EFFICIENT RNA SWITCHES AND RELATED EXPRESSION SYSTEMS
The present invention provides novel RNA switches that are based on modified hammerhead ribozymes with improved activities. Also provided are expression vectors and related expression systems for regulating transgene expression in various clinical or industrial applications.
1 . A type III variant of a type I cis-acting hammerhead ribozyme, comprising the 5′ and 3′ ends of its sequence in stem III.
2 . The hammerhead ribozyme variant of claim 1 , wherein the type I cis-acting hammerhead ribozyme is ribozyme N107, N117, or a natural Schistosoma mansoni hammerhead ribozyme.
3 . The hammerhead ribozyme variant of claim 1 , comprising a sequence (5′-3′) accgg ugcgu ccugga uucca cugcu aucca uucgu gaggu gcagg uacau ccagc ugacg agucc caaau aggac gaaac gcgcc ggu (SEQ ID NO:8).
4 . The hammerhead ribozyme variant of claim 1 , further comprising one or more additional modifications of the sequence of the type I cis-acting hammerhead ribozyme, wherein the additional modification is (a) optimization of stem III of the resulting type III ribozyme or (b) modifications of stem I and loop Ito facilitate formation of the “UAC” bulge at stem I and stabilize the tertiary interactions between the “UAC” bulge and loop II.
5 . The hammerhead ribozyme variant of claim 4 , wherein the optimized stem III comprises from about 4 to about 8 pair pairs, and has a calculated annealing energy of about −9 kcal/mol.
6 . The hammerhead ribozyme variant of claim 5 , wherein the two sequences on the opposite sides of the optimized stem III respectively contain residues (5′-3′/3′-5′) acgcgc/ugcgcg or acgccg/ugcggc.
7 . The hammerhead ribozyme variant of claim 5 , comprising a sequence (5′-3′) as shown in SEQ ID NO:9 or SEQ ID NO:10.
8 . The hammerhead ribozyme variant of claim 4 , wherein the two sequences on the opposite sides of the modified stem I, immediately 5′ to the “UAC” bulge, are perfectly matched.
9 . The hammerhead ribozyme variant of claim 8 , wherein the two sequences on the opposite sides of the modified stem I, immediately 5′ to the “UAC” bulge, respectively contain residues (5′-3′/3′-5′) cc/gg, cgc/gcg, cgcg/gcgc, or cgcgc/gcgcg.
10 . The hammerhead ribozyme variant of claim 4 , wherein the altered loop I comprises a sequence (5′-3′) auuc, uucg, gaaa, ucac, or agaggaggc.
11 . The hammerhead ribozyme variant of claim 4 , comprising both an optimized stem III and a modified stem I.
12 . The hammerhead ribozyme variant of claim 11 , comprising a sequence (5′-3′) shown in any one of SEQ ID NOs:11-17.
13 . The hammerhead ribozyme variant of claim 4 , comprising an optimized stem III, a modified stem I and an altered loop I.
14 . The hammerhead ribozyme variant of claim 13 , comprising a sequence (5′-3′) shown in any one of SEQ ID NOs:18-22.
15 . An expression vector, comprising a target gene sequence that is operably fused to an hammerhead ribozyme-coding sequence, wherein the hammerhead ribozyme-coding sequence is inserted into the target gene at its 3′-UTR or 5′-UTR, and wherein the hammerhead ribozyme is a type III variant of a type I cis-acting hammerhead ribozyme that comprises the 5′ and 3′ ends of its sequence in Stem III.
16 . The expression vector of claim 15 , further comprising one or more transcriptional regulatory sequences that regulate transcription of the target gene in a mammalian cell.
17 . The expression vector of claim 15 , which is a DNA vector.
18 . The expression vector of claim 15 , wherein the hammerhead ribozyme comprises one or more additional modifications of the sequence of the type I cis-acting hammerhead ribozyme, wherein the additional modification is (a) optimization of stem III to have a calculated annealing energy of about −9 kcal/mol or (b) modifications of stem I and loop I to facilitate formation of the “UAC” bulge at stem I and stabilize the tertiary interactions between the “UAC” bulge and loop II.
19 . The expression vector of claim 18 , wherein the hammerhead ribozyme comprises a sequence (5′-3′) shown in any one of SEQ ID NOs:9-22.
20 . The expression vector of claim 15 , wherein the target gene encodes erythropoietin (Epo).
21 . An engineered mammalian cell harboring the expression vector of claim 15 .
22 . A method for inducing expression of a target gene in a cell, comprising (a) constructing an expression vector of claim 15 , (b) introducing the expression vector into the cell, and (c) contacting the cell with an RNase H-independent antisense oligonucleotide that is complementary to the hammerhead ribozyme-coding sequence in the expression vector;
thereby inducing expression of the target gene in the cell.
23 . The method of claim 22 , wherein the RNase H-independent antisense oligonucleotide is a morpholino oligonucleotide.
24 . The method of claim 22 , wherein the cell is a mammalian cell.
25 . The method of claim 22 , wherein the expression vector is an adeno-associated virus (AAV) vector.
26 . The method of claim 22 , wherein the hammerhead ribozyme encoded by the vector comprises one or more additional modifications of the sequence of the type I cis-acting hammerhead ribozyme, wherein the additional modification is (a) optimization of stem III to have a calculated annealing energy of about −9 kcal/mol or (b) modifications of stem I and loop I to facilitate formation of the “UAC” bulge at stem I and stabilize the tertiary interactions between the “UAC” bulge and loop II.
27 . The method of claim 26 , wherein the hammerhead ribozyme encoded by the vector comprises a sequence (5′-3′) shown in any one of SEQ ID NOs:9-22.
28 . The method of claim 22 , wherein the target gene encodes erythropoietin (Epo).
29 . The method of claim 22 , wherein the RNase H-independent antisense oligonucleotide is a modified morpholino.
30 . The method of claim 29 , wherein the modified morpholino is an octa-guanidine dendrimer-coupled morpholino.
31 . The method of claim 22 , wherein the RNase H-independent antisense oligonucleotide comprises an oligonucleotide sequence as shown in any one of SEQ ID NOs:38-46.
32 . The method of claim 22 , wherein the cell is present in a subject in need of the polypeptide encoded by the target gene.
33 . The method of claim 22 , wherein the cell is obtained from the subject prior to introduction of the expression vector into the cell.
34 . The method of claim 33 , further comprising, subsequent to introducing the expression vector into the cell, reintroducing the cell into the subject.
35 . The method of claim 33 , wherein the RNase H-independent antisense oligonucleotide is administered to the subject subsequent to reintroducing the cell into the subject.