE 8
In Vitro Cleavage of KDR RNA by Hammerhead Ribozymes
[0222] In this non-limiting example, hammerhead ribozymes targeted
against sites 726, 527, 3702 and 3950 within KDR RNA were
synthesized as described above. RNA cleavage reactions were carried
out in vitro essentially as described under Example 7.
[0223] Referring to FIGS. 14 and 15, all four ribozymes were able
to cleave their cognate target RNA efficiently in a
sequence-specific manner.
EXAMPLE 9
In Vitro Cleavage of RNA by Hammerhead Ribozymes Targeted Against
Cleavage Sites that are Homologous Between KDR and flt-1 mRNA
[0224] Given that flt-1 and KDR mRNAs are highly homologous in
certain regions, some ribozyme target sites are also homologous
(see Table X). In this case, a single ribozyme will target both
flt-1 and KDR mRNAs. Hammerhead ribozyme (FLT/KDR-I) targeted
against one of the homologous sites between flt-1 and KDR (flt-1
site 3388 and KDR site 3151) was synthesized as described above.
Ribozymes with either a 3 bp stem II or a 4 bp stem II were
synthesized. RNA cleavage reactions were carried out in vitro
essentially as described under Example 7.
[0225] Referring to FIG. 16, FLT/KDR-I ribozyme with either a 3 or
a 4 bp stem II was able to cleave its target RNA efficiently in
vitro.
EXAMPLE 10
Effect of Multiple Ribozymes Targeted Against Both flt-1 and KDR
RNA on Cell Proliferation
[0226] Since both flt-1 and KDR receptors of VEGF are involved in
angiogenesis, the inhibition of the expression of both of these
genes can be an effective approach to inhibit angiogenesis.
[0227] Human microvascular endothalial cells were treated with
hammerhead ribozymes targeted against sites flt-1 4229 alone, KDR
527 alone, KDR 726 alone, KDR 3950 alone, flt-1 4229+KDR 527, flt-1
4229+KDR 726 or flt-1 4229+KDR 3950. As shown in FIG. 17, all the
combinations of active ribozymes (A) caused significant inhibition
of VEGF-mediated induction of cell proliferation. No significant
inhibition of cell proliferation was observed when the cells were
treated with a catalytically inactive (I) hammerhead ribozymes.
Additionally, cells treated with ribozymes targeted against both
flt-1 and KDR RNAs-flt-1 4229+KDR 527; flt-1 4229+KDR 726; flt-1
4229+KDR 3950, were able to cause a greater inhibition of
VEGF-mediated induction of cell proliferation when compared with
individual ribozymes targeted against either flt-1 or KDR RNA (see
flt-1 4229 alone; KDR 527 alone; KDR 726 alone; KDR 3950 alone).
This strongly suggests that treatment of cells with multiple
ribozymes can be a more effective means of inhibition of gene
expression.
Animal Models
[0228] There are several animal models in which the
anti-angiogenesis effect of nucleic acids of the present invention,
such as enzymatic nucleic acids, directed against VEGF-R mRNAs can
be tested. Typically a corneal model has been used to study
angiogenesis in rat and rabbit since recruitment of vessels can
easily be followed in this normally avascular tissue (Pandey et
al., 1995 Science 268: 567-569). In these models, a small Teflon or
Hydron disk pretreated with an angiogenesis factor (e.g. bFGF or
VEGF) is inserted into a pocket surgically created in the cornea.
Angiogenesis is monitored 3 to 5 days later. Enzymatic nucleic
acids directed against VEGF-R mRNAs are delivered in the disk as
well, or dropwise to the eye over the time course of the
experiment. In another eye model, hypoxia has been shown to cause
both increased expression of VEGF and neovascularization in the
retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92:
905-909; Shweiki et al., 1992 J. Clin. Invest. 91: 2235-2243).
[0229] In human glioblastomas, it has been shown that VEGF is at
least partially responsible for tumor angiogenesis (Plate et al.,
1992 Nature 359, 845). Animal models have been developed in which
glioblastoma cells are implanted subcutaneously into nude mice and
the progress of tumor growth and angiogenesism is studied (Kim et
al., 1993 supra; Millauer et al., 1994 supra).
[0230] Another animal model that addresses neovascularization
involves Matrigel, an extract of basement membrane that becomes a
solid gel when injected subcutaneously (Passaniti et al., 1992 Lab.
Invest. 67: 519-528). When the Matrigel is supplemented with
angiogenesis factors such as VEGF, vessels grow into the Matrigel
over a period of 3 to 5 days and angiogenesis can be assessed.
Again, nucleic acids directed against VEGF-R mRNAs are delivered in
the Matrigel.
[0231] Several animal models exist for screening of anti-angiogenic
agents. These include corneal vessel formation following corneal
injury (Burger et al., 1985 Cornea 4: 35-41; Lepri, et al., 1994 J
Ocular Pharmacol. 10: 273-280; Ormerod et al., 1990 Am. J. Pathol.
137: 1243-1252) or intracorneal growth factor implant (Grant et
al., 1993 Diabetologia 36: 282-291; Pandey et al. 1995 supra;
Zieche et al., 1992 Lab. Invest. 67: 711-715), vessel growth into
Matrigel matrix containing growth factors (Passaniti et al., 1992
supra), female reproductive organ neovascularization following
hormonal manipulation (Shweiki et al., 1993 Clin. Invest. 91:
2235-2243), several models involving inhibition of tumor growth in
highly vascularized solid tumors (O'Reilly et al., 1994 Cell 79:
315-328; Senger et al., 1993 Cancer and Metas. Rev. 12: 303-324;
Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al., 1993
supra), and transient hypoxia-induced neovascularization in the
mouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92:
905-909).
[0232] The cornea model, described in Pandey et al. supra, is the
most common and well characterized anti-angiogenic agent efficacy
screening model. This model involves an avascular tissue into which
vessels are recruited by a stimulating agent (growth factor,
thermal or alkalai burn, endotoxin). The corneal model utilizes the
intrastromal corneal implantation of a Teflon pellet soaked in a
VEGF-Hydron solution to recruit blood vessels toward the pellet
which can be quantitated using standard microscopic and image
analysis techniques. To evaluate their anti-angiogenic efficacy,
nucleic acids are applied topically to the eye or bound within
Hydron on the Teflon pellet itself. This avascular cornea as well
as the Matrigel (see below) provide for low background assays.
While the corneal model has been performed extensively in the
rabbit, studies in the rat have also been conducted.
[0233] The mouse model (Passaniti et al., supra) is a non-tissue
model which utilizes Matrigel, an extract of basement membrane
(Kleinman et al., 1986) or Millipore.RTM. filter disk, which can be
impregnated with growth factors and anti-angiogenic agents in a
liquid form prior to injection. Upon subcutaneous administration at
body temperature, the Matrigel or Millipore.RTM. filter disk forms
a solid implant. VEGF embedded in the Matrigel or Millipore.RTM.
filter disk is used to recruit vessels within the matrix of the
Matrigel or Millipore.RTM. filter disk which can be processed
histologically for endothelial cell specific vWF (factor VIII
antigen) immunohistochemistry, Trichrome-Masson stain, or
hemoglobin content. Like the cornea, the Matrigel or Millipore.RTM.
filter disk are avascular; however, it is not tissue. In the
Matrigel or Millipore.RTM. filter disk model, nucleic acids are
administered within the matrix of the Matrigel or Millipore.RTM.
filter disk to test their anti-angiogenic efficacy. Thus, delivery
issues in this model, as with delivery of nucleic acids by
Hydron-coated Teflon pellets in the rat cornea model, may be less
problematic due to the homogeneous presence of the nucleic acid
within the respective matrix.
[0234] These models offer a distinct advantage over several other
angiogenic models listed previously. The ability to use VEGF as a
pro-angiogenic stimulus in both models is highly desirable since
the instant nucleic acid molecules target only VEGFr mRNA. In other
words, the involvement of other non-specific types of stimuli in
the cornea and Matrigel models is not advantageous from the
standpoint of understanding the pharmacologic mechanism by which
the anti-VEGFr mRNA nucleic acid molecules produce their effects.
In addition, the models allow for testing the specificity of the
anti-VEGFr mRNA nucleic acids by using either a- or bFGF as a
pro-angiogenic factor. Vessel recruitment using FGF should not be
affected in either model by anti-VEGFr mRNA nucleic acid molecules.
Other models of angiogenesis including vessel formation in the
female reproductive system using hormonal manipulation (Shweiki et
al., 1993 supra); a variety of vascular solid tumor models which
involve indirect correltations with angiogenesis (O'Reilly et al.,
1994 supra; Senger et al., 1993 supra; Takahasi et al., 1994 supra;
Kim et al., 1993 supra); and retinal neovascularization following
transient hypoxia (Pierce et al., 1995 supra) were not selected for
efficacy screening due to their non-specific nature, although there
is a correlation between VEGF and angiogenesis in these models.
[0235] Other model systems to study tumor angiogenesis is reviewed
by Folkman, 1985 Adv. Cancer. Res. 43, 175.
Use of Murine Models
[0236] For a typical systemic study involving 10 mice (20 g each)
per dose group, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14
days continuous administration), approximately 400 mg of enzymatic
nucleic acid, formulated in saline is used. A similar study in
young adult rats (200 g) requires over 4 g. Parallel
pharmacokinetic studies involve the use of similar quantities of
enzymatic nucleic acid further justifying the use of murine
models.
Enzymatic Nucleic Acids and Lewis Lung Carcinoma and B-16 Melanoma
Murine Models
[0237] Identifying a common animal model for systemic efficacy
testing of enzymatic nucleic acid is an efficient way of screening
enzymatic nucleic acid for systemic efficacy.
[0238] The Lewis lung carcinoma and B-16 murine melanoma models are
well accepted models of primary and metastatic cancer and are used
for initial screening of anti-cancer agents. These murine models
are not dependent upon the use of immunodeficient mice, are
relatively inexpensive, and minimize housing concerns. Both the
Lewis lung and B-16 melanoma models involve subcutaneous
implantation of approximately 10.sup.6 tumor cells from
metastatically aggressive tumor cell lines (Lewis lung lines 3LL or
D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively,
the Lewis lung model can be produced by the surgical implantation
of tumor spheres (approximately 0.8 mm in diameter). Metastasis
also can be modeled by injecting the tumor cells directly
intravenously. In the Lewis lung model, microscopic metastases can
be observed approximately 14 days following implantation with
quantifiable macroscopic metastatic tumors developing within 21-25
days. The B-16 melanoma exhibits a similar time course with tumor
neovascularization beginning 4 days following implantation. Since
both primary and metastatic tumors exist in these models after
21-25 days in the same animal, multiple measurements can be taken
as indices of efficacy. Primary tumor volume and growth latency as
well as the number of micro- and macroscopic metastatic lung foci
or number of animals exhibiting metastases can be quantitated. The
percent increase in lifespan can also be measured. Thus, these
models provide suitable primary efficacy assays for screening
systemically administered enzymatic nucleic acids and enzymatic
nucleic acid formulations.
[0239] In the Lewis lung and B-16 melanoma models, systemic
pharmacotherapy with a wide variety of agents usually begins 1-7
days following tumor implantation/inoculation with either
continuous or multiple administration regimens. Concurrent
pharmacokinetic studies can be performed to determine whether
sufficient tissue levels of ribozymes can be achieved for
pharmacodynamic effect to be expected. Furthermore, primary tumors
and secondary lung metastases can be removed and subjected to a
variety of in vitro studies (i.e. target RNA reduction).
[0240] Flt-1, KDR and/or flk-1 protein levels can be measured
clinically or experimentally by FACS analysis. Flt-1, KDR and/or
flk-1 encoded mRNA levels are assessed by Northern analysis,
RNase-protection, primer extension analysis and/or quantitative
RT-PCR. Enzymatic nucleic acids that block flt-1, KDR and/or flk-1
protein encoding mRNAs and therefore result in decreased levels of
flt-1, KDR and/or flk-1 activity by more than 20% in vitro can be
identified.
[0241] Enzymatic nucleic acids and/or genes encoding them are
delivered by either free delivery, liposome delivery, cationic
lipid delivery, adeno-associated virus vector delivery, adenovirus
vector delivery, retrovirus vector delivery or plasmid vector
delivery in these animal model experiments (see above).
[0242] Patients can be treated by locally administering nucleic
acids targeted against VEGF-R by direct injection. Routes of
administration include, but are not limited to, intravascular,
intramuscular, subcutaneous, intraarticular, aerosol inhalation,
oral (tablet, capsule or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery.
EXAMPLE 11
Ribozyme-Mediated Inhibition of Angiogenesis In Vivo
[0243] The purpose to this study was to assess the anti-angiogenic
activity of hammerhead ribozymes targeted against fit-1 4229 site
in the rat cornea model of VEGF induced angiogenesis (see above).
These ribozymes have either active or inactive catalytic core and
either bind and cleave or just bind to VEGF-R mRNA of the flt-1
subtype. The active ribozymes, that are able to bind and cleave the
target RNA, have been shown to inhibit (.sup.125I-labeled) VEGF
binding in cultured endothelial cells and produce a dose-dependent
decrease in VEGF induced endothelial cell proliferation in these
cells (see Examples 3-5 above). The catalytically inactive forms of
these ribozymes, wherein the ribozymes can only bind to the RNA but
cannot catalyze RNA cleavage, fail to show these characteristics.
The ribozymes and VEGF were co-delivered using the filter disk
method: Nitrocellulose filter disks (Millipore.RTM.) of 0.057
diameter were immersed in appropriate solutions and were surgically
implanted in rat cornea as described by Pandey et al., supra. This
delivery method has been shown to deliver rhodamine-labeled free
ribozyme to scleral cells and, in all likelihood cells of the
pericorneal vascular plexus. Since the active ribozymes show cell
culture efficacy and can be delivered to the target site using the
disk method, it is essential that these ribozymes be assessed for
in vivo anti-angiogenic activity.
[0244] The stimulus for angiogenesis in this study was the
treatment of the filter disk with 30 .mu.M VEGF which is implanted
within the cornea's stroma. This dose yields reproducible
neovascularization stemming from the pericorneal vascular plexus
growing toward the disk in a dose-response study 5 days following
implant. Filter disks treated only with the vehicle for VEGF show
no angiogenic response. The ribozymes were co-adminstered with VEGF
on a disk in two different ribozyme concentrations. One concern
with the simultaneous administration is that the ribozymes will not
be able to inhibit angiogenesis since VEGF receptors can be
stimulated. However, Applicant has observed that in low VEGF doses,
the neovascular response reverts to normal suggesting that the VEGF
stimulus is essential for maintaining the angiogenic response.
Blocking the production of VEGF receptors using simultaneous
administration of anti-VEGF-R mRNA ribozymes could attenuate the
normal neovascularization induced by the filter disk treated with
VEGF.
Materials and Methods:
1. Stock Hammerhead Ribozyme Solutions:
[0245] a. flt-1 4229 (786 .mu.M)--Active [0246] b. fit-1 4229 (736
.mu.M)--Inactive
[0247] 2. Experimantal Solutions/Groups: TABLE-US-00004 Group 1
Solution 1 Control VEGF solution: 30 .mu.M in 82 mM Tris base Group
2 Solution 2 flt-1 4229 (1 .mu.g/.mu.L) in 30 .mu.M VEGF/82 mM Tris
base Group 3 Solution 3 flt-1 4229 (10 .mu.g/.mu.L) in 30 .mu.M
VEGF/82 mM Tris base Group 4 Solution 4 No VEGF, flt-1 4229 (10
.mu.g/.mu.L) in 82 mM Tris base Group 5 Solution 5 No VEGF, No
ribozyme in 82 mM Tris base
[0248] 10 eyes per group, 5 animals (Since they have similar
molecular weights, the molar concentrations should be essentially
similar).
[0249] Each solution (VEGF and RIIBOZYMES) were prepared as a
2.times. solution for 1:1 mixing for final concentrations above,
with the exception of solution 1 in which VEGF was 2.times. and
diluted with ribozyme diluent (sterile water).
3. VEGF Solutions
[0250] The 2.times.VEGF solution (60 .mu.M) was prepared from a
stock of 0.82 .mu.g/.mu.L in 50 mM Tris base. 200 .mu.L of VEGF
stock was concentrated by speed vac to a final volume of 60.8
.mu.L, for a final concentration of 2.7 .mu.g/.mu.L or 60 .mu.M.
Six 10 .mu.L aliquots were prepared for daily mixing. 2.times.
solutions for VEGF and Ribozyme was stored at 4.degree. C. until
the day of the surgery. Solutions were mixed for each day of
surgery. Original 2.times. solutions were prepared on the day
before the first day of the surgery.
4. Surgical Solutions:
[0251] Anesthesia:
[0252] stock ketamine hydrochloride 100 mg/mL
[0253] stock xylazine hydrochloride 20 mg/mL
[0254] stock acepromazine 10 mg/mL
[0255] Final anesthesia solution: 50 mg/mL ketamine, 10 mg/mL
xylazine, and 0.5 mg/mL acepromazine
[0256] 5% povidone iodine for opthalmic surgical wash
[0257] 2% lidocaine (sterile) for opthalmic administration (2 drops
per eye)
[0258] sterile 0.9% NaCl for opthalmic irrigation
5. Surgical Methods:
[0259] Standard surgical procedure was performed as described in
Pandey et al., supra. Filter disks were incubated in 1 .mu.L of
each solution for approximately 30 minutes prior to
implantation.
6. Experimental Protocol:
[0260] The animal corneas were treated with the treatment groups as
described above. Animals were allowed to recover for 5 days after
treatment with daily observation (scoring 0-3). On the fifth day
animals were euthanized and digital images of each eye was obtained
for quantitaion using Image Pro Plus. Quantitated neovascular
surface areas were analyzed by ANOVA followed by two post-hoc tests
including Dunnets and Tukey-Kramer tests for significance at the
95% confidence level. Dunnets provide information on the
significance between the differences within the means of treatments
vs. controls while Tukey-Kramer provide information on the
significance of differences within the means of each group.
[0261] Results are graphically represented in FIG. 18. As shown in
FIG. 18, flt-1 4229 active hammerhead ribozyme at both
concentrations was effective at inhibiting angiogenesis, while the
inactive ribozyme did not show any significant reduction in
angiogenesis. A statistically signifiant reduction in neovascular
surface area was observed only with active ribozymes. This result
clearly shows that the ribozymes are capable of significantly
inhibiting angiogenesis in vivo. Specifically, the mechanism of
inhibition appears to be by the binding and cleavage of target RNA
by ribozymes.
EXAMPLE 12
Bioactivity of Anti-Angiogenesis Ribozymes Targeting flt-1 and kdr
RNA
Materials and Methods
[0262] Ribozymes: Hammerhead ribozymes and controls designed to
have attenuated activity (attenuated controls) were synthesized and
purified as previously described above. The attenuated ribozyme
controls maintain the binding arm sequence of the parent ribozyme
and thus are still capable of binding to the mRNA target. However,
they have two nucleotide changes in the core sequence that
substantially reduce their ability to carry out the cleavage
reaction. Ribozymes were designed to target Flt-1 or KDR mRNA sites
conserved in human, mouse, and rat. In general, ribozymes with
binding arms of seven nucleotides were designed and tested. If,
however, only six nucleotides surrounding the cleavage site were
conserved in all three species, six nucleotide binding arms were
used. A subset of ribozyme and attenuated control sequences and
modifications are listed in Table XII. Data are presented herein
for 2'-NH.sub.2 uridine modified ribozymes in cell proliferation
studies and for 2'-C-allyl uridine modified ribozymes in RNAse
protection, in vitro cleavage and corneal studies.
[0263] In vitro ribozyme cleavage assays: In vitro RNA cleavage
rates on a 15 nucleotide synthetic RNA substrate were measured as
previously described above.
[0264] Cell culture: Human dermal microvascular endothelial cells
(HMVEC-d, Clonetics Corp.) were maintained at 37.degree. C. in
flasks or plates coated with 1.5% porcine skin gelatin (300 bloom,
Sigma) in Growth medium (Clonetics Corp.) supplemented with 10-20%
fetal bovine serum (FBS, Hyclone). Cells were grown to confluency
and used up to the seventh passage. Stimulation medium consisted of
50% Sigma 99 media and 50% RPMI 1640 with L-glutamine and
additional supplementation with 10 .mu.g/mL
Insulin-Transferrin-Selenium (Gibco BRL) and 10% FBS. Cell growth
was stimulated by incubation in Stimulation medium supplemented
with 20 ng/mL of either VEGF.sub.165 or bFGF. VEGF.sub.165 (165
amino acids) was selected for cell culture and animal studies
because it is the predominant form of the four native forms of VEGF
generated by alternative mRNA splicing. Cell culture assays were
carried out in triplicate.
Ribozyme and Ribozyme/LIPOFECTAMINE.TM. Formulations:
[0265] Cell culture: Ribozymes or attenuated controls (50-200 nM)
were formulated for cell culture studies and used immediately.
Formulations were carried out with LIPOFECTAMINE.TM. (Gibco BRL) at
a 3:1 lipid to phosphate charge ratio in serum-free medium
(OPTI-MEM.TM., Gibco BRL) by mixing for 20 minutes at room
temperature. For example, a 3:1 lipid to phosphate charge ratio was
established by complexing 200 nM ribozyme with 10.8 .mu.g/.mu.L
LIPOFECTAMINE.TM. (13.5 .mu.M DOSPA).
[0266] In vivo: For corneal studies, lyophilized ribozyme or
attenuated controls were resuspended in sterile water at a final
stock concentration of 170 .mu.g/.mu.L (highest dose). Lower doses
(1.7-50 .mu.g/.mu.L) were prepared by serial dilution in sterile
water.
[0267] Proliferation assay: HMVEC-d were seeded (5.times.10.sup.3
cells/well) in 48-well plates (Costar) and incubated 24-30 hours in
Growth medium at 37.degree. C. After removal of the Growth medium,
cells were treated with 50-200 nM LIPOFECTAMINE.TM. complexes of
ribozyme or attenuated controls for 2 hours in OPTI-MEM.TM.. The
ribozyme/control-containing medium was removed and the cells were
washed extensively in 1.times.PBS. The medium was then replaced
with Stimulation medium or Stimulation medium supplemented with 20
ng/mL VEGF.sub.165 or bFGF. After 48 hours, the cell number was
determined using a Coulter.TM. cell counter. Data are presented as
cell number per well following 48 h of VEGF stimulation.
[0268] RNAse protection assay: HMVEC-d were seeded
(2.times.10.sup.5 cells/well) in 6-well plates (Costar) and allowed
to grow 32-36 hours in Growth medium at 37.degree. C. Cells were
treated with LIPOFECTAMINE.TM. complexes containing 200 nM ribozyme
or attenuated control for 2 hours as described under "Proliferation
Assay" and then incubated in Growth medium containing 20 ng/mL
VEGF.sub.165 for 24 hours. Cells were harvested and an RNAse
protection assay was carried out using the Ambion Direct Protect
kit and protocol with the exception that 50 mM EDTA was added to
the lysis buffer to eliminate the possibility of ribozyme cleavage
during sample preparation. Antisense RNA probes targeting portions
of Flt-1 and KDR were prepared by transcription in the presence of
[.sup.32P]-UTP. Samples were analyzed on polyacrylamide gels and
the level of protected RNA fragments was quantified using a
Molecular Dynamics Phosphorimager. The levels of Flt-1 and KDR were
normalized to the level of cyclophilin (human cyclophilin probe
template, Ambion) in each sample. The coefficient of variation for
cyclophilin levels was 11% [265940 cpm.+-.29386 (SD)] for all
conditions tested here (i.e. in the presence of either active
ribozymes or attenuated controls). Thus, cyclophilin is useful as
an internal standard in these studies.
Rat Corneal Pocket Assay of VEGF-Induced Angiogenesis:
[0269] Animal guidelines and anesthesia. Animal housing and
experimentation adhered to standards outlined in the 1996 Guide for
the Care and Use of Laboratory Animals (National Research Council).
Male Sprague Dawley rats (250-300 g) were anesthetized with
ketamine (50 mg/kg), xylazine (10 mg/kg), and acepromazine (0.5
mg/kg) administered intramuscularly (im). The level of anesthesia
was monitored every 2-3 min by applying hind limb paw pressure and
examining for limb withdrawal. Atropine (0.4 mg/kg, im) was also
administered to prevent potential corneal reflex-induced
bradycardia.
[0270] Preparation of VEGF soaked disk. For corneal implantation,
0.57 mm diameter nitrocellulose disks, prepared from 0.45 .mu.m
pore diameter nitrocellulose filter membranes (Millipore
Corporation), were soaked for 30 min in 1 .mu.L of 30 .mu.M
VEGF.sub.165 in 82 mM Tris HCl (pH 6.9) in covered petri dishes on
ice.
[0271] Corneal surgery. The rat corneal model used in this study
was a modified from Koch et al. Supra and Pandey et al., supra.
Briefly, corneas were irrigated with 0.5% povidone iodine solution
followed by normal saline and two drops of 2% lidocaine. Under a
dissecting microscope (Leica MZ-6), a stromal pocket was created
and a presoaked filter disk (see above) was inserted into the
pocket such that its edge was 1 mm from the corneal limbus.
[0272] Intraconjunctival injection of test solutions. Immediately
after disk insertion, the tip of a 40-50 .mu.m OD injector
(constructed in our laboratory) was inserted within the
conjunctival tissue 1 mm away from the edge of the corneal limbus
that was directly adjacent to the VEGF-soaked filter disk. Six
hundred nanoliters of test solution (ribozyme, attenuated control
or sterile water vehicle) were dispensed at a rate of 1.2 .mu.L/min
using a syringe pump (Kd Scientific). The injector was then
removed, serially rinsed in 70% ethanol and sterile water and
immersed in sterile water between each injection. Once the test
solution was injected, closure of the eyelid was maintained using
microaneurism clips until the animal began to recover gross motor
activity. Following treatment, animals were warmed on a heating pad
at 37.degree. C.
[0273] Animal treatment groups/experimental protocol. Ribozymes
targeting Flt-1 site 4229 and KDR mRNA site 726 were tested in the
corneal model along with their attenuated controls. Five treatment
groups were assigned to examine the effects of five doses of each
test substance over a dose range of 1-100 .mu.g on VEGF-stimulated
angiogenesis. Negative (30 .mu.M VEGF soaked filter disk and
intraconjunctival injection of 600 nL sterile water) and no
stimulus (Tris-soaked filter disk and intraconjunctival injection
of sterile water) control groups were also included. Each group
consisted of five animals (10 eyes) receiving the same
treatment.
[0274] Quantitation of angiogenic response. Five days after disk
implantation, animals were euthanized following im administration
of 0.4 mg/kg atropine and corneas were digitally imaged. The
neovascular surface area (NSA, expressed in pixels) was measured
postmortem from blood-filled corneal vessels using computerized
morphometry (Image Pro Plus, Media Cybernetics, v2.0). The
individual mean NSA was determined in triplicate from three regions
of identical size in the area of maximal neovascularization between
the filter disk and the limbus. The number of pixels corresponding
to the blood-filled corneal vessels in these regions was summated
to produce an index of NSA. A group mean NSA was then calculated.
Data from each treatment group were normalized to VEGF/ribozyme
vehicle-treated control NSA and finally expressed as percent
inhibition of VEGF-induced angiogenesis.
[0275] Statistics. After determining the normality of treatment
group means, group mean percent inhibition of VEGF-induced
angiogenesis was subjected to a one-way analysis of variance. This
was followed by two post-hoc tests for significance including
Dunnett's (comparison to VEGF control) and Tukey-Kramer (all other
group mean comparisons) at alpha=0.05. Statistical analyses were
performed using JMP v.3.1.6 (SAS Institute).
Results
[0276] Ribozyme-mediated reduction of VEGF-induced cell
proliferation: Ribozyme cleavage of Flt-1 or KDR mRNA should result
in a decrease in the density of cell surface VEGF receptors. This
decrease should limit VEGF binding and consequently interfere with
the mitogenic signaling induced by VEGF. To determine if cell
proliferation was impacted by anti-Flt-1 and/or anti-KDR ribozyme
treatment, proliferation assays using cultured human microvascular
cells were carried out. Ribozymes included in the proliferation
assays were initially chosen by their ability to decrease the level
of VEGF binding to treated cells (see FIG. 8). In these initial
studies, ribozymes targeting 20 sites in the coding region of each
mRNA were screened. The most effective ribozymes against two sites
in each target (Table XII), Flt-1 sites 1358 and 4229 and KDR sites
726 and 3950, were included in the proliferation assays reported
here (FIG. 19). In addition, attenuated analogs of each ribozyme
were used as controls (Table XII). These attenuated controls are
still capable of binding to the mRNA target since the binding arm
sequence is maintained. However, these controls have two nucleotide
changes in the core sequence that substantially reduce their
ability to carry out the cleavage reaction.
[0277] The antiproliferative effect of active ribozymes targeting
two lead sites on each VEGF receptor mRNA is shown in FIG. 19. The
active ribozymes tested decreased the relative proliferation of
HMVEC-d after VEGF stimulation, an effect that increased with
ribozyme concentration. This concentration dependency was not
observed following treatment with the attenuated controls designed
for these sites. In fact, little or no change in cell growth was
noted following treatment with the attenuated controls, even though
these controls can still bind to the specific target sequences. At
200 nM, there was a distinct "window" between the
anti-proliferative effects of each ribozyme and its attenuated
control; a trend also observed at lower doses. This window of
inhibition of proliferation (56-77% based on total cells/well)
reflects the contribution of ribozyme-mediated activity. In
comparison, no effect of anti-Flt-1 or anti-KDR ribozymes was noted
on bFGF-stimulated cell proliferation (FIGS. 19C, 19F). Moreover,
an irrelevant, but active, ribozyme whose binding sequence is not
found in either Flt-1 or KDR mRNA had no effect in this assay (FIG.
19B). These data are consistent with the basic ribozyme mechanism
in which binding and cleavage are necessary components. Although
the relative surface distribution of Flt-1 and KDR receptors in
this cell type is not known, the antiproliferative effects of these
ribozymes indicate that, at least in cell culture, both receptors
are functionally coupled to proliferation.
[0278] Specific reduction of Flt-1 or KDR mRNA by ribozyme
treatment: To confirm that anti-Flt-1 and anti-KDR ribozymes reduce
their respective mRNA targets, cellular levels of Flt-1 or KDR were
quantified using an RNAse protection assay with specific Flt-1 or
KDR probes. For each target, one ribozyme/attenuated control pair
was chosen for continued study. Data from a representative
experiment are shown in FIG. 20. Exposure of HMVEC-d to active
ribozyme targeting Flt-1 site 4229 decreased Flt-1 mRNA, but not
KDR mRNA. Likewise, treatment with the active ribozyme targeting
KDR site 726 decreased KDR, but not Flt-1 mRNA. Both ribozymes
decreased the level of their respective target RNA by greater than
50%. The degree of reduction associated with the corresponding
attenuated controls was not greater than 13%.
In Vitro Activity of Anti-Flt and Anti-KDR Ribozymes.
[0279] To confirm further the necessity of an active ribozyme core,
in vitro cleavage activities were determined for the Flt-1 site
4229 ribozyme and the KDR site 726 ribozyme as well as their paired
attenuated controls. The first order rate constants calculated from
the time-course of short substrate cleavage for the anti-Flt-1
ribozyme and its attenuated control were 0.081.+-.0.0007 min.sup.-1
and 0.001.+-.6.times.10.sup.-5 min.sup.-1, respectively. For the
anti-KDR ribozyme and its paired control, the first order rate
constants were 0.434.+-.0.024 min.sup.-1 and
0.002.+-.1.times.10.sup.-4 min.sup.-1, respectively. Although the
attenuated controls retain a very slight level of cleavage activity
under these optimized conditions, the decrease in in vitro cleavage
activity between each active ribozyme and its paired attenuated
control is about two orders of magnitude. Thus, an active core is
essential for cleavage activity in vitro and is also necessary for
ribozyme activity in cell culture.
[0280] Ribozyme-mediated reduction of VEGF-induced angiogenesis in
vivo. To assess whether ribozymes targeting VEGF receptor mRNA
could impact the complex process of angiogenesis, prototypic
anti-Flt-1 and KDR ribozymes that were identified in cell culture
studies were screened in a rat corneal pocket assay of VEGF-induced
angiogenesis. In this assay, corneas implanted with VEGF-containing
filter disks exhibited a robust neovascular response in the corneal
region between the disk and the corneal limbus (from which the new
vessels emerge). Disks containing a vehicle solution elicited no
angiogenic response. In separate studies, intraconjunctival
injections of sterile water vehicle did not affect the magnitude of
the VEGF-induced angiogenic response. In addition, ribozyme
injections alone did not induce angiogenesis.
[0281] The dose-related effects of anti-Flt-1 or KDR ribozymes on
the VEGF-induced angiogenic response were then examined. FIGS. 21
and 22 illustrate the quantified antiangiogenic effect of the
anti-Flt-1 (site 4229) and KDR (site 726) ribozymes and their
attenuated controls over a dose range from 1 to 100 .mu.g,
respectively. For both ribozymes, the maximal antiangiogenic
response (48 and 36% for anti-Flt-1 and KDR ribozymes,
respectively) was observed at a dose of 10 .mu.g.
[0282] The anti-Flt-1 ribozyme produced a significantly greater
antiangiogenic response than its attenuated control at 3 and 10
.mu.g (p<0.05; FIG. 21). Its attenuated control exhibited a
small but significant antiangiogenic response at doses above 10
.mu.g compared to vehicle treated VEGF controls (p<0.05; FIG.
21). At its maximum, this response was not significantly greater
than that observed with the lowest dose of active anti-Flt-1
ribozyme. The anti-KDR ribozyme significantly inhibited
angiogenesis from 3 to 30 .mu.g (p<0.05; FIG. 22). The anti-KDR
attenuated control had no significant effect at any dose
tested.
EXAMPLE 13
In Vivo Inhibition of Tumor Growth and Metastases by VEGF-R
Ribozymes
[0283] A. Lewis Lung Carcinoma Mouse Model: Ribozymes were
chemically synthesized as described above. The sequence of
ANGIOZYME.TM. bound to its target RNA is shown in FIG. 26.
[0284] The tumors in this study were derived from a cell line
(LLC-HM) which gives rise to reproducible numbers of spontaneous
lung metastases when propagated in vivo. The LLC-HM line was
obtained from Dr. Michael O'Reilly, Harvard University. Tumor
neovascularization in Lewis lung carcinoma has been shown to be
VEGF-dependent. Tumors from mice bearing LLC-HM (selected for the
highly metastatic phenotype by serial propagation) were harvested
20 days post-inoculation. A tumor brei suspension was prepared from
these tumors according to standard protocols. On day 0 of the
study, 0.5.times.10.sup.6 viable LLC-HM tumor cells were injected
subcutaneously (sc) into the dorsum or flank of previously
untreated mice (100 .mu.L injectate). Tumors were allowed to grow
for a period of 3 days prior to initiating continuous intravenous
administration of saline or 30 mg/kg/d ANGIOZYME.TM. via Alzet
mini-pumps. One set of animals was dosed from days 3 to 17,
inclusive. Tumor length and width measurements and volumes were
calculated according to the formula:
Volume=0.5(length)(width).sup.2. At post-inoculation day 25,
animals were euthanized and lungs harvested. The number of lung
macrometastatic nodules was counted. It should be noted that
metastatic foci were quantified 8 days after the cessation of
dosing. Ribozyme solutions were prepared to deliver to another set
of animals 100, 10, 3, or 1 mg/kg/day of ANGIOZYME.TM. via Alzet
mini-pumps. A total of 10 animals per dose or saline control group
were surgically implanted on the left flank with osmotic mini-pumps
pre-filled with the respective test solution three days following
tumor inoculation. Pumps were attached to indwelling jugular vein
catheters.
[0285] FIG. 27 shows the antitumor effects of ANGIOZYME.TM.. There
is a statistically significant inhibition (p<0.05) of primary
LLC-HM tumor growth in tumors grown in the flank regions compared
to saline control. ANGIOZYME.TM. significantly reduced (p<0.05)
the number of lung metastatic foci in animals inoculated either in
the flank regions. FIG. 28 illustrates the dose-dependent
anti-metastatic effect of ANGIOZYME.TM. compared to saline
control.
[0286] B. Mouse Colorectal Cancer Model. KM12L4a-16 is a human
colorectal cancer cell line. On day 0 of the study,
0.5.times.10.sup.6 KM12L4a-16 cells were implanted into the spleen
of nude mice. Three days after tumor inoculation, Alzet minipumps
were implanted and continuous subcutaneous delivery of either
saline or 12, 36 or 100 mg/kg/day of ANGIOZYME.TM. was initiated.
On day 5, the spleens containing the primary tumors were removed.
On day 18, the Alzet minipumps were replaced with fresh pumps so
that delivery of saline or ANGIOZYME.TM. was continuous over a 28
day period from day 3 to day 32. Animals were euthanized on day 41
and the liver tumor burden was evaluated.
[0287] Following treatment with 100 mg/kg/day of ANGIOZYME.TM.,
there was a significant reduction in the incidence and median
number of liver metastasis (FIGS. 29 and 30). In saline-treated
animals, the median number of metastases was .gtoreq.101. However,
at the high dose of ANGIOZYME.TM. (100 mg/kg/day), the median
number of metastases was zero.
EXAMPLE 14
Effect of ANGIOZYME.TM. Alone or in Combination with
Chemotherapeutic Agents in the Mouse Lewis Lung Carcinoma Model
Methods
[0288] Tumor inoculations. Male C57/BL6 mice, age 6 to 8 weeks,
were inoculated subcutaneously in the flank with 5.times.10.sup.5
LLC-HM cells from brei preparations made from tumors grown in
mice.
[0289] Ribozymes and controls. The ribozyme and controls tested in
this study are given in Table XIII. RPI.4610, also known as
ANGIOZYME.TM., is an anti-Flt-1 ribozyme that targets site 4229 in
the human Flt-1 receptor mRNA (EMBL accession no. X51602). The
controls tested include RPI.13141, an attenuated version of
RPI.4610 in which four nucleotides in the catalytic core are
changed so that the cleavage activity is dramatically decreased.
RPI.13141, however, maintains the base composition and binding arms
of RPI.4610 and so is still capable of binding to the target site.
The second control (RPI.13030) also has changes to the catalytic
core (three) to inhibit cleavage activity, but in addition the
sequence of the binding arms has been scrambled so that it can no
longer bind to the target sequence. One nucleotide in the arm of
RPI.13030 is also changed to maintain the same base composition as
RPI.4610.
[0290] Ribozyme administrations. Ribozymes and controls were
resuspended in normal saline. Administration was initiated seven
days following tumor inoculation. Animals either received a daily
subcutaneous injection (30 mg/kg test substance) from day 7 to day
20 or were instrumented with an Alzet osmotic minipump (12
.mu.L/day flow rate) containing a solution of ribozyme or control.
Subcutaneous infusion pumps delivered the test substances (30
mg/kg/day) from day 7 to 20 (14-day pumps, 420 mg/kg total test
substance) or days 7-34 (28-day pumps, 840 mg/kg total test
substance). Where indicated, chemotherapeutic agents were given in
combination with ribozyme treatment. Cyclophosphamide was given by
ip administration on days 7, 9 and 11 (125 mg/kg). Gemcitabine was
given by intraperitoneal administration on days 8, 11 and 14 (125
mg/kg). Untreated, uninstrumented animals were used as comparison.
Five animals were included in each group.
Results
[0291] The antiangiogenic ribozyme, ANGIOZYME.TM., was tested in a
model of Lewis lung carcinoma alone and in combination with two
chemotherapeutic agents. Previously (see above), 30 mg/kg/day
ANGIOZYME.TM. alone was determined to inhibit both primary tumor
growth and lung metastases in a highly metastatic variant of Lewis
lung (continuous 14-day intraveneous delivery via Alzet
minipump).
[0292] In this study, 30 mg/kg/day ANGIOZYME.TM. delivered either
as a daily subcutaneous bolus injection or as a continuous infusion
from an Alzet minipump resulted in a delay in tumor growth (FIG.
23). On average, tumor growth to 500 mm.sup.3 was delayed by
approximately 7 days in animals being treated with ANGIOZYME.TM.
compared to an untreated group. Growth of tumors in animals being
treated with either of two attenuated controls was delayed by only
approximately 2 days.
[0293] ANGIOZYME.TM. delivered by subcutaneous bolus was also
tested in combination with either Gemcytabine or cyclophosphamide
(FIG. 24). Tumor growth delay increased by about 3 days in the
presence of combination therapy with ANGIOZYME.TM. and Gemcytabine
over the effects of either treatment alone. The combination of
ANGIOZYME.TM. and cyclophosphamide did not increase tumor growth
delay over that of cyclophosphamide alone, however, suboptimal
doses of cyclophosphamide were not included in this study. Neither
of the attenuated controls increased the effect of the
chemotherapeutic agents.
[0294] The effect of ANGIOZYME.TM. on metastases to the lung was
also determined in the presence and absence of additional
chemotherapeutic treatment. Macrometastases to the lungs were
counted in two animals in each treatment group on day 20. Data for
the daily subcutaneous administration of 30 mg/kg ANGIOZYME.TM.
alone or with Gemcytabine or cyclophosphamide is given in FIG. 25.
In the presence of ANGIOZYME.TM., with or without a
chemotherapeutic agent, the lung metastases were reduced to zero.
Treatment with either Gemcytabine or cyclophosphamide alone (mean
number of metastases 4.5 and 4, respectively) were not as effective
as ANGIOZYME.TM. alone or when used in combination with
ANGIOZYME.TM.. Neither of the attenuated controls increased the
effect of the chemotherapeutic agents.
[0295] The effect on metastases to the lung was also determined
following continuous treatment with ANGIOZYME.TM.. At day 20, an
average of approximately 8 macrometastases were noted in the
treatment groups which had been instrumented with Alzet minipumps
(either 14- or 28-day pumps). This is a decrease in metastases of
approximately 50% from the untreated group. Since ANGIOZYME.TM.
delivered by a daily subcutaneous bolus resulted in zero metastases
(FIG. 4) in the two animals counted, it is possible that the
additional burden of being instrumented with the minipump
contributes to a slightly decreased response to ANGIOZYME.TM..
EXAMPLE 15
Phase I/II Study of Repetitive Dose ANGIOZYME.TM. Targeting the
FLT-1 Receptor of VEGF
[0296] A ribozyme therapeutic agent ANGIOZYME.TM., was assessed by
daily subcutaneous administration in a phase I/II trial for 31
patients with refractory solid tumors. Demographic information
relating to patients enrolled in the study are shown in Table XX.
The primary study endpoint was to determine the safety and maximum
tolerated dose of ANGIOZYME.TM.. Secondary endpoints assessed
ANGIOZYME.TM. pharmacokinetics and clinical response. Patients were
treated in four cohorts of three patients at doses of 10, 30, 100,
and 300 mg/m.sup.2/day. Following the dose escalation phase, an
additional 15 evaluable patients were entered in an expanded cohort
at 100 mg/m2/day. Patients were dosed for a minimum of 29
consecutive days with 24-hour pharmacokinetic analyses on Day 1 and
29. Clinical response was assessed monthly.
[0297] Results The data from 20 patients indicated that
ANGIOZYME.TM. was well tolerated, with no systemic adverse events.
FIG. 31 shows the plasma concentration profile of ANGIOZYME.TM.
after a single SC (sub-cutaneous) dose of 10, 30, 100, or 300
mg/m.sup.2. The pharmacokinetic parameters of ANGIOZYME.TM. after
SC bolus administration are outlined in Table XXI. An MTD (maximum
tolerated dose) could not be established. One patient in the 300
mg/m.sup.2/d group experienced a grade 3 injection site reaction.
Patients in the other groups experienced intermittent grade 1 and
grade 2 injection site reactions with erythema and induration. No
systemic or laboratory toxicities were observed. Pharmacokinetic
analyses demonstrated dose-dependent plasma concentrations with
good bioavailability (70-90%), t1/2=209-384 min, and no
accumulation after repeated doses. To date, 17/28 (61%) of
evaluable patients have had stable disease for periods of one to
six months and two patients (nasopharyngeal squamous cell carcinoma
and melanoma) had minor clinical responses. The patient with
nasopharyngeal carcinoma demonstrated central tumor necrosis as
indicated by MRI. The longest period of treatment thus far has been
8 months for two patients at 100 mg/m.sup.2/d (breast, peritoneal
mesothelioma).
EXAMPLE 16
In Vivo Inhibition of Neovascularization in an Ocular Animal Model
by VEGF-R Ribozymes
[0298] Summary of the Mouse Model: A mouse model of proliferative
retinopathy (Aiello et al., 1995, Proc. Natl. Acad. Sci. USA 92:
10457-10461; Robinson et al., 1996, Proc. Natl. Acad. Sci. USA 93:
4851-4856; Pierce et al., 1996, Archives of Ophthalmology 114:
1219-1228) in which neovascularization of the mouse retina is
induced by exposure of 7-day old mice to 75% oxygen followed by a
return to normal room air. The initial period in high oxygen causes
an obliteration of developing blood vessels in the retina. Exposure
to room air five days later is perceived as hypoxia by the now
underperfused retina. The result is an immediate upregulation of
VEGF mRNA and VEGF protein (between 6-12 hours) followed by an
extensive retinal neovascularization that peaks in approximately 5
days. Although this model is more representative of retinopathy of
prematurity than diabetic retinopathy, it is an accepted small
animal model in which to study neovascular pathophysiology of the
retina. In fact, intravitreal injection of certain antisense DNA
constructs targeting VEGF mRNA have been found to be antiangiogenic
in this model, as were soluble VEGF receptor chimeric proteins
designed to bind VEGF in the vitreous humor (Aiello et al., 1995,
Proc. Natl. Acad. Sci. USA 92: 10457-10461; Robinson et al., 1996,
Proc. Natl. Acad. Sci. USA 93: 4851-4856; Pierce et al., 1996,
Archives of Ophthalmology 114: 1219-1228).
[0299] Summary of experiment: The effect of an anti-KDR/Flk-1
ribozyme on the peak level of neovascularization was tested in the
mouse model described above. As shown in FIG. 34A, P7 mice were
removed from the hyperoxic chamber and the mice received two
intraocular injections (P12 and P13) in the right eye of 10 .mu.g
RPI.4731, the anti-KDR/Flk-1 ribozyme. The left eye of each mouse
was treated as a control and received intraocular injections of
saline. Five days after being exposed to room air, neovascular
nuclei in the retina of both eyes were counted. Data are presented
in FIG. 34B. There was a significant decrease in retinal
neovascularization (approximately 40%) compared to the control,
saline-injected eyes.
[0300] RPI.4731 Sequence and Chemical Composition: TABLE-US-00005
(SEQ ID NO: 13488) 5'-u.sub.sa.sub.sc.sub.s a.sub.sau ucU GAu Gag
gcg aaa gcc Gaa Aag aca aB-3'
[0301] where: [0302] uppercase G, A=ribonucleotides [0303]
lowercase=2'-OMe [0304] U=2'-C-allyl uridine [0305] B=inverted
abasic nucleotide [0306] S=phosphorothioate linkage Indications
[0307] 1) Tumor angiogenesis: Angiogenesis has been shown to be
necessary for tumors to grow into pathological size (Folkman, 1971,
PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer
Res and Treatment 38, 109-119). In addition, it allows tumor cells
to travel through the circulatory system during metastasis.
Increased levels of gene expression of a number of angiogenic
factors such as vascular endothelial growth factor (VEGF) have been
reported in vascularized and edema-associated brain tumors (Berkman
et al., 1993 J Clini. Invest. 91, 153). A more direct demonstration
of the role of VEGF in tumor angiogenesis was demonstrated by Jim
Kim et al., 1993 Nature 362, 841 wherein, monoclonal antibodies
against VEGF were successfully used to inhibit the growth of
rhabdomyosarcoma, glioblastoma multiforme cells in nude mice.
Similarly, expression of a dominant negative mutated form of the
flt-1 VEGF receptor inhibits vascularization induced by human
glioblastoma cells in nude mice (Millauer et al., 1994, Nature 367,
576). Specific tumor/cancer types that can be targeted using the
nucleic acid molecules of the invention include but are not limited
to the tumor/cancer types described under Diagnosis in Table
XX.
[0308] 2) Ocular diseases: Neovascularization has been shown to
cause or exacerbate ocular diseases including but not limited to,
macular degeneration, neovascular glaucoma, diabetic retinopathy,
myopic degeneration, and trachoma (Norrby, 1997, APMIS 105,
417-437). Aiello et al., 1994 New Engl. J. Med. 331, 1480, showed
that the ocular fluid, of a majority of patients suffering from
diabetic retinopathy and other retinal disorders, contains a high
concentration of VEGF. Miller et al., 1994 Am. J. Pathol. 145, 574,
reported elevated levels of VEGF mRNA in patients suffering from
retinal ischemia. These observations support a direct role for VEGF
in ocular diseases. Other factors, including those that stimulate
VEGF synthesis, may also contribute to these indications.
[0309] 3) Dermatological Disorders: Many indications have been
identified which may be angiogenesis dependent including, but not
limited to, psoriasis, verruca vulgaris, angiofibroma of tuberous
sclerosis, pot-wine stains, Sturge Weber syndrome,
Kippel-Trenaunay-Weber syndrome, and Osler-Weber-Rendu syndrome
(Norrby, supra). Intradermal injection of the angiogenic factor
b-FGF demonstrated angiogenesis in nude mice (Weckbecker et al.,
1992, Angiogenesis: Key principles-Science-Technology-Medicine, ed
R. Steiner) Detmar et al., 1994 J. Exp. Med. 180, 1141 reported
that VEGF and its receptors were over-expressed in psoriatic skin
and psoriatic dermal microvessels, suggesting that VEGF plays a
significant role in psoriasis.
[0310] 4) Rheumatoid arthritis: Immunohistochemistry and in situ
hybridization studies on tissues from the joints of patients
suffering from rheumatoid arthritis show an increased level of VEGF
and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341).
Additionally, Koch et al., 1994 J. Immunol. 152, 4149, found that
VEGF-specific antibodies were able to significantly reduce the
mitogenic activity of synovial tissues from patients suffering from
rheumatoid arthritis. These observations support a direct role for
VEGF in rheumatoid arthritis. Other angiogenic factors including
those of the present invention may also be involved in
arthritis.
Combination Therapies
[0311] Gemcytabine and cyclophosphamide are non-limiting examples
of chemotherapeutic agents that can be combined with or used in
conjunction with the nucleic acid molecules (e.g. ribozymes and
antisense molecules) of the instant invention. Those skilled in the
art will recognize that other anti-angiogenic and/or anti-cancer
compounds and therapies can be similarly be readily combined with
the nucleic acid molecules of the instant invention (e.g. ribozymes
and antisense molecules) and are hence within the scope of the
instant invention. Such compounds and therapies are well known in
the art (see for example Cancer: Principles and Pranctice of
Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and
Rosenberg, S. A., J. B. Lippincott Company, Philadelphia, USA;
incorporated herein by reference) and include, without limitations,
folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine
analogs, adenosine analogs, topoisomerase I inhibitors,
anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum
analogs, alkylating agents, nitrosoureas, plant derived compounds
such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase
inhibitors, taxols, radiation therapy, surgery, nutritional
supplements, gene therapy, radiotherapy, for example 3D-CRT,
immunotoxin therapy, for example ricin, and monoclonal antibodies.
Specific examples of chemotherapeutic compounds that can be
combined with or used in conjuction with the nucleic acid molecules
of the invention include, but are not limited to, Paclitaxel;
Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine;
Tomaxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan;
Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin
C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine;
L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan;
Ifosfamide; 4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan
(CAMPTOSAR.RTM., CPT-11, Camptothecin-11, Campto) Tamoxifen,
Herceptin; IMC C225; ABX-EGF: and combinations thereof.
Diagnostic Uses
[0312] The nucleic acid molecules of this invention (e.g.,
enzymatic nucleic acid) can be used as diagnostic tools to examine
genetic drift and mutations within diseased cells or to detect the
presence of flt-1, KDR and/or flk-1 RNA in a cell. The close
relationship between enzymatic nucleic acid activity and the
structure of the target RNA allows the detection of mutations in
any region of the molecule which alters the base-pairing and
three-dimensional structure of the target RNA. By using multiple
enzymatic nucleic acids described in this invention, one can map
nucleotide changes which are important to RNA structure and
function in vitro, as well as in cells and tissues. Cleavage of
target RNAs with enzymatic nucleic acid can be used to inhibit gene
expression and define the role (essentially) of specified gene
products in the progression of disease. In this manner, other
genetic targets can be defined as important mediators of the
disease. These experiments can lead to better treatment of the
disease progression by affording the possibility of combinational
therapies (e.g., multiple enzymatic nucleic acids targeted to
different genes, enzymatic nucleic acids coupled with known small
molecule inhibitors, or intermittent treatment with combinations of
enzymatic nucleic acids and/or other chemical or biological
molecules). Other in vitro uses of enzymatic nucleic acids of the
invention are well known in the art, and include detection of the
presence of mRNAs associated with flt-1, KDR and/or flk-1 related
condition. Such RNA is detected by determining the presence of a
cleavage product after treatment with a ribozyme using standard
methodology.
[0313] In a specific example, enzymatic nucleic acids which can
cleave only wild-type or mutant forms of the target RNA are used
for the assay. The first enzymatic nucleic acid is used to identify
wild-type RNA present in the sample and the second enzymatic
nucleic acid is used to identify mutant RNA in the sample. As
reaction controls, synthetic substrates of both wild-type and
mutant RNA are cleaved by both enzymatic nucleic acids to
demonstrate the relative enzymatic nucleic acid efficiencies in the
reactions and the absence of cleavage of the "non-targeted" RNA
species. The cleavage products from the synthetic substrates also
serve to generate size markers for the analysis of wild-type and
mutant RNAs in the sample population. Thus, each analysis requires
two enzymatic nucleic acids, two substrates and one unknown sample
which is combined into six reactions. The presence of cleavage
products is determined using an RNAse protection assay so that
full-length and cleavage fragments of each RNA can be analyzed in
one lane of a polyacrylamide gel. It is not absolutely required to
quantify the results to gain insight into the expression of mutant
RNAs and putative risk of the desired phenotypic changes in target
cells. The expression of mRNA whose protein product is implicated
in the development of the phenotype (i.e., flt-1, KDR and/or flk-1)
is adequate to establish risk. If probes of comparable specific
activity are used for both transcripts, then a qualitative
comparison of RNA levels is adequate and decreases the cost of the
initial diagnosis. Higher mutant form to wild-type ratios is
correlated with higher risk whether RNA levels are compared
qualitatively or quantitatively. The use of enzymatic nucleic acid
molecules in diagnostic applications contemplated by the instant
invention is more fully described in George et al., U.S. Pat. Nos.
5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332,
Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,
International PCT publication No. WO 00/24931, Breaker et al.,
International PCT Publication Nos. WO 00/26226 and 98/27104, and
Sullenger et al., International PCT publication No. WO
99/29842.
Additional Uses
[0314] Potential usefulness of sequence-specific enzymatic nucleic
acid molecules of the instant invention have many of the same
applications for the study of RNA that DNA restriction
endonucleases have for the study of DNA (Nathans et al., 1975 Ann.
Rev. Biochem. 44:273). For example, the pattern of restriction
fragments can be used to establish sequence relationships between
two related RNAs, and large RNAs can be specifically cleaved to
fragments of a size more useful for study. The ability to engineer
sequence specificity of the enzymatic nucleic acid molecule is
ideal for cleavage of RNAs of unknown sequence. Applicant describes
the use of nucleic acid molecules to down-regulate gene expression
of target genes in bacterial, microbial, fungal, viral, and
eukaryotic systems including plant, or mammalian cells.
[0315] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0316] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and compositions described herein as presently
representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art,
which are encompassed within the spirit of the invention, are
defined by the scope of the claims.
[0317] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. Thus, such additional embodiments are
within the scope of the present invention and the following
claims.
[0318] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments, optional features, modification and variation of the
concepts herein disclosed may be resorted to by those skilled in
the art, and that such modifications and variations are considered
to be within the scope of this invention as defined by the
description and the appended claims.
[0319] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0320] Other embodiments are within the claims that follow.
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lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
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"Sequence Listing" is available in electronic form from the USPTO
web site
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from the USPTO upon request and payment of the fee set forth in 37
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0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070042029A1).
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* * * * *
References