Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1992-06-09
1993-12-21
Rollins, John W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 6, 435 691, 435 703, 435 712, 435 9131, 4351721, 4351723, 4352523, 43525233, C07H 1512, C12Q 168, C12P 2106, C12P 2102
Patent
active
052722627
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for the production of catalytic RNA (ribozymes) in cells, including mammalian and bacterial cells. More particularly, the invention relates to synthetic genes which encode a ribozyme, to mammalian and bacterial cells having such genes transformed therein, and to the ribozyme containing expression products of such bacteria. The invention also includes the in vitro and therapeutic use of such expression products.
BACKGROUND OF THE INVENTION
One form of gene expression impairment by RNA-RNA duplex formation has been termed "antisense" inhibition. Exploitation of antisense gene regulation could lead to potent anti-viral therapy. A serious limitation of the antisense approach, especially as it applies to anti-viral activity, is that it is stoichiometric and may require large molar excesses of anti-sense versus target RNA to be effective.
Within recent years, discoveries of ribozymes, e.g., RNAs with enzymatic activities have led to the development of antisense molecules which not only form RNA-RNA hybrids, but catalytically cleave the covalent phosphodiester linkages and turn over large numbers of substrate molecules. Ribozymes can now be targeted to virtually any RNA transcript, and efficient cleavage can be readily achieved in vitro. See, Kim, S. H., et al. Proc. Natl Acad. Sci. U.S.A. 84:8788-8792 (1987); Haseloff, J., et al., Nature 234:585-591 (1988); PCT published application WO/89/05852; Cech, T.R. JAMA 260:3030-3034 (1988); PCT published application WO/88/04300; Jeffries, A. G., et al., Nucleic Acids Research 17:1371-1377 (1989).
U.S. Pat. No. 5,144,019 and application Ser. No. 401,613 describe stable, catalytically efficient ribozymes useful, inter alia, to cleave HIV-1 RNA or any other viral or endogenous cellular RNA in vitro and in vivo, mammalian cells transformed with such ribozymes, vectors useful to accomplish such transformation and the use for human therapy of such ribozymes whether produced synthetically or as expressed by such transformed cells. See Chang, et al. Clinical Biotechnology 2:23-31 (1990) which is incorporated herein by reference.
SUMMARY OF THE INVENTION
The production of large amounts of specifically targeted ribozymes for therapeutic use is problematical. This invention provides a novel method for the large scale production of any ribozyme by expression of a stable RNA molecule into which catalytic RNA has been inserted. Any stable RNA can be utilized. In the preferred practice of the invention, E. Coli is transformed by Tac promoter driven 4.5 S RNA having a catalytic RNA sequence inserted therein. High levels of expression of such fusion RNAs can be achieved by varying the temperature of growth, using mutants of E. Coli or other bacteria which are defective in certain enzymatic activities such as RNAse III, inducing expression with isopropyl thio .beta. galactopyranoside (IPTG) in an appropriate host, preparing a set of nested deletions within the 4.5 S or similar gene to eliminate undesirable folding and by other standard molecular biological techniques.
The invention also includes synthetic genes which encode catalytic RNA, microorganisms transformed with such genes, the catalytic RNA containing expression products of such genes and the in vitro and therapeutic use of such expression products.
DESCRIPTION OF THE FIGURES
FIG. 1 illustrates a vector containing a Tac promoter and a ribozyme fused to an E. Coli 4.5 S RNA gene.
FIG. 2 illustrates the vector shown by FIG. 1 with the transcriptional unit containing the ribozyme depicted by FIG. 1.
FIG. 3 is a Northern analysis showing the expression of an anti-HIV-1 gag ribozyme fused with 4.5 S RNA (lanes a and b) and 4.5 S RNA alone (lane c).
FIG. 4 depicts an ethidium bromide stained gel of an acid-phenol extract of small RNAs overproduced by E. Coli transformants in accordance with the invention.
FIG. 5 depicts a preparative gel including ethidium stained RNAs as shown by FIG. 4. The arrowhead points to the fusion ribozymes.
FIG. 6 is a composite depiction of the RNA folding of the
REFERENCES:
Chem. Abst. 110 (21):187321k, 1988.
Chem. Abst. 112(7):51284j 1989.
M. Baer, et al. Science, 228:999-1002, 1985.
Cameron et al., PNAS 86:9139-9143.
Rossi John J.
Taylor Nerida
City of Hope
Irons Edward S.
Rollins John W.
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