Use of RNA trans-splicing for generation of interfering RNA molecules
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).
1 . 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.
2 . The cell of claim 1 wherein the splice region comprises a 3′ splice region.
3 . The cell of claim 1 wherein the splice region comprises a 5′ splice region.
4 . The cell of claim 2 wherein the 3′ splice region comprises at least one of a branch point and a 3′ splice acceptor site.
5 . The cell of claim 2 wherein the 3′ splice region further comprises a pyrimidine tract.
6 . The cell of claim 2 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′ splice region
7 . The cell of claim 3 wherein said nucleic acid molecule further comprises a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′ splice site.
8 . The cell of claim 3 wherein the nucleic acid molecule further comprises a 5′ donor site.
9 . 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.
10 . The method of claim 9 wherein the splice region comprises a 3′ splice region.
11 . The method of claim 9 wherein the splice region comprises a 5′ splice region.
12 . The method of claim 10 wherein the 3′ splice region comprises at least one of a branch point and a 3′ splice acceptor site.
13 . The method of claim 10 wherein the 3′ splice region further comprises a pyrimidine tract.
14 . The method of claim 10 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′ splice region
15 . The method of claim 11 wherein said nucleic acid molecule further comprises a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′ splice site.
16 . The method of claim 11 wherein the nucleic acid molecule further comprises a 5′ donor site.
17 . 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.
18 . The nucleic acid of claim 17 wherein the splice region comprises a 3′ splice region.
19 . The nucleic acid of claim 17 wherein the splice region comprises a 5′ splice region.
20 . The nucleic acid of claim 18 wherein the 3′ splice region comprises at least one of a branch point and a 3′ splice acceptor site.
21 . The nucleic acid of claim 18 wherein the 3′ splice region further comprises a pyrimidine tract.
22 . The nucleic acid of claim 18 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′ splice region
23 . The nucleic acid of claim 19 wherein said nucleic acid molecule further comprises a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′ splice site.
24 . The nucleic acid of claim 19 wherein the nucleic acid molecule further comprises a 5′ donor site.
25 . 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.
26 . The nucleic acid of claim 25 wherein the splice region comprises a 3′ splice region.
27 . The nucleic acid of claim 25 wherein the splice region comprises a 5′ splice region.
28 . The nucleic acid of claim 26 wherein the 3′ splice region comprises at least one of a branch point and a 3′ splice acceptor site.
29 . The nucleic acid of claim 26 wherein the 3′ splice region further comprises a pyrimidine tract.
30 . The nucleic acid of claim 26 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′ splice region
31 . The nucleic acid of claim 27 wherein said nucleic acid molecule further comprises a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′ splice site.
32 . The nucleic acid of claim 27 wherein the nucleic acid molecule further comprises a 5′ donor site.