High-efficiency reconstitution of RNA molecules
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.
1. 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.
2. 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.
3. The system of claim 2 , further comprising:
(d) a fourth synthetic nucleic acid molecule comprising, from 5′ to 3′, a sequence encoding:
a fifth dimerization domain comprising a fifth RNA hairpin, wherein the fifth 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 fifth stem and the region of the non-complementary RNA sequence of the fifth RNA hairpin forms a fifth loop;
a third branch point sequence;
a third polypyrimidine tract;
a third splice acceptor;
an RNA molecule encoding a second middle portion of the target protein, comprising a splice junction at a 5′-end of the RNA molecule encoding the second middle portion of the target protein and a splice junction at a 3′-end of the RNA molecule encoding the second middle portion of the target protein;
a third splice donor; and
a sixth dimerization domain comprising a sixth RNA hairpin, wherein the sixth 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 sixth stem and the region of the non-complementary RNA sequence of the sixth RNA hairpin forms a sixth loop,
wherein the sixth loop hybridizes with the fourth loop,
wherein the fourth loop does not hybridize to the third loop,
wherein the fifth loop hybridizes with the third loop, and
wherein the fifth and sixth loops avoid intramolecular annealing.
4. The system of claim 1 , wherein the first and second loops comprise hypodiverse sequences.
5. The system of claim 1 , wherein the first, second, or both dimerization domains do not comprise a cryptic splice acceptor.
6. The system of claim 1 , wherein the target protein is a protein associated with disease, or a therapeutic protein.
7. The system of claim 6 , wherein the disease is a monogenic disease, a recessive genetic disease, a disease caused by a mutation in a gene greater than 4500 nt, or a combination thereof.
8. The system of claim 6 , wherein the therapeutic protein is a toxin.
9. The system of claim 6 , wherein the disease is a retinal disorder, a blood cell disorder, a primary immunodeficiency disease or disorder, a monogenetic disorder, a mucopolysaccaridosis disorder, a cancer, or a neurological disorder.
10. The system of claim 1 , wherein the target protein is encoded by a coding sequence of at least 4500 nucleotides.
11. The system of claim 6 , wherein the disease is selected from: Duchenne muscular dystrophy; Becker muscular dystrophy; Dysferlinopathy; Cystic fibrosis; Usher's Syndrome 1B; Stargardt disease 1; Von Willebrand disease; Marfan Syndrome; Von Recklinghausen disease; sickle cell anemia; hemophilia; hemophilia A; hemophilia B; Alpha-Thalassemia; Beta-Thalassemia; Delta-Thalassemia; von Willebrand Disease; pernicious anemia; Fanconi anemia; Thrombocytopenia purpura; thrombophilia; T-B+SCID; T-B-SCID; WHIM syndrome; IL-7 receptor severe combined immune deficiency (SCID); Adenosine deaminase deficiency SCID; Purine nucleoside phosphorylase deficiency; Wiskott-Aldrich syndrome; Chronic granulomatous disease; Leukocyte adhesion deficiency; HIV disease; Glycogen storage disease type IA; Retinal Dystrophy; X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection, and neoplasia (XMEN); Metachromatic leukodystrophy (MLD); Adrenoleukodystrophy (ALD); Hunter syndrome; Hurler syndrome; Scheie syndrome; Sanfilippo syndrome A, B, C, and D; Morquio syndrome A; Morquio syndrome B; Maroteaux-Lamy syndrome; Sly syndrome; Natowicz syndrome; Alpha mannosidosis; Nieman Pick disease types A, B, and C; Polycystic kidney disease; Tay Sachs Disease; Gaucher disease; Huntington's disease; Neurofibromatosis types 1 and 2; Familial hypercholesterolemia; Chronic myeloid leukemia; Acute myeloid leukemia; Osteosarcoma; Colorectal cancer; Gastric cancer; Melanoma; Prostate cancer; Cervical cancer; Glioblastoma; Alzheimer's disease; Multiple sclerosis; X-linked adrenoleukodystrophy; AACD deficiency; Batten disease; Canavan disease; Giant axonal neuropathy; Leber's hereditary optic neuropathy; MPS IIIA; Parkinson's disease; Pompe disease; and Spinal muscular atrophy type 1.
12. The system of claim 1 , wherein:
the first synthetic nucleic acid molecule further comprises a downstream intronic splice enhancer (DISE) 3′ to the splice donor and 5′ to the first dimerization domain, an intronic splice enhancer (ISE) 3′ to the splice donor and 5′ to the first dimerization domain, or both a DISE and ISE;
the second synthetic nucleic acid molecule further comprises an ISE 3′ to the second dimerization domain and 5′ to the branch point sequence; or
any combination thereof.
13. The system of claim 1 , wherein:
the synthetic first and second nucleic acid molecules, when introduced into a cell, recombine such that the RNA molecule encoding the N-terminal portion of the target protein and the RNA molecule encoding the C-terminal portion of the target protein combine into a full-length coding sequence of the target protein.
14. The system of claim 1 , wherein the synthetic first nucleic acid molecule is part of a first viral vector, and
wherein the second nucleic acid molecule is part of a second viral vector.
15. The system of claim 14 , wherein the first and second viral vectors are AAVs.
16. The system of claim 1 , wherein:
the first dimerization domain and the second dimerization domain are each no more than 1000 nt, and
the system has a recombination efficiency of at least 20%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90%.
17. The system of claim 1 , wherein the target protein is selected from: Dystrophin; Dysferlin; Myosin VIIA; Fibrillin 1; Neurofibromatosis-1; ß-globin chain of hemoglobin; Clotting factor I; Clotting factor II; Clotting factor III; Clotting factor IV; Clotting factor V; Clotting factor VI; Clotting factor VII; Clotting factor VIII; Clotting factor IX; Clotting factor X; Clotting factor XI; Clotting factor XII; Clotting factor XIII; HBA1; HBA2; HBB; HBD; von Willebrand factor; MTHFR; FANCA; FANCC; FANCD 2 ; FANCG; FANCJ; ADAMTS13; Factor V Leiden Prothrombin; IL-2RG; JAK3; IL-2 receptor gamma chain; IL-4 receptor gamma chain; IL-7 receptor gamma chain; IL-9 receptor gamma chain; IL-15receptor gamma chain; IL-21 receptor gamma chain; RAG1; RAG2; CXCR4; IL7 receptor; ADA; PNP; WAS; CYBA; CYBB; NCF1; NCF2; NCF4; Beta-2 integrin; C-C chemokine receptor type 5 (CCR5); MSRB1; CSCR4; P17; PSIP1; CCR5; DMD; G6Pase; CEP290; ABCA4; MAGT1; arylsulfatase A (ARSA); ABCD1; IDS; IDUA; SGSH; NAGLU; HGSNAT; GNS; GALNS; GLB1; ARSB; GUSB; HYAL1; MAN2B1; SMPD1; NPC1; NPC2; CFTR; PKD-1; PDK-2; PDK-3; HEXA; GBA; HTT; NF-1; NF2; APOB; LDLR; LDLRAP1; PCSK9; BCR-ABL; ASXL1; RUNX2; EPHA1; PD-1; Androgen receptor; E6; E7; CD; NGF; MBP; WASP; AADC; CLN2; ASPA; GAN; MT-ND4; SUMF1; GAD; NTRN; TH; CH1; GDNF; GAA; SMN; and thymidine kinase.
18. A method of expressing a protein in a cell, comprising:
introducing the system of claim 1 into a cell; and
expressing system in the cell.
19. The method of claim 18 , wherein the cell is in a subject, and introducing comprises administering a therapeutically effective amount of the system to the subject.
20. The method of claim 18 , wherein the method treats a genetic disease caused by a mutation in a gene encoding the target protein in the subject, wherein the method results in expression of functional target protein in the subject.
21. The method of claim 20 , wherein:
the genetic disease is Duchenne muscular dystrophy and the target protein is dystrophin;
the genetic disease is hemophilia A and the target protein is Coagulation Factor VIII;
the genetic disease is Stargardt disease and the target protein is ABCA4;
the genetic disease is Retinal Dystrophy and the target protein is CEP290;
the genetic disease is Dysferlinopathy and the target protein is Dysferlin; or the genetic disease is Usher syndrome and the target protein is MYO7A.
22. The system of claim 1 , wherein the first synthetic nucleic acid molecule further comprises a first promoter 5′ to the sequence encoding the RNA molecule encoding the N-terminal portion of the target protein; and
wherein the second synthetic nucleic acid molecule further comprises a second promoter 5′ to the sequence encoding the second dimerization domain.
23. The system of claim 2 , wherein the first nucleic synthetic molecule further comprises a first promoter 5′ to the sequence encoding the RNA molecule encoding the N-terminal portion of the target protein molecule,
wherein the second synthetic nucleic acid molecule further comprises a second promoter 5′ to the sequence encoding the second dimerization domain, and
wherein the third synthetic nucleic acid molecule further comprises a third promoter 5′ to the sequence encoding the fourth dimerization domain.
24. The system of claim 3 , wherein the first nucleic synthetic molecule further comprises a first promoter 5′ to the sequence encoding the RNA molecule encoding the N-terminal portion of the target protein molecule,
wherein the second synthetic nucleic acid molecule further comprises a second promoter 5′ to the sequence encoding the second dimerization domain,
wherein the third synthetic nucleic acid molecule further comprises a third promoter 5′ to the sequence encoding the fourth dimerization domain, and
wherein the fourth synthetic nucleic acid molecule further comprises a fourth promoter 5′ to the sequence encoding the fifth dimerization domain.
25. The method of claim 18 , wherein the first synthetic nucleic acid molecule in a) further comprises a first promoter 5′ to the sequence encoding the RNA molecule encoding the N-terminal portion of the target protein, and wherein the second synthetic nucleic acid molecule in b) further comprises a second promoter 5′ to the sequence encoding the second dimerization domain.
26. The system of claim 1 , wherein the first RNA hairpin is a plurality of the first RNA hairpins, and
wherein the second RNA hairpin is a plurality of the second RNA hairpins.
27. 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.