ERbB-4 targeted ribozymes

Details ERbB-4 targeted ribozymes ERbB-4 targeted ribozymes

2000-06-23

2003-05-20

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C435S455000, C435S458000, C536S023100, C536S024500

Reexamination Certificate

active

06566104

ABSTRACT:

INTRODUCTION
This invention relates to methods for inhibition of growth of transformed cells, and treatment and diagnosis of diseases and conditions related to ErbB-4 expression.
The epidermal growth factor (EGF) receptors have been implicated in human cancer more frequently than any other family of growth factor receptors. The EGF receptor gene is often amplified or overexpressed in squamous cell carcinoma and glioblastomas [Jenkins et al. (1989)
Cancer Genet. Cytogenet.
39:253]. Similarly, ErbB-4 is overexpressed in adenocarcinomas of the stomach, breast and ovary.
The epidermal growth factor receptor (EGFR/ErbB) family is a group of tyrosine kinases that is frequently overexpressed in a variety of carcinomas [Gullick, W. J. (1991)
Br. Med. Bull.
47:87-98; Hynes, N. E. and Stern, D. F. (1994)
Biochem. Biophys. Acta
1198:165-184; Lemoine, N. R. et al. (1992)
Br. J. Cancer
66:1116-1121]. This class I subfamily of receptors is comprised of four members: EGFR [Xu, Y. H. et al. (1984)
Nature
309:806-810], HER2/ErbB-2
eu [Schechter, A. L. et al. (1984)
Nature
312:513-516], HER3/ErbB-3 [Kraus, M. H. et al.
Proc. Natl. Acad. Sci. USA
86:9193-9197; Plowman, G. D. et al. (1990)
Proc. Natl. Acad. Sci. USA
87:4905-4909], and HER4/ErbB-4 [Plowman, G. D. et al. (1993)
Proc. Natl. Acad. Sci. USA
90:1746-1750]. Data from numerous laboratories suggest that the EGFR family members may play a complex role in signaling [Wada, T. et al. (1990)
Cell
61:1339-1347; Goldman, R. et al. (1990)
Biochemistry
29:11024-11028; Caraway, K. L. and Cantley L. C. (1994)
Cell
78:5-8]. Most human breast cancer cells express more than one of the EGF family receptors, and different combinations of receptors can heterodimerize or homodimerize. These receptor interactions lead to the activation of multiple signaling pathways and contribute to the pathogenicity and tumorigenicity of breast cancer [Earp, S. H. et al. (1995)
Breast Cancer Resarch and Treatment].
A number of growth factors, classified as EGF-like ligands, have been identified that bind and stimulate the kinase activity of EGF-family receptors. EGF, transforming growth factor &agr; (TGF&agr;), amphiregulin (AR), heparin-binding EGF(HB-EGF), and betacellulin (BTC) have been described as specific for EGFR [Savage, C. R. et al. (1972)
J. Biol. Chem.
241:7612-7621; Marquardt, H. et al. (1983)
Science
223:1079-1082; Shoyab, M. et al. (1989)
Science
243:1079-1082; Higashiyama, S. et al. (1991)
Science
251: 936-939; shing, Y. et al. (1993)
Science
259:1604-1607]. Several differentially spliced variants, named heregulin (HRG) also known as neuregulin (NRG), or neu differentiation factor (NDF) [Holmes, W. E. et al. (1992)
Science
256:1205-1210; Wen, D. et al. (1992)
Cell
69:559-572], acetylcholine-receptor inducing activity (ARIA) [Falls, D. G. et al. (1993)
Cell
72:801-815], glial growth factor (GGF) [Marchionni, M. A. et al. (1993)
Nature
(London)362: 312-318] and gp30 [Lupu, R. et al. (1990)
Science
249:1552-1555], were initially identified as candidate neu ligands by their ability to induce neu tyrosine phosphorylation [Peles, E. and Yarden, Y. (1993)
Bioassays
15:815-824]. However, recent results demonstrate that ErbB-3 and ErbB-4 are primary receptors for heregulin [Plowman, G. D. et al. (1993)
Nature
366:473-475; Carraway, K. L. III et al. (1994)
J. Biol. Chem.
269: 14303-14306]. Activation of ErbB-2 by HRG is thought to occur through transphosphorylation resulting from heterodimerization with either ErbB-3 or ErbB-4 [Tzahar, E. et al. (1994)
J. Biol. Chem.
269:40:25226-25223; Peles, E. et al. (1993)
EMBO J.
12:961-971; Sliwkowski, M. X. et al. (1994)
J. Biol. Chem.
269: 14661-14665]. Most recently, betacellulin was also shown to activate the ErbB-4 receptor in a Ba/F3 system [Riesell, D. J. et al. (1996)
Oncogene
12: 245-353].
Amplification and/or overexpression of EGFR and ErbB-2 are clearly important factors in neoplastic transformation of breast epithelium [Jardines, L. et al. (1993)
Pathobiology
61:268-282]. Elevated ErbB-4 levels have been found in certain breast cancer cell lines [Plowman, G. D. et al. (1993)
Proc. Natl. Acad. Sci. USA
90:1746-1750], but little is known about the expression or the clinical significance of ErbB-4 receptors in the diagnosis and prognosis of human breast cancer.
SUMMARY OF THE INVENTION
To investigate the biological significance of ErbB-4 in human breast cancer, we used molecular targeting of the ErbB-4 mRNA by ribozymes. We describe the generation of three ribozymes (Rz6, Rz21, Rz29) targeted to specific sites within the ErbB-4 mRNA open reading frame. We demonstrate that all three ErbB-4 ribozymes cleave ErbB-4 mRNA precisely and efficiently under physiological conditions in this cell free system. We also illustrate the intracellular efficacy and specificity of the ErbB-4 ribozymes in a model system (32D cell system). 32D cells are a murine hematopoietic IL3-dependent cell line that does not express detectable levels of endogenous EGF-family receptors. Overexpression of ErbB-4 receptors in 32D cells (32D/ErbB-4) abrogated IL-3-dependence by stimulation with NRG. We show that two of the ErbB-4 ribozymes (Rz6 and Rz29) were able to down-regulate ErbB-4 expression and were capable of abolishing the neuregulin-induced mitogenic effect in 32D/ErbB-4 cells. These results demonstrate that ribozyme Rz29 and Rz6 are biologically functional ribozymes.
Therefore, this invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding specific sites in ErbB-4. In particular, applicants describe the selection and function of ribozymes capable of cleaving this RNA and their use to reduce activity of ErbB-4 in various tissues to treat the diseases discussed herein, more particularly, breast cancer. Such ribozymes are also useful for diagnostic applications.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro [Jefferies, et al. (1989)
Nucleic Acid Res.
17:1371].
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic protion of the RNA which acts to cleave the target RNA. Thus, the ribozyme 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 a ribozyme 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 effective concentration of ribozyme necessary to effect 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, T. M. et al. (1992)
Proc. Natl. Acad. Sci. USA
89:7305-7309]

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