Ribozyme treatment of diseases or conditions related to levels of intercellular adhesion molecule-1 (ICAM-1)

FIELD OF THE INVENTION 
The present invention relates to therapeutic compositions and methods for 
the treatment or diagnosis of diseases or conditions related to ICAM-1 
levels, such as transplant rejection, cancer, rheumatoid arthritis, 
asthma, reperfusion injury, and inflammatory or autoimmune disorders. For 
example, such treatments will be useful for transplant rejection, 
myocardial ischemia, stroke, psoriasis, and Kawasaki disease. 
BACKGROUND OF THE INVENTION 
The following is a brief description of the physiological role of ICAM-1. 
The discussion is not meant to be complete and is provided only for 
understanding of the invention that follows. This summary is not an 
admission that any of the work described below is prior art to the claimed 
invention. 
Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose 
expression is induced by inflammatory mediators. ICAM-1 is required for 
adhesion of leukocytes to endothelial cells and for several immunological 
functions including antigen presentation, immunoglobulin production and 
cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell 
recognition and activity during transplant rejection and in animal models 
of rheumatoid arthritis, asthma and reperfusion injury. 
Cell--cell adhesion plays a pivotal role in inflammatory and immune 
responses (Springer et al., 1987 Ann. Rev. Immunol. 5, 223-252). Cell 
adhesion is required for leukocytes to bind to and migrate through 
vascular endothelial cells. In addition, cell--cell adhesion is required 
for antigen presentation to T cells, for B cell induction by T cells, as 
well as for the cytotoxicity activity of T cells, NK cells, monocytes or 
granulocytes. Intercellular adhesion molecule-1 (ICAM-1) is a 110 
kilodalton member of the immunoglobulin superfamily that is involved in 
all of these cell--cell interactions (Simmons et al., 1988 Nature (London) 
331, 624-627). 
ICAM-1 is expressed on only a limited number of cells and at low levels in 
the absence of stimulation (Dustin et al., 1986 J. Immunol. 137, 245-254). 
Upon treatment with a number of inflammatory mediators 
(lipopolysaccharide, .gamma.-interferon, tumor necrosis factor-.alpha., or 
interleukin-1), a variety of cell types (endothelial, epithelial, 
fibroblastic and hematopoietic cells) in a variety of tissues express high 
levels of ICAM-1 on their surface (Sringer et. al. supra; Dustin et al., 
supra; and Rothlein et al., 1988 J. Immunol. 141, 1665-1669). Induction 
occurs via increased transcription of ICAM-1 mRNA (Simmons et al., supra). 
Elevated expression is detectable after 4 hours and peaks after 16-24 
hours of induction. 
ICAM-1 induction is critical for a number of inflammatory and immune 
responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to 
cytokine-activated endothelial cells (Boyd, 1988 Proc. Natl. Acad. Sci. 
USA 85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331). 
Thus, ICAM-1 expression may be required for the extravasation of immune 
cells to sites of inflammation. Antibodies to ICAM-1 also block T cell 
killing, mixed lymphocyte reactions, and T cell-mediated B cell 
differentiation, suggesting that ICAM-1 is required for these cognate cell 
interactions (Boyd et al., supra). The importance of ICAM-1 in antigen 
presentation is underscored by the inability of ICAM-1 defective murine B 
cell mutants to stimulate antigen-dependent T cell proliferation (Dang et 
al., 1990 J. Immunol. 144, 4082-4091). Conversely, murine L cells require 
transfection with human ICAM-1 in addition to HLA-DR in order to present 
antigen to human T cells (Altmann et al., 1989 Nature (London) 338, 
512-514). In summary, evidence in vitro indicates that ICAM-1 is required 
for cell--cell interactions critical to inflammatory responses, cellular 
immune responses, and humoral antibody responses. 
SUMMARY OF THE INVENTION 
This invention relates to ribozymes, or enzymatic RNA molecules, directed 
to cleave mRNA species encoding ICAM-1. In particular, applicant describes 
the selection and function of ribozymes capable of cleaving this RNA and 
their use to reduce levels of ICAM-1 in various tissues to treat the 
diseases discussed herein. Such ribozymes are also useful for diagnostic 
uses. 
Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to 
inflammatory or autoimmune disorders. ICAM-1 function can be blocked 
therapeutically using monoclonal antibodies. Ribozymes have the advantage 
of being generally immunologically inert, whereas significant neutralizing 
anti-IgG responses can be observed with some monoclonal antibody 
treatments. Antisense DNA molecules have been described that block ICAM-1 
expression (Chiang et al., 1991 J. Biol. Chem. 266, 18162-18171). However, 
ribozymes may show greater perdurance or lower effective doses than 
antisense molecules due to their catalytic properties and their inherent 
secondary and tertiary structures. Such ribozymes, with their catalytic 
activity and increased site specificity (as described below), represent 
more potent and safe therapeutic molecules than antisense 
oligonucleotides. 
Applicant indicates that these ribozymes are able to inhibit expression of 
ICAM-1 and that the catalytic activity of the ribozymes is required for 
their inhibitory effect. Those of ordinary skill in the art, will find 
that it is clear from the examples described that other ribozymes that 
cleave target ICAM-1 encoding mRNAs may be readily designed and are within 
the invention. 
Six 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 will destroy 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. 
The enzymatic nature of a ribozyme is advantageous over other technologies, 
such as antisense technology (where a nucleic acid molecule simply binds 
to a nucleic acid target to block its translation) since the concentration 
of ribozyme necessary to affect a therapeutic treatment is lower than that 
of an antisense oligonucleotide. This advantage reflects the ability of 
the ribozyme to act enzymatically. Thus, a single ribozyme molecule is 
able to cleave many molecules of target RNA. In addition, the ribozyme 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 completely eliminate 
catalytic activity of a ribozyme. Similar mismatches in antisense 
molecules do not prevent their action (Woolf et al., 1992 Proc. Natl. 
Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a 
ribozyme is greater than that of an antisense oligonucleotide binding the 
same RNA site. 
In preferred embodiments of this invention, the enzymatic nucleic acid 
molecule is formed in a hammerhead or hairpin motif, but may also be 
formed in the motif of a hepatitis delta virus, group I intron or RNaseP 
RNA (in association with an RNA guide sequence). Examples of such 
hammerhead motifs are described by Rossi et al., 1992 Aids Research and 
Human Retroviruses, 8, 183, of hairpin motifs by Hampel et al., "RNA 
Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which 
is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, 
Hampel and Tritz, 1989 Biochemistry, 28, 4929 and Hampel et al., 1990 
Nucleic Acids Res.earch 18, 299, and an example of the hepatitis delta 
virus motif is described by Perrotta and Been, 1992, Biochemistry 31, 16, 
of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, of the 
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) and of the Group I intron by Cech et al., U.S. Pat. No. 
4,987,071. 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. 
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 a target ICAM-1 encoding mRNA 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 cells as required. 
Alternatively, the ribozymes can be expressed from DNA vectors that are 
delivered to specific cells. 
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 enzymatic nucleic acid 
motifs. (e.g., of the hammerhead or the hairpin structure) are used for 
exogenous delivery. The simple structure of these molecules increases the 
ability of the enzymatic nucleic acid to invade targeted regions of the 
mRNA structure. However, these catalytic RNA molecules can also be 
expressed within cells from eukaryotic promoters (e.g., Scanlon 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, 5531-5534; Ojwang et al., 1992 Proc. 
Natl. Acad. Sci. USA, 89, 10802-10806; Chen et al., 1992 Nucleic Acids 
Res., 20, 4581-1589; Sarver et al., 1990 Science, 247, 1222-1225). Those 
skilled in the art realize that any ribozyme can be expressed in 
eukaryotic cells from the appropriate DNA vector. The activity of such 
ribozymes can be augmented by their release from the primary transcript by 
a second 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, 5125-5130; Ventura et al., 1993 Nucleic 
Acids Res., 21, 3249-55). 
Thus, in a first aspect, the invention features ribozymes that inhibit 
ICAM-1 production. These chemically or enzymatically synthesized RNA 
molecules contain substrate binding domains that bind to accessible 
regions of their target mRNAs. The RNA molecules also contain domains that 
catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes 
of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the 
target ICAM-1 encoding mRNAs, preventing translation and protein 
accumulation. In the absence of the expression of the target gene, a 
therapeutic effect may be observed. 
By "inhibit" is meant that the activity or level of ICAM-1 encoding mRNA is 
reduced below that observed in the absense of the ribozyme, and preferably 
is below that level observed in the presence of an inactive RNA molecule 
able to bind to the same site on the mRNA, but unable to cleave that RNA. 
Such ribozymes are useful for the prevention of the diseases and conditions 
discussed above, and any other diseases or conditions that are related to 
the level of ICAM-1 activity in a cell or tissue. By "related" is meant 
that the inhibition of ICAM-1 mRNA and thus reduction in the level of 
ICAM-1 will relieve to some extent the symptoms of the disease or 
condition. 
Ribozymes are added directly, or can be complexed with cationic lipids, 
packaged within liposomes, or otherwise delivered to target cells. The RNA 
or RNA complexes can be locally administered to relevant tissues or cells 
ex vivo or in vivo by injection or through the use of a catheter, infusion 
pump or stent, with or without their incorporation in biopolymers. In 
preferred embodiments, the ribozymes have binding arms which are 
complementary to the sequences in Tables II, III, VI-IX. Examples of such 
ribozymes are shown in Tables IV-VIII and X. Examples of such ribozymes 
consist essentially of sequences defined in these Tables. By "consists 
essentially of" is meant that the active ribozyme contains an enzymatic 
center equivalent to those in the examples, and binding arms able to bind 
mRNA such that cleavage at the target site occurs. Other sequences may be 
present which do not interfere with such cleavage. 
In another aspect of the invention, ribozymes that cleave target molecules 
and inhibit ICAM-1 activity are expressed from transcription units 
inserted into DNA, RNA, or viral vectors. Preferably, the recombinant 
vectors capable of expressing the ribozymes are locally delivered as 
described above, and transiently persist in target cells. Once expressed, 
the ribozymes cleave the target mRNA. The recombinant vectors are 
preferably DNA plasmids or adenovirus vectors. However, other mammalian 
cell vectors that direct the expression of RNA may be used for this 
purpose. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The drawings will first briefly be described.