Chimeric DNA/RNA ribozymes containing propanediol

Details Chimeric DNA/RNA ribozymes containing propanediol Chimeric DNA/RNA ribozymes containing propanediol

2000-08-09

2002-04-30

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C536S023100, C536S023720, C536S024500, C536S025300

Reexamination Certificate

active

06379931

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to ribozymes, which are molecules containing catalytic RNA sequences that are capable of cleaving RNA sequences in a substrate.
U.S. Pat. No. 5,149,796 describes chimeric DNA-RNA-DNA-RNA-DNA ribozymes of formulas I and II:
3′ X-aaag-Y-aguaguc-Z 5′ (5′ Z-cugauga-Y-gaaa-X 3′)  I.
3′ X-caaag-Y-aguaguc-Z 5′ (5′ Z-cugauga-Y-gaaac-X 3′)  II.
in which X, Y and Z are DNA sequences and cugauga, gaaa and gaaac are catalytic RNA sequences. The flanking X and Z components may be any DNA sequences that allow base pairing with the substrate RNA at appropriate positions adjacent to the substrate cleavage site. Y may be any DNA sequence that base pairs inter se in the manner required for catalytic cleavage of the substrate by the RNA sequences.
The X and Z sequences may be substituted at the respective 3′ and 5′ ends with ligands to facilitate cell entry, targeting within the cell and ultimate stability of the catalysts.
The chimeric DNA-RNA-DNA-RNA-DNA ribozymes can be synthesized in known manner by commercially available DNA synthesizers.
The patent discloses specific chimeric ribozymes which are targeted to cleave HIV-1 RNA sequences and states that the chimeric ribozymes are administered by known delivery agents and systems such as liposomes, defective viral particles, viral capsids, and standard DNA/RNA transfective procedures.
SUMMARY OF THE INVENTION
This invention provides chimeric DNA-RNA-(Pr)
n
-RNA-DNA ribozymes which are capable of binding and cleaving an RNA substrate and which comprise the sequences of formula III or IV:
5′ Z-cugaugag-(Pr)
n
-cgaaa-X 3′  III.
3′X-aaagc-(Pr)
n
-gaguaguc-Z-R-Z-cugaugag-(Pr)
n
-cgaaa-X 3′  IV.
in which
X and Z comprise DNA sequences of at least six 2′-deoxy-ribonucleotide residues or modified 2′-deoxyribonucleotide residues that base pair with the RNA substrate at positions adjacent to the RNA cleavage site;
cugaugag and cgaaa are RNA sequences in which c, u, g and a are, respectively, residues of the ribonucleotides cytidylic acid, uridylic acid, guanylic and adenylic acid or, in the case of c and u, modified residues of c and u in which 2′-hydroxy is replaced with 2′-methoxy or other uncharged group such as 2′-allyloxy or 2′-fluoro;
Pr is a residue —P(O)(OH)—O—CH
2
CH
2
CH
2
—O— derived from the C3 spacer reagent 3-(4,4′-dimethoxytrityloxy)propane-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;
n is at least 1, preferably 1-50, more preferably 2-10:
R is a residue —O—CH
2
—C(CH
2
OH)(CH
3
)—CH
2
—O— derived from the bridging reagent 2-(dimethoxytrityl-O-methyl)-2-methyl-1,3-bis-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-propane.
Examples of modified deoxyribonucleotides which can be present in Z and X include phosphorothioates and methlyphosphonates. There is no upper limit on the length of Z and X, but they will generally be about 6-50 residues, preferably about 10-20 residues, more preferably about 12-15 residues.
Z and X can each be substituted at the respective 5′ and 3′ ends with ligands to facilitate cell entry, targeting within the cell, and stability of the ribozymes and to facilitate capture and detection in in vitro assays. Such ligands include other nucleotides, proteins, carbohydrates, lipids, steroid hormones, cholesterol, amino linkers, Pr spacers, dyes such as fluoroscein and rhodamine, capture reagents such as biotin, and others.
The ribozymes can be encapsulated into liposomes for delivery into cells in vitro or in vivo.
A specific ribozyme of the invention is:
5′AGCTTTATTcugaugag(Pr)
4
cgaaaGGCTTAAG3′  (Seq. ID 1)
which contains 5′AGCTTTATTcugaugag3′ (SED ID NO. 1) and 5′cgaaaGGCTTAAG3′ (SEQ ID NO. 2)
where
c, u, g and a are, respectively, residues of the ribonucleotides cytidylic acid, uridylic acid, guanylic acid and adenylic acid;
C, T, G and A are, respectively, residues of the 2′-deoxyribonucleotides deoxycytidylic acid, deoxythymidylic acid, deoxyguanylic acid and deoxyadenylic acid
FIGURE DESCRIPTION
FIG.
1
. The ribozyme containing SEQ ID NO. 1 and SEQ ID NO. 2 is targeted to cleave an HIV-1 RNA in the U5 region, as illustrated in FIG.
1
.
Ribozymes of this invention can be designed and used for the same purposes and in the same ways that other ribozymes are used, as disclosed in U.S. Pat. No. 5,149,796, cited above, and as reviewed in Rossi, “Ribozymes, genomics and therapeutics,”
Chemistry and Biology,
February 1999, vol. 6, no. 2, R33-R37, which are incorporated herein by reference. Thus, ribozymes of the invention can be used in the field of functional genomics as tools for studying gene functions and identifying potential new therapeutic targets for the treatment of disease. They can also be designed and used to treat RNA-mediated diseases such as HIV-1 infection or other diseases associated with altered expression or mutant forms of a gene or genes, including genetic diseases linked to allelic polymorphisms, and various cancers such as chronic myelogenous leukemia, breast cancer, and bladder cancer.
For example, use of a DNA-RNA chimeric ribozyme to study the pathogenetic role of the bcr-abl gene in Ph
1
+ leukemogenesis, and potentially to treat patients with Ph
1
+ chronic myelogenous leukemia, is disclosed in Snyder et al., “Ribozyme Inhibition of bcr-abl Gene Expression,”
Blood
82:2:600-605 (1992). As another example, a DNA-RNA chimeric ribozyme targeted to cleave mRNA for proliferating cell nuclear antigen (PCNA), and its use to reduce stent-induced stenosis in a porcine model, is disclosed in Frimerman et al.,
Circulation
99;697-703 (1999). As another example, a DNA-RNA chimeric ribozyme targeted to cleave mRNA for leukocyte-type 12 lipoxygenase (12-LO), and its use to study the specific effects of the 12-LO gene pathway in vascular disease, is disclosed in Gu et al.,
Circ. Res.
77:14-20 (1995). Ribozymes of this invention having the same RNA-binding flanking sequences as the ribozymes of the Snyder et al., Frimerman et al., and Gu et al. papers can be synthesized and used for the purposes disclosed in those papers. The disclosures of those papers are incorporated herein.
These examples are not intended to limit the invention. A ribozyme of the invention can be designed and synthesized to target any RNA which contains an NUH target sequence, where N is any ribonucleotide (C, U, G or A) and H is A, C or U, in an accessible binding site. Methods for identifying accessible binding sites in RNA are discussed in the Rossi review article cited above.


REFERENCES:
patent: 5144019 (1992-09-01), Rossi et al.
patent: 5149796 (1992-09-01), Rossi et al.
patent: 5496698 (1996-03-01), Draper et al.
patent: 5500357 (1996-03-01), Taira et al.
patent: 5695938 (1997-12-01), Rossi et al.
patent: 5700923 (1997-12-01), Goodchild et al.
patent: 5965720 (1999-10-01), Gryaznov et al.
patent: 5972704 (1999-10-01), Draper et al.
patent: WO 91/03162 (1991-03-01), None
patent: WO 94/13688 (1994-06-01), None
patent: WO 95/06764 (1995-03-01), None
patent: WO 96/15240 (1996-05-01), None
Eugen Uhlmann et al., Antisense Oligonucleotides: A New Therapeutic Principle, Chemical Reviews, vol. 90, No. 4, Jun. 1990 pp. 543-584.*
Friz Benseler et al., Hammerhead like Molecules Containing Non-Nucleoside Linkers Are Active RNA Catalysts, J. Am. Chem. Soc., vol. 115, No. 18, 1993 pp. 8483-8484.*
B. Dropulić et al., “Functional Characterization of a U5 Ribozyme: Intracellular Suppression of Human Immunodeficiency Virus Type 1 Expression,” Journal of Virology 66(3):1432-1441, (Mar. 1992).
M. Koizumi et al., “Design and Anti-HIV-1 Activity of Hammerhead and Hairpin Ribozymes Containing a Stable Loop,” Nucleosides & Nucleotides 17(1-3):207-218, 1998.
Rossi, “Ribozymes, genomics and therapeutics,”Chemistry and Biology, Feb. 1999, vol. 6, No. 2, R33-R37.
Rossi, “Making Ribozymes Work in Cells,”Current Biology, 1994, vol. 4, No. 5, p

Affiliated with

Also associated with

Rating

Chimeric DNA/RNA ribozymes containing propanediol does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Chimeric DNA/RNA ribozymes containing propanediol, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chimeric DNA/RNA ribozymes containing propanediol will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2885351