Claims for Patent: 9,297,013

Summary for Patent: 9,297,013

Title:	pRNA multivalent junction domain for use in stable multivalent RNA nanoparticles
Abstract:	Trifurcate RNA junction domains derived from phi29 pRNA are described that assemble with high affinity and that are stable in vitro and in vivo. Further expansion of trifurcated RNA domains to multiple way junction scaffolds via creative designs enable an array of toolkit to construct nanoparticle architectures with diverse shapes and angles. The scaffolds can be used to form RNA nanoparticles having a wide variety of uses, including promotion of RNA crystallization, creation of RNA aptamer with high affinity to mimic antibody, delivery of therapeutic and/or diagnostic agents such as biologically active RNA-based moieties, including siRNA, ribozymes, aptamers, and others.
Inventor(s):	Guo; Peixuan (Lexington, KY)
Assignee:	University of Cincinnati (Cincinnati, OH)
Application Number:	13/992,714
Patent Claims:	1. A multivalent RNA junction scaffold, comprising: a multiple RNA oligomer-formed polymer complex, wherein said polymer complex is configured to promote an array of thermodynamically and stoichiometrically stable RNA nanoparticles, wherein said multivalent RNA junction scaffold comprises a branched junction domain, said branched junction domain comprises groups of RNA polynucleotide helical regions, each said helical region has defined number of RNA nucleotide base pairs that form Watson-Crick bonds, wherein said groups of RNA polynucleotide helical regions comprise at least one unpaired RNA nucleotide base that is situated between at least two helical regions, and 3 unpaired RNA nucleotides situated at a 3' end of at least one helical region. 2. The multivalent RNA junction scaffold according to claim 1, wherein said RNA oligomer further comprises sequences for at least one biologically active moiety. 3. The multivalent RNA junction scaffold according to claim 1, wherein said RNA oligomer-formed polymer complex further comprises RNA branches with selective RNA or DNA sticky ends or palindrome sequences therein. 4. The multivalent RNA junction scaffold according to claim 1, wherein said oligomer-formed polymer complex is a trimer, a tetramer, a pentamer, a hexamer, a heptamer, or an octamer. 5. The multivalent RNA nanoparticle according to claim 2, wherein said bioactive moiety is heterogeneous. 6. The multivalent RNA nanoparticle according to claim 2, wherein at least one of said bioactive moiety is selected from the followings: SEQ. ID. NO: 1: folate-DNA strand (5' folate-CTC CCG GCC GCC ATG GCC GCG GGA TT 3'), SEQ. ID. NO: 2: Survivin siRNA antisense strand (5' UGA CAG AUA AGG AAC CUG CUU 3'), SEQ. ID. NO: 3: scramble control of Survivin siRNA antisense strand (5' AUA GUG GGA CCA AUC AAG CUU 3'), SEQ. ID. NO: 4: MG binding aptamer -1 (5' GGA UCC CGA CUG GCG AGA GCC AGG UAA CGA AUG GAU CC 3'), SEQ. ID. NO: 5: MG binding aptamer -2 strand 1 (5' AUG GUA ACG AAU GA 3'), SEQ. ID. NO: 6: MG binding aptamer -2 strand 2 (5' CAA UCC GAC AU 3'), SEQ. ID. NO: 7: STV binding aptamer (5' CGA CCA GAA UCA UGC AAG UGC GUA AGA UAG UCG CGG GUC G 3'), SEQ. ID. NO: 8: working HBV ribozyme (5' CAA AUU CUU UAC UGA UGA GUC CGU GAG GAC GAA ACG GGU C 3'), SEQ. ID. NO: 9: disabled HBV ribozyme (5' CAA AUU CUU UAC UAA UGA GUC CGU GAG GAC GAA ACG GGU C 3'), SEQ. ID. NO: 10: firefly Luciferase siRNA 1 antisense strand (5' GUU GGC ACC AGC AGC GCA C 3'), SEQ. ID. NO: 11: firefly Luciferase siRNA 2 antisense strand (5' UCG AAG UAC UCA GCG UAA G 3'), SEQ. ID. NO: 12: firefly Luciferase siRNA 3 antisense strand (5' GCC CAU AUC GUU UCA UAG C 3'), SEQ. ID. NO: 13: firefly Luciferase siRNA 4 antisense strand (5' GUA GAU GAG AUG UGA CGA A 3'), SEQ. ID. NO: 14: scramble control of firefly Luciferase siRNA antisense strand (5'GUC GGU UUC GUG AAG GAG A 3'). 7. The multivalent RNA junction scaffold according to claim 2, wherein said bioactive moiety is drugs, markers, fluorescent dyes, chemicals, siRNAs, ribozymes, riboswitches, aptamer or other functionalities. 8. The multivalent RNA junction scaffold according to claim 2, wherein said multifunctional bioactive moiety is therapeutics for treatment of cancers, viral infections, genetic diseases and other ailing. 9. The multivalent RNA junction scaffold according to claim 2, wherein said multifunctional bioactive moiety is for detection or diagnosis of cancers, viral infections, genetic diseases and other ailing. 10. The multivalent RNA junction scaffold according to claim 2, wherein at least one of said multifunctional bioactive moiety is used as an RNA antibody for detection or diagnosis of cancers, viral infections, genetic diseases and other ailing. 11. The multivalent RNA junction scaffold according to claim 1, wherein said multiple RNA junction scaffold is a trifurcate junction domain comprising: a. an a.sub.3WJ RNA polynucleotide; b. a b.sub.3WJ RNA polynucleotide; and c. a c.sub.3WJ RNA polynucleotide. 12. The multivalent RNA junction scaffold according to claim 1, wherein said multiple RNA junction scaffold is a X-motif comprising: d. an a.sub.X-RNA polynucleotide; e. a b.sub.X-RNA polynucleotide; f. a c.sub.X-RNA polynucleotide; and g. a d.sub.X-RNA polynucleotide. 13. The multivalent RNA junction scaffold according to claim 11, wherein said trifurcate junction domain comprising at least: an a.sub.3WJ RNA polynucleotide comprises the SEQ. ID. NO: 15 (5'-UUG CCA UGU GUA UGU GGG-3'); a b.sub.3WJ RNA polynucleotide comprises the SEQ. ID. NO: 16 (5'-CCC ACA UAC UUU GUU GAU CC-3'); a c.sub.3WJ RNA polynucleotide comprises the SEQ. ID. NO: 17 (5'-GGA UCA AUC AUG GCA A-3'). 14. The multivalent RNA junction scaffold according to claim 12, wherein said X-motif comprising: a. an a.sub.X-RNA polynucleotide comprises the SEQ. ID. NO:18 (5'-UUG CCA UGU GUA UGU GGG UUC CAG CAC-3') b. a b.sub.X-RNA polynucleotide comprising the SEQ. ID. NO: 19 (5'-GUG CUG GAA CUG ACU GC-3') c. a c.sub.X-RNA polynucleotide comprising the SEQ. ID. NO: 20 (5'-GCA GUC AGC CCA CAU ACU UUG UUG AUC C-3'); and d. a d.sub.X-RNA polynucleotide comprising SEQ. ID. NO: 21 (5'-GGA UCA AUC AUG GCA A-3'). 15. The multivalent RNA junction scaffold according to claim 13, wherein said trifurcate RNA junction domain comprising: a. a first helical region RNA polynucleotide comprising 8 RNA nucleotide base pairs, said RNA polynucleotide forms canonical Watson-Crick bonds; b. a second helical region RNA polynucleotide comprising (i) 9 RNA nucleotide base pairs, (ii) at least one unpaired RNA nucleotide base that is situated between the first helical region and the second helical region, and (iii) 3 unpaired RNA nucleotides situated at a 3' end of said second helical region RNA polynucleotide; and c. a third helical region RNA polynucleotide comprising 8 RNA nucleotide base pairs which form canonical Watson-Crick bonds. 16. The multivalent RNA junction scaffold according to claim 13, wherein said trifurcate RNA junction domain comprising: a. a first helical region RNA polynucleotide comprising 6 RNA nucleotide base pairs, said RNA polynucleotide forms canonical Watson-Crick bonds; b. a second helical region RNA polynucleotide comprising (i) 9 RNA nucleotide base pairs, (ii) at least one unpaired RNA nucleotide base that is situated between the first helical region and the second helical region, and (iii) 3 unpaired RNA nucleotides situated at a 3' end of said second helical region RNA polynucleotide; and c. a third helical region RNA polynucleotide comprising 6 RNA nucleotide base pairs which form canonical Watson-Crick bonds. 17. A multipartite RNA nanoparticle, comprising an array of thermodynamically and stoichiometrically stable, multifunctional bioactive moiety crystalline, wherein said crystalline is self-assembled by a multivalent RNA junction scaffold, wherein said multivalent RNA junction scaffold is a multiple RNA oligomer formed polymer complex, and comprises a branched junction domain, said branched junction domain comprises groups of RNA polynucleotide helical regions, each said helical region has defined number of RNA nucleotide base pairs that form Watson-Crick bonds, wherein said groups of RNA polynucleotide helical regions comprise at least one unpaired RNA nucleotide base that is situated between at least two helical regions, and 3 unpaired RNA nucleotides situated at a 3' end of at least one helical region. 18. The multipartite RNA nanoparticle according to claim 17, wherein said RNA crystalline structure is formed by bottom-up self-assembly. 19. The multipartite RNA nanoparticle according to claim 17, wherein said RNA junction scaffold core is a polygon formed by said multiple RNA oligomer. 20. The multipartite RNA nanoparticle according to claim 17, wherein said RNA crystalline structure is further arranged by alternating the orientation of at least one said RNA polynucleotide's neighboring molecule. 21. The multipartite RNA nanoparticle according to claim 17, wherein said RNA crystalline structure is further arranged to form 1D and 2D sheets by controlling said RNA polynucleotides' numbers in a given unit of said scaffold. 22. The multipartite RNA nanoparticle according to claim 17, wherein said RNA oligomer-formed polymer complex further comprises RNA branches with selective RNA or DNA sticky ends or palindrome sequences therein. 23. The multipartite RNA nanoparticle according to claim 17, wherein said RNA oligomers-formed polymer complex is a trimer, a tetramer, a pentamer, a hexamer, a heptamer or an octamer. 24. A method of making a multipartite RNA nanoparticle, comprising admixing in substantially equimolar amounts of RNA polynucleotides, wherein said RNA polynucleotides contain a collection of RNA junction domains to form a multivalent RNA junction scaffold comprising a branched junction domain, said branched junction domain comprises groups of RNA polynucleotide helical regions, each said helical region has defined number of RNA nucleotide base pairs that form Watson-Crick bonds, wherein said groups of RNA polynucleotide helical regions comprise at least one unpaired RNA nucleotide base that is situated between at least two taro helical regions, and 3 unpaired RNA nucleotides situated at a 3' end of at least one helical region, wherein said multipartite RNA nanoparticle promotes the assembly of an array of bioactive moiety into crystalline structure. 25. A method of delivering a biological active moiety to a cell, comprising contacting the cell with a multipartite RNA nanoparticle, wherein said multipartite RNA nanoparticle is assembled by an RNA junction scaffold comprising a branched junction domain, said branched junction domain comprises groups of RNA polynucleotide helical regions, each said helical region has defined number of RNA nucleotide base pairs that form Watson-Crick bonds, whey said groups of RNA polynucleotide helical regions comprise at least one unpaired RNA nucleotide base that is situated between at least two helical regions, and 3 unpaired RNA nucleotides situated at a 3' end of at least one helical region. 26. A method of delivering a therapeutic agent or a detectable labeling agent, or both, comprising administering to the subject a multipartite RNA nanoparticle, wherein said multipartite RNA nanoparticle is assembled by an RNA junction scaffold comprising a branched junction domain, said branched junction domain comprises groups of RNA polynucleotide helical regions, each said helical region has defined number of RNA nucleotide base pairs that form Watson-Crick bonds, wherein said groups of RNA polynucleotide helical regions comprise at least one unpaired RNA nucleotide base that is situated between at least two helical regions, and 3 unpaired RNA nucleotides situated at a 3' end of at least one helical region. 27. A method of using branched trifurcate or four way X-motif junction domain as a scaffold to produce stable multivalent RNA aptamers, wherein said stable multivalent RNA aptamers recognize at least one given substrate, comprising: a. attaching a collection of random sequences to pRNA trifurcate or four way X-motif junction domain; b. selecting said random sequences to identify those that having high binding affinity to said substrate; and c. conjugating said identified aptamer or an otherwise available aptamer to pRNA trifurcate or four way X-motif junction domain, wherein said available aptamer mimics at least one antibody to said at least one substrate. 28. The method according to claim 27, wherein said stable aptamer carries multivalent variants that recognize both cytotoxic T lymphocytes and different tumor cells. 29. The method according to claim 27, wherein said given substrate is an antigen, a protein, or a cell surface marker. 30. The method according to claim 27, wherein said identified aptamer provides advantages over traditional antibody fragments. 31. The trifurcate junction domain according to claim 11, wherein said domain is used for therapeutic delivery, diagnosis of diseases, promotion of RNA crystallization, or creation of stable RNA aptamer. 32. The trifurcate junction domain according to claim 11 is a trifurcate junction domain of DNA packaging RNA selected from the followings: phages PZA, phi15, BS32, B103, Nf, M2Y and GA-1, wherein said trifurcate junction domain is used for therapeutic delivery, diagnosis of diseases, promotion of RNA crystallization, or creation of stable RNA aptamer. 33. The trifurcate junction domain in claim 11 is selected from the followings: pRNA, 5s rRNA, HCV, Alu SRP, Hammerhead ribozyme, 16s H34-H35-H38, 23s H75-H76-H79, 23s H83-H84-H85, G-Riboswitch (Type I), TPP Riboswitch (Type II), and M-box Riboswitch (Type II), wherein said trifurcate junction domain is used for therapeutic delivery, diagnosis of diseases, promotion of RNA crystallization, or creation of stable RNA aptamer. 34. The trifurcate junction domain in claim 11 is selected from the followings: Family A: rRNA (16S H20-21-22; 16S H22-23-23a; 16S H25-25-26a; 16S H34-35-38; 23S H3-4-23; 23S H5H6H7; 23S H48-X-60; 23S H49-59.1-X; 23S H75-76-79; 23S H99-100-101), Family B: rRNA (16S H28-29-43; 16S H32-33-34; 16S H33-33a-33b; 23S H33-34-35; 23S H49-50-51; 23S H83-84-85), Family C: rRNA (16S H4-5-15; 16S H30-31-32; 16S H35-36-37; 16S H38-39-40; 23S H2-3-24; 23S H18-19-20; 23S H32-33-35; 23S H90-91-92); L11 rRNA; 5S rRNA; Alu domain; S domain; HH; G-riboswitch; P4P6; Twort Intron; S-dom RNaseP B-typ, or Unclassified family: Packaging RNA from: phi29; B103; SF5; and MN/NF phages. 35. The four way junction motif in claim 12 is selected from the followings: Family H: 1U9S_78 Ribonuclease P_A; 2A2E_70 Ribonuclease P_A; 1NBS_89 Ribonuclease P_B; 2A64_90 Ribonuclease P_B; 1M50_13 Hairpin ribozyme; 1S72_1827 23S rRNA; 2AW4_1771 23S rRNA; 2J01_1771 23S rRNA, Family cH: 1KH6_4 HCV IRES; 2AVY_16S rRNA; 2J00_141 16S rRNA; 1NKW_2621 23S rRNA; 1S72_2678 23S rRNA; 2AW4_2642 23S rRNA; 2J01_2642 23S rRNA; 3F2Q_7 Riboswitch (FMN); 3F2Q_31 Riboswitch (FMN); 1NKW_1457 23S rRNA; 2AW4_1443 23S rRNA, Family cL: 2AVY_568 16S rRNA; 2J00_568 16S rRNA; 1NKW_1282 23S rRNA; 1S72_1373 23S rRNA; 2AW4_1269 23S rRNA; 2J01_1269 23S rRNA; 1EFW_6 Transfer RNA; 1EHZ_6 Transfer RNA; 1N78_506 Transfer RNA; 1QRS_6 Transfer RNA; 1U08_6 Transfer RNA; 2GIS_7 Riboswitch (SAM I), Family cK: 2AVY_114 16S rRNA; 2J00_114 16S rRNA; 1NKW_2263 23S rRNA; 1S72_2318 23S rRNA: 2AW4_2284 23S rRNA; 2J01_2284 23S rRNA; 1NKW_1360 23S rRNA; 1S72_1452 23S rRNA; 2AW4_1346 23S rRNA; 2J01_1347 23S rRNA; 2AVY_18 16S rRNA; 2J00_18 16S rRNA, Family .pi.: 1U9S_118 Ribonuclease P_A; 2A2E_110 Ribonuclease P_A, Family cW: 1NKW_1682 23S rRNA; 1S72_1743 23S rRNA; 2AW4_1665 23S rRNA; 2J01_1665 23S rRNA, Family .psi.: 1S72_42 23S rRNA; 1NKW_1824 23S rRNA; 1S72_1888 23S rRNA; 2AW4_1832 23S rRNA: 2J01_1832 23S rRNA: 1NKW_244 23S rRNA; 2AW4_267 23S rRNA, Family X: 1NKW_608 23S rRNA; 2AW4_600 23S rRNA; 2J01_600 23S rRNA, or Family cX: 2IHX_166 Sarcoma virus; 2AVY_942 16S rRNA; 2J00_940 16S rRNA.

Details for Patent 9,297,013

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2031-06-08
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2031-06-08
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2031-06-08