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US 12,448,636 B2Granted
High-efficiency reconstitution of RNA molecules
AssigneeThe Salk Institute for Biological Studies
InventorsLukas Christoph Bachmann, Samuel Lawrence Pfaff
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Abstract
Provided herein are synthetic RNA molecules for reconstitution of RNA molecules, including compositions and methods of using these molecules. For example, such molecules can be used to deliver a protein coding sequence over two or more viral vectors (such as AAVs), resulting in reconstitution of the full-length protein in a cell. Such methods can be used to deliver a therapeutic protein, for example to treat a genetic disease or cancer.
Claims27
A system for expressing a target protein, comprising:
- (a) a first synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- an RNA molecule encoding an N-terminal portion of the target protein, comprising a splice junction at a 3′-end of the RNA molecule encoding the N-terminal portion of the target protein;
- a splice donor;
- and
- a first dimerization domain comprising a first RNA hairpin, wherein the first RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a first stem and the region of the non-complementary RNA sequence forms a first loop;
- and
- (b) a second synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- a second dimerization domain comprising a second RNA hairpin, wherein the second RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a second stem and the region of the non-complementary RNA sequence of the second RNA hairpin forms a second loop;
- a branch point sequence;
- a polypyrimidine tract;
- a splice acceptor;
- and
- an RNA molecule encoding a C-terminal portion of the target protein, comprising a splice junction at a 5′-end of the RNA molecule encoding the C-terminal portion of the target protein,
- wherein the first loop hybridizes with the second loop, and
- wherein the first and second loops avoid intramolecular annealing.
12 more elements
A system for expressing a target protein, comprising:
- (a) a first synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- an RNA molecule encoding an N-terminal portion of the target protein, comprising a splice junction at a 3′-end of the RNA molecule encoding the N-terminal portion of the target protein;
- a first splice donor;
- and
- a first dimerization domain comprising a first RNA hairpin, wherein the first RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a first stem and the region of the non-complementary RNA sequence forms a first loop;
- (b) a second synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- a second dimerization domain comprising a second RNA hairpin, wherein the second RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a second stem and the region of the non-complementary RNA sequence of the second RNA hairpin forms a second loop;
- a first branch point sequence;
- a first polypyrimidine tract;
- a first splice acceptor;
- an RNA molecule encoding a middle portion of the target protein, comprising a splice junction at a 5′-end of the RNA molecule encoding the middle portion of the target protein and a splice junction at a 3′-end of the RNA molecule encoding the middle portion of the target protein;
- a second splice donor;
- and
- a third dimerization domain comprising a third RNA hairpin, wherein the third RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a third stem and the region of the non-complementary RNA sequence of the third RNA hairpin forms a third loop;
- and
- (c) a third synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- a fourth dimerization domain comprising a fourth RNA hairpin, wherein the fourth RNA hairpin comprises RNA complementary sequences separated by a region of a non-complementary RNA sequence, wherein base pairing between the complementary sequences forms a fourth stem and the region of the non-complementary RNA sequence of the fourth RNA hairpin forms a fourth loop;
- a second branch point sequence;
- a second polypyrimidine tract;
- a second splice acceptor;
- and
- an RNA molecule encoding a C-terminal portion of the target protein, comprising a splice junction at a 5′-end of the RNA molecule encoding the C-terminal portion of the target protein,
- wherein the first loop hybridizes with the second loop,
- wherein the third loop hybridizes with the fourth loop, and
- wherein the first, second, third, and fourth loops avoid intramolecular annealing.
22 more elements
A system for expressing a target protein, comprising:
- (a) a first synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- a first RNA molecule encoding an N-terminal portion of the target protein, the first RNA molecule comprising, at a 3′ end, a splice junction;
- a splice donor;
- and
- a first dimerization domain comprising an aptamer that binds to an aptamer target;
- and
- (b) a second synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
- a second dimerization domain comprising an aptamer that binds to the aptamer target bound by the first dimerization domain;
- a branch point sequence;
- a polypyrimidine tract;
- a splice acceptor;
- and
- a second RNA molecule encoding a C-terminal portion of the target protein, the second RNA molecule comprising, at a 5′ end, a splice junction,
- wherein the aptamers of the first and second dimerization domains bind the aptamer target in single-stranded regions that avoid intramolecular annealing.
11 more elements
Dependencies are inferred from the claim text.