Cell ablation using trans-splicing ribozymes
AssigneesThe General Hospital Corporation, President and Fellows of Harvard College
InventorsJames Haseloff, Andrea Brand, Norbert Perrimon, Howard M. Goodman
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Abstract
The design of new ribozymes capable of self-catalyzed trans-splicing which are based upon the catalytic core of a Group I intron are described. Using this design, it is possible to construct ribozymes capable of efficiently splicing a new 3' exon sequence into any chosen target RNA sequence in a highly precise manner. A method of cell ablation is also described that provides a toxic product to a host cell in vivo in a targetted, regulated manner utilizing novel trans-splicing ribozymes of the invention. Inactive pro-ribozyme forms are also described.
Claims11
A method for in vitro trans-splicing, such method comprising the steps of:
- (1) providing in a trans-splicing reaction mixture, a polynucleotide molecule encoding a trans-splicing Group I ribozyme, the sequence of said ribozyme being a fusion RNA, the sequence of said fusion RNA comprising:
- (a) a first RNA sequence, which hybridizes to a target RNA that encodes a transcription activator protein, and
- (b) a second RNA sequence, which is to be trans-spliced into said target RNA;
- wherein said polynucleotide molecule is operably linked to a transcription regulatory element which is specifically recognized by said transcription activator protein, such that association of said transcription activator protein with said transcription regulatory element results in activation of transcription of said polynucleotide molecule and production of said trans-splicing ribozyme;
- (2) providing in said trans-splicing reaction mixture, said target RNA;
- and
- (3) allowing said polynucleotide molecule to be transcribed and trans-splicing ribozyme to catalyze trans-splicing of said second RNA sequence into said target RNA.
A method for inactivating the activity of a target RNA in vitro, said method comprising:
- (1) providing to a trans-splicing reaction mixture, a polynucleotide molecule encoding a trans-splicing Group I ribozyme, the sequence of said ribozyme being a fusion RNA the sequence of said fusion RNA comprising:
- (a) a first RNA sequence, which hybridizes to a target RNA that encodes a transcription activator protein, and
- (b) a second RNA sequence, which is to be trans-spliced into said target RNA;
- wherein said polynucleotide molecule is operably linked to a transcription regulatory element which is specifically recognized by said transcription activator protein, such that association of said transcription activator protein with said transcription regulatory element results in activation of transcription of said polynucleotide molecule and production of said trans-splicing ribozyme;
- and wherein said ribozyme possesses catalytic activity against said target RNA, said catalytic activity resulting in the inactivation of the functioning of said target RNA;
- (2) providing said target RNA to said reaction mixture;
- and
- (3) providing conditions that allow said polynucleotide molecule to be transcribed and said trans-splicing ribozyme to catalyze trans-splicing of said second RNA sequence into said target RNA.
Dependencies are inferred from the claim text.