Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-12-23
2002-06-25
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091310, C435S325000, C435S366000, C435S375000, C536S063000
Reexamination Certificate
active
06410224
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to NF-&kgr;B levels, such as restenosis, rheumatoid arthritis, asthma, inflammatory or autoimmune disorders and transplant rejection.
BACKGROUND OF THE INVENTION
The following is a brief description of the physiological role of NF-&kgr;B. 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.
The nuclear DNA-binding activity, NF-&kgr;B, was first identified as a factor that binds and activates the immunoglobulin &kgr; light chain enhancer in B cells. NF-&kgr;B now is known to activate transcription of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-&kgr;B has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-&kgr;B is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-&kgr;B (encoded by the nf-&kgr;B2 or nf-&kgr;B1 genes, respectively) are generated from the precursors NF-&kgr;B1 (p105) or NF-&kgr;B2 (p100). The p65 subunit of NF-&kgr;B (now termed Rel A ) is encoded by the rel A locus.
The roles of each specific transcription-activating complex now are being elucidated in cells (N. D. Perkins, et al., 1992
Proc. Natl Acad. Sci USA
89, 1529-1533). For instance, the heterodimer of NF-&kgr;B1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NF-&kgr;B2/RelA heterodimers (p49/p65) actually inhibit transcription (H. B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)). Conversely, heterodimers of NF-&kgr;B2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-&kgr;B1/RelA (p50/p65) heterodimers have little effect (J. Liu, N. D. Perkins, R. M. Schmid, G. J. Nabel,
J. Virol.
1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-&kgr;B1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993
Mol. Cell. Biol.
13, 3802-3810). Thus, the promiscuous role initially assigned to NF-&kgr;B in transcriptional activation (M. J. Lenardo, D. Baltimore, 1989
Cell
58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic “knock-out” mice of individual members of the rel family. Such “knock-outs” show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.
A number of specific inhibitors of NF-&kgr;B function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-&kgr;B binding sites, and over expression of the natural inhibitor MAD-3 (an I&kgr;B family member). These agents have been used to show that NF-&kgr;B is required for induction of a number of molecules involved in inflammation, as described below.
NF-&kgr;B is required for phorbol ester-mediated induction of IL-6 (I. Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993
Mol. Cell. Biol.
13, 6137-46).
NF-&kgr;B is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993
Mol. Cell. Biol.
13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994
J. Exp. Med.
179, 503-512) on endothelial cells.
NF-&kgr;B is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).
The above studies suggest that NF-&kgr;B is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators. Two recent papers point to another connection between NF-&kgr;B and inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-&kgr;B. The glucocorticoid receptor and p65 both act at NF-&kgr;B binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994
J. Biol. Chem.
269, 6185-6192). Glucocorticoid receptor inhibits NF-&kgr;B-mediated induction of IL-6 (Ray and Prefontaine, 1994
Proc. Natl Acad. Sci USA
91, 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor (Id.).
SUMMARY OF THE INVENTION
This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding Rel A protein (p65). In particular, applicant describes the selection and function of ribozymes capable of cleaving this RNA and their use to reduce activity of NF-&kgr;B in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic applications.
Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Antisense DNA molecules have been described that block NF-&kgr;B activity. See Narayanan et al., supra. 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 the activity of NF-&kgr;B 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 rel A 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 mismatch
Draper Kenneth G.
McSwiggen James
Stinchcomb Dan T.
McDonnell & Boehnen Hulbert & Berghoff
Ribozyme Pharmaceuticals Inc.
Wang Andrew
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