Use of RNA trans-splicing for generation of interfering RNA molecules
OwnerMariano Garcia-Blancoinventor
Life of this patent
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Abstract
Methods and compositions for generating novel nucleic acid molecules through trans-splicing that function to reduce the level of expression of a target RNA. The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and mediate a trans-splicing reaction resulting in the generation of primary microRNAs (pri-miRNAs), which are processed in the cell to molecules, referred to as mature miRNA duplex or short interfering RNAs (siRNAs), capable of producing gene silencing by RNA interference (RNAi).
Claims32
A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:
- a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell;
- b) a splice region;
- c) a spacer region that separates the splice region from the target binding domain;
- and
- d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence is designed to form a stem-loop structure;
- wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
A method of producing a chimeric RNA molecule in a cell, wherein said RNA is capable of gene silencing by RNA interference, comprising:
- contacting a target pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
- a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell;
- b) a splice region;
- c) a spacer region that separates the splice region from the target binding domain;
- and
- d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence is designed to form a stem-loop structure;
- under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.
A nucleic acid molecule comprising:
- a) one or more target binding domains that target binding of the nucleic acid molecule to a target pre-mRNA expressed within the cell;
- b) a splice region;
- c) a spacer region that separates the splice region from the target binding domain;
- and
- d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence is designed to form a stem loop structure;
- wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
A nucleic acid molecule comprising:
- a) a splice region;
- b) a spacer region that separates the splice region from the target binding domain;
- and
- c) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence is designed to form a stem loop structure;
- wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
Dependencies are inferred from the claim text.