Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1997-05-08
2002-12-03
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091310, C435S320100, C435S325000, C536S023100, C536S023200, C536S024500
Reexamination Certificate
active
06489163
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of selective inhibition of androgen receptor. The invention further relates generally to the field of gene therapy, and particularly gene therapy in the treatment of prostatic cancer.
BACKGROUND OF THE INVENTION
The prostate gland is an androgen-dependent organ and continues to grow with age. This leads to enlarged prostate in older men with consequent pathological manifestations. Androgen receptor is the principal mediator of prostatic growth.
A hammerhead ribozyme is a small RNA capable of cleaving a target RNA in a catalytic manner in the presence of a divalent cation (Pyle, 1993). Naturally occurring hammerhead ribozymes were discovered in certain plant viroids and viruses (Forster and Symons, 1987). The hammerhead ribozyme acts in “cis” during viral replication by the rolling circle mechanism. However a hammerhead ribozyme was engineered to cleave in “trans” against other RNAs (Uhlenbeck, 1987). A hammerhead ribozyme consists of antisense segments (stems I and III) and a catalytic domain (stem II). It can be designed to target specific mRNAs by selecting sequences flanking the catalytic element. The only requirement for the target substrate is the sequence HUX (H can be any nucleotide, X is A, C or U), where cleavage occurs after X (Haseloff and Gerlach, 1988). Hammerhead cleavage produces RNA products with 5′ hydroxyl and 2′, 3′ cyclic phosphate termini (Buzayan, et al., 1986; Prody, et al., 1986). A hammerhead ribozyme has potential therapeutic applications, e.g., it inactivates specific RNAs in vivo, such as HIV-1 gene expression (Sarver, et al., 1990; Ojwang, et al, 1992; Yu, et al., 1993), RNAs responsible for other viral infections (Chen, et al., 1992; Sullenger and Cech, 1993; Tang, et al., 1994) and the RNA transcripts of other genes (Scanlon, et al., 1991; Kashani-Sahetet, et al., 1992; Lange, et al., 1993; Ha and Kim, 1994; Kobayashi, et al., 1994; Sioud, et al., 1994; Jarvis, et al., 1996; Ohta, et al., 1996; Sioud, 1996).
Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid/thyroid hormone receptor superfamily (Evans, 1988; Beato, 1989). AR plays an important role in the coordination of the male-specific sexual phenotype and in the development of the male-reproductive organs such as the prostate gland (Quiley, et al., 1995). AR is expressed in various cells and tissues (Chang, et al., 1995; Roy and Chatterjee, 1995). It has also been considered as an etiologic factor for human benign prostatic hyperplasia (Brolin, et al., 1992; Wilding, 1992; Lepor, et al., 1993). Furthermore, AR gene mutations are involved in primary and secondary prostate cancer (Newmark, et al., 1992; Culig, et al., 1993; Suzuki, et al., 1993; Taplin, et al., 1995). A high expression of AR in recurrent prostate cancer cells and metastatic prostate cancer cells has also been observed (Taplin, et al., Viskarpi, et al., 1995; Umeki, et al., 1996). However, how the AR regulates differentiation and development of the male reproductive organs and its role in prostatic diseases are not known.
Clinical treatment of prostatic cancer has included the use of surgical techniques to remove enlarged prostate tissue, or the use of enzyme inhibitors such as PROSCAR™. PROSCAR inhibits 5-alpha reductase, which is the enzyme that converts testosterone to dihydrotestosterone. The abolition of testesterone itslf to induce androgen action limits the use and effectiveness of this therapy. These approaches are thus undesirable in many patients. Need continues to exist in the medical arts for a therapy that provides a more targeted approach to treatment of this pathology.
Androgen receptor plays a central role in the development, differentiation and maintenance of the male reproductive organs (Coffey, 1988; Griffin et al., 1989; Migeon et al., 1994). It is also involved in prostate disorders and other diseases (Edward, 1992; Macke et al., 1993; Qingley et al., 1995). The molecular mechanisms whereby AR regulates the physiological and pathological events are not clearly understood (Wilding, 1992; Lapor and Lawson, 1993). Hence, there has been no significant development of clinical approaches for treatment of prostate and related disorders.
SUMMARY OF THE INVENTION
The present invention describes inactivation of AR gene expression by engineering hammerhead ribozymes to cleave specific sites in AR mRNA. The present in vitro studies of hammerhead ribozymes reveal a high efficiency of such cleavage activity. The hammerhead ribozymes suppress AR mRNA expression in cultured cells.
Included in the present invention are hammerhead ribozymes that can selectively and efficiently degrade human androgen receptor messenger RNA. Also part of the present invention are expression vectors containing the gene for a ribozyme that, when introduced into a human prostate cancer cell, is capable of abolishing the androgen receptor mediated transactivation of a reporter gene. Targeting of the ribozyme gene into specific tissues of transgenic mice can be done to produce tissue-specific inactivation of androgen receptor. Therapeutic use of the ribozymes of the present invention to suppress androgen action in human clinical conditions such as the prostatic hyperplasia may be accomplished in vivo of the present invention.
The following provides three selection criteria in identifying and designing synthetic ribozyme of the present invention:
Based on these three selection criteria, the inventors designed three hammerhead ribozymes and tested their effectiveness in the in vitro endonuclease assay. One of these ribozymes, HR-2 was found to be particularly highly effective in selectively degrading the androgen receptor mRNA. This androgen receptor degrading ribozyme is more active than all ribozymes reported in the literature.
After eliminating sequence regions that can potentially form secondary structures or have significant homology to heterologous mRNAs, the inventors chose three structural domains of the AR mRNA with high AU contents as targets for the hammerhead ribozyme.
Hammerhead ribozymes are composed of two functionally distinct components; (i) the central catalytic core usually containing about 24 nucleotides with a conserved stem loop structure, and (ii) two variable specifier sequences on both 5′ and 3′ sides of the catalytic core that are complementary to the target RNA. The three targeted areas on the AR mRNA that were selected correspond to (i) transactivation domain of the rat AR (ribozyme, R-1), (ii) transactivation domain of the human AR (ribozyme, H-1), and (iii) the DNA binding domain of the human AR with 95% homology to the rat AR (ribozyme, HR-2). All three of these ribozymes contained 9-12 nt long specifier arms on each side of the catalytic core. Both ribozymes and truncated AR targets were cloned into the Bluescript vector and were transcribed with either T7 or T3 RNA polymerase for the in vitro endonuclease assay. At an equimolar enzyme-substrate ratio and at 37° C., R1 and H1 required ~4 hr for 75 to 100% cleavage of the substrate. The ribozyme HR-2 required less than 30 min for complete cleavage of the target substrate. The HR-2 ribozyme was also effective at a E:S ratio as low as 1:50. A mutant HR-2 containing two base substitutions within the catalytic core was enzymatically inactive and the wild type HR-2 did not act on substrates corresponding to R-1 and H-1, substantiating the specificity of the ribozyme function.
The inventors examined the effectiveness of the HR-2 in AR
(−)
PC3 (prostate cancer derived) cells transfected with the AR expression vector and a reporter construct containing MMTV-CAT. In this transfection assay an expression vector containing the HR-2 ribozyme was able to inhibit the AR mediated transactivation of the MMRV-promoter in a dose-dependent manner with a more than 95% inhibition at an AR:HR-2 ratio of 100. These results indicate that ribozymes can be an effective means for inactivating androgen action and are useful as a therapeutic agent when del
Chen Shuo
Roy Arun K
Board of Regents , The University of Texas System
LeGuyader John L.
Schmidt M
Williams, Morgan and Amerson
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