Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-23
2003-08-12
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091310, C435S029000, C435S320100, C536S024500
Reexamination Certificate
active
06605429
ABSTRACT:
A. BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to methods for using hairpin ribozymes to inactivate target RNA molecules. The present invention specifically provides methods for identifying, isolating, and characterizing unknown genes and gene products responsible for detectable phenotypic traits or for characterizing unknown phenotypic effects of known genes, and methods of inactivating target RNAs. Compared to other known ribozymes, the hairpin ribozyme has been discovered to be uniquely effective as a randomized antisense tool.
2. Related Art
A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes. See Castanotto et al. (1994)
Advances in Pharmacology
25:289-317 for a general review of the properties of different ribozymes.
The general features of hairpin ribozymes are described e.g., in Hampel et al. (1990)
Nucl. Acids Res.
18:299-304; Hampel et al. (1990) European Patent Publication No. 0 360 257; U.S. Pat. No. 5,254,678, issued Oct. 19, 1993; Wong-Staal et al., WO 94/26877; Ojwang et al. (1993)
Proc. Natl. Acad. Sci. USA
90:6340-6344; Yamada et al. (1994)
Human Gene Therapy
1:39-45; Leavitt et al. (1995)
Proc. Natl. Acad. Sci. USA
92:699-703; Leavitt et al. (1994)
Human Gene Therapy
5:1151-1120; and Yamada et al. (1994)
Virology
205:121-126; see FIG.
1
. Hairpin ribozymes typically cleave one of two target sequences, NNNNN*GUCNNNNNNNN or NNNNN*GUANNNNNNNN (* denotes the cleavage site, and N can be any nucleotide). See, De Young et al. (1995)
Biochemistry
34:15785-15791. The products of the cleavage reaction are a 5′ fragment terminating in a 2′,3′ cyclic phosphate and a 3′ fragment bearing a newly formed 5′-OH. The reaction is reversible; ribozymes also catalyze the formation of phosphodiester bonds. See generally, Buzayan et al. (1986)
Nature
323:349-352; Gerlach et al. (1986)
Virology
151:172-185; Hampel et al. (1989)
Biochemistry
28:4929-4933; Gerlach et al. (1989)
Gene
82:43-52; Feldstein et al. (1989)
Gene
82:53-61; and Hampel et al. Australian Patent No. AU-B-41594/89.
Ribozymes can be used to engineer RNA molecules prior to reverse transcription and cloning, in a manner similar to the DNA endonuclease “restriction” enzymes. The production of specific ribozymes which target particular sequences is taught in the art (see, e.g., Yu et al. (1993)
Proc. Natl. Acad. Sci. USA
90:6340-6344 and Dropulic et al. (1992)
J. Virol.
66(3):1432-1441; Wong-Staal et al., WO 94/26877). Ribozymes which cleave or ligate a particular RNA target sequence can be expressed in cells to prevent or promote expression and translation of RNA molecules comprising the target sequence. For instance, expression of hairpin ribozymes which specifically cleave human immunodeficiency (HIV) RNAs prevent replication of the virus in cells. See, Yu et al. (1993)
Proc. Natl. Acad. Sci. USA
90:6340-6344; Yamada et al. (1994)
Virology
205:121-126; Yamada et al. (1994)
Gene Therapy
1:38-45; Yu et al. (1995)
Virology
206:381-386; Yu et al. (1995)
Proc. Nat. Acad. Sci.
92:699-703; and Wong-Staal et al. WO 94/26877 (PCT/US94/05700). The trans-splicing activity of ribozymes can be used to repair defective mRNA transcripts within cells and restore gene expression. Sullenger and Cech (1994)
Nature
371:619-622. Quasi-random ribozyme expression vectors were reportedly used to clone target specific ribozymes. Macjak and Draper (1993)
J. Cell. Biochem
. Supplement 17E, S206:202. A hammerhead ribozyme library comprising a randomized recognition sequence was used for in vitro selection of ribozymes which actively cleave a specific target RNA (Lieber and Strauss (1995)
Mol. Cell. Biol.
15:540-551; patent publication 96/01314); ribozymes selected by this method were then expressed in tissue culture cells (id.) and in transgenic mice (Lieber and Kay (1996)
J. Virol.
70:3153-3158). In addition, hammerhead ribozyme libraries comprising a randomized catalytic region have been used to select ribozymes that efficiently cleave a specific target RNA. Patent publication WO 92/01806. A library of the ribozyme form of the group I intron of
Tetrahymena thermophila
having a partially randomized recognition sequence was used for in vitro selection of ribozymes which actively cleave a specific target RNA. Campbell and Cech (1995)
RNA
1:598-609.
However, even when both the sequence of the cleavage sites of a specific target RNA and the recognition sequences of ribozymes that cleave that specific RNA are known, targeted cleavage of RNA in vivo has been difficult to achieve (See, e.g., Ojwang et al. (1992)
Proc. Natl. Acad. Sci. USA
89:10802-10806), in part for the following reasons: (a) The target site may be hidden within the folds of secondary structure in the substrate RNA, or by interaction with RNA binding molecules. (b) The substrate RNA and the ribozyme may not be present in the same cellular compartment. (c) The ribozyme may be inhibited or inactivated in vivo, either because it is degraded, or because it assumes a secondary structure in vivo that is incompatible with catalytic activity, or because of interactions with cellular molecules. The observed biological effects in some instances can be attributed to simple binding of the ribozyme, as opposed to binding and cleavage. (d) The ribozyme is not produced in sufficient quantities.
The present invention addresses these and other problems.
B. SUMMARY OF THE INVENTION
In the present invention, a hairpin ribozyme gene library having a randomized target recognition sequence, packaged in a vector which is suitable for high level transduction and expression in a wide variety of cells, is used to identify, isolate, and characterize unknown genes and gene products responsible for detectable phenotypic traits, and to characterize unknown phenotypic effects of known genes. In a preferred embodiment, the vector is an adeno-associated virus vector (AAV). The ribozyme gene is preferably operably linked to a transcriptional promoter that allows for optimal inhibition of target RNA expression in vivo, such as a pol III promoter. The result is an unexpectedly high level of expression of the ribozyme gene products. This efficient expression in turn makes possible the in vivo or in vitro selection of ribozyme genes that are active in vivo, even when the target site is not known.
In one embodiment, the invention comprises a method of correlating expression of a nucleic acid that encodes a hairpin ribozyme with the appearance or loss of a detectable phenotype which results from the inhibition or expression of a cellular gene not previously known to result in said phenotype, which involves generating transduced cell clones which express at least one reporter gene or otherwise selectable marker and one or more ribozyme genes from a library of hairpin ribozyme-encoding nucleic acids having randomized target recognition sequences, detecting a phenotypic difference between a transduced cell that expresses said hairpin ribozyme, and a cell of the parental cell line that does not express said hairpin ribozyme, deconvoluting if necessary, isolating and sequencing the ribozyme present in transduced cells that express a selected phenotype. The hairpin ribozyme-encoding nucleic acid is operably linked to an inducible or constitutive promoter. In this and other embodiments, the cells can be eukaryotic, particularly mammalian cells, and the cellular gene can be from the genome of the transformed cell.
In another embodiment, the invention comprises a method of determining unknown phenotypic effects of a coding nucleic acid of known sequence, comprising: simultaneously expressing within a same cell a coding nucleic acid of known sequence and also a hairpin ribozyme that recognizes at least one GUC site present in said coding nucleic acid of known sequence; and then detecting phenotypic differences between cells that simul
Barber Jack R.
Tritz Richard
Welch Peter
Yei Soonpin
Horlick Kenneth R.
Immusol Incorporated
Townsend and Townsend / and Crew LLP
Wilder Cynthia
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