Claims for Patent: 5,780,272

Summary for Patent: 5,780,272

Title:	Intron-mediated recombinant techniques and reagents
Abstract:	The present invention makes available methods and reagents for novel manipulation of nucleic acids. As described herein, the present invention makes use of the ability of intronic sequences, such as derived from group I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and ligation of discontinuous nucleic acid molecules. For example, novel genes and gene products can be generated by admixing nucleic acid constructs which comprise exon nucleic acid sequences flanked by intron sequences that can direct trans-splicing of the exon sequences to each other. The flanking intronic sequences can, by intermolecular complementation, form a reactive complex which promotes the transesterification reactions necessary to cause the ligation of discontinuous nucleic acid sequences to one another, and thereby generate a recombinant gene comprising the ligated exons.
Inventor(s):	Jarrell; Kevin A. (Boston, MA)
Assignee:	President and Fellows of Harvard College (Cambridge, MA)
Application Number:	08/488,015
Patent Claims:	1. A purified preparation of a reverse-splicing intron, which reverse-splicing intron comprises: a first segment comprising a 5' portion of a group II intron, which 5' portion includes an exon binding site not naturally present in said group II intron; and a second segment comprising a 3' portion of a group II intron, which 3' portion includes a domain V motif, a branch site acceptor forming a phosphodiester bond with a 5' end of said first segment, and a nucleophilic group at a 3' end of said second segment for transesterifying a phosphodiester bond of a ribonucleic acid, wherein said first and second segments together form an autocatalytic y-branched intron which catalyzes integration of at least the first segment of the reverse-splicing intron into a substrate ribonucleic acid by a reverse-splicing reaction. 2. The reverse-splicing intron of claim 1, wherein said 5' portion of the group II intron comprises intron domains V and VI, and said 3' portion of the group II intron comprises intron domains I-III. 3. The reverse-splicing intron of claim 2, which reverse-splicing intron is represented by the general formula: where (IVS 1-3) represents a 5' portion of a group II intron, (IVS5,6) represents a 3' portion of a group II intron, which portion includes a branch site acceptor, and 2'-5' represents a phosphodiester bond formed between a branch site acceptor of (IVS5,6) and the 5' end of (IVS1-3), (IVS1-3) and (IVS5,6) otherwise being discontinuous with each other, wherein (IVS 1-3) and (IVS5,6) together form an autocatalytic Y-branched intron which catalyzes integration of the (IVS1-3) fragment into a substrate ribonucleic acid by a reverse-splicing reaction. 4. The reverse-splicing intron of claim 1, wherein said first and second segments are contiguous, via a covalent bond other than the phosphodiester bond formed with said branch site acceptor, and form a y-branched lariat. 5. The reverse-splicing intron of claims 2 or 4, which reverse-splicing intron is represented by the general formula: ##STR2## wherein (IVS1-3) represents a 5' portion of a group II intron, (IVS5,6) represents a 3' portion of a group II intron, which portion includes a branch site acceptor, '- ' represents a phosphodiester bond formed between a branch site acceptor of (IVS5,6) and the 5' end of (IVS1-3), and A represents a phosphodiester bond between a 3' end of (IVS1-3) and a 5' end of (IVS5,6), wherein (IVS1-3) and (IVS5,6) together form an autocatalytic Y-branched lariat which catalyzes integration of the reverse-splicing intron into a substrate ribonucleic acid by a reverse-splicing reaction. 6. The reverse-splicing intron of claims 1, 2 or 4, wherein said exon binding site is selected to provide specific integration into the substrate ribonucleic acid after a selected intron binding site, which intron binding site is from 3-16 nucleotides in length. 7. The reverse-splicing intron of claim 6, wherein said intron binding site is from 5-7 nucleotides in length. 8. The reverse-splicing intron of claim 1, wherein said exon binding site comprises an EBS1 and an EBS2. 9. A purified preparation of a reverse-splicing construct, which reverse-splicing construct comprises two or more fragments of autocatalytic introns and catalyzes integration of at least a portion of the reverse-splicing construct into a substrate ribonucleic acid by a reverse-splicing reaction, wherein the portion of the intron sequences that provides sequence specificity for the substrate ribonucleic acid differs in structure and specificity from the naturally-occurring sequence of the autocatalytic intron. 10. The preparation of claim 9, wherein the autocatalytic intron fragments are selected from group II introns. 11. The preparation of claim 10, wherein the reverse-splicing construct comprises a 5' portion of a group II intron including intron domains V and VI, and a 3' portion of a group II intron including intron domains I-III. 12. The preparation of claim 10, wherein the reverse-splicing construct comprises, from a group I intron, an internal guide sequence, a GTP-binding site, wherein said group I intron fragments reconstitute a functional intron through intermolecular complementation. 13. The preparation of claim 9, wherein the autocatalytic intron fragments are selected from group I introns. 14. A library of reverse-splicing introns comprising a variegated population of purified y-branched group II introns, which variegated population is characterized as including at least 25 different y-branched group II introns of unique specificity. 15. The library of reverse-splicing introns of claim 14, wherein the variegated population includes at least 100 different y-branched group II introns of unique specificity. 16. The library of reverse-splicing introns of claim 14, wherein the variegated population includes from 10.sup.3 to 10.sup.5 different y-branched group II introns of unique specificity. 17. The library of reverse-splicing introns of claim 14, wherein the y-branched group II introns include: a first segment comprising a 5' portion of a group II intron, which 5' portion includes an exon binding site not naturally present in said group II intron; and a second segment comprising a 3' portion of a group II intron, which 3' portion includes a domain V motif, a branch site acceptor forming a phosphodiester bond with a 5' end of said first segment, and a nucleophilic group at a 3' end of said second segment for transesterifying a phosphodiester bond of a ribonucleic acid, wherein said first and second segments together form an autocatalytic y-branched intron which catalyzes integration of at least the first segment of the reverse-splicing intron into a substrate ribonucleic acid by a reverse-splicing reaction. 18. The library of reverse-splicing introns of claim 17, wherein said 5' portion of the group II intron comprises intron domains V and VI, and said 3' portion of the group II intron comprises intron domains I-III. 19. The library of reverse-splicing intron of claim 17, wherein said first and second segments are contiguous, via a covalent bond other than the phosphodiester bond formed with said branch site acceptor, and form a y-branched lariat. 20. The library of reverse-splicing intron of claims 14, 18 or 19, which reverse-splicing intron is represented by the general formula: ##STR3## wherein (IVS1-3) represents a 5' portion of a group II intron, (IVS5,6) represents a 3' portion of a group II intron, which portion includes a branch site acceptor, '- ' represents a phosphodiester bond formed between a branch site acceptor of (IVS5,6) and the 5' end of(IVS1-3), and A, if present, represents a phosphodiester bond between a 3' end of (IVS1-3) and a 5' end of (IVS5,6), wherein (IVS1-3) and (IVS5,6) together form an autocatalytic Y-branched intron which catalyzes integration of the reverse-splicing intron into a substrate ribonucleic acid by a reverse-splicing reaction. 21. A method for generating a chimeric ribonucleic acid by trans-splicing, comprising admixing two or more splicing constructs under trans-splicing reaction conditions, which splicing constructs comprise a ribonucleic acid represented by the general formula (3' IVS)-EX-(5' IVS), wherein EX represents an exonic ribonucleic acid sequence which is intended to be present in a chimeric ribonucleic acid, said exonic sequence having a 5' exon end and a 3' exon end, (3' IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exonic sequence by a phosphodiester bond, and (5' IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exonic sequence by a phosphodiester bond, with the proviso that at least one of (3' IVS) and (5' IVS) is present on each splicing construct; and the intron fragments for at least a portion of said splicing constructs are not from intronic sequences which naturally flank the corresponding covalently attached exonic sequence; wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid. 22. The method of claim 21, wherein at least a portion of said exonic sequences are spliced to each other in predetermined order. 23. The method of claim 21, wherein said 3' and 5' intron fragments of said splicing constructs comprise group II intron fragments including, i) an exon binding site, and ii) a branch site acceptor comprising an activated nucleophile for forming a phosphodiester bond with a 5' intron end of said 5' intron fragment and for cleaving said 5' intron fragment from the 3' end of said exonic sequence. 24. The method of claim 23, wherein said group II intron fragments further comprise at least a portion of a group II domain V sufficient to reconstitute said functional intron. 25. The method of claim 23, wherein said trans-splicing reaction conditions further comprises admixing with said splicing constructs at least a portion of a domain V of a group II intron sufficient to interact with said 3' and 5' intron fragments and reconstitute said functional intron. 26. The method of claim 21, wherein said 3' and 5' intron fragments of said splicing constructs comprise group I intron fragments including an internal guide sequence, a GTP-binding site, and a 3' terminal G located in said 3' intron fragment immediately adjacent said 5' exon end of said exonic sequence. 27. The method of claim 21, wherein said 3' and 5' intron fragments of said splicing constructs comprise nuclear pre-mRNA intron fragments including a 5' splice junction sequence, a 3' splice junction sequence, and a branchpoint sequence; and said trans-splicing reaction conditions include admixing, with said splicing constructs, adenosine triphosphate (ATP) and small nuclear ribonucleoproteins (snRNPs). 28. The method of claim 27, wherein said snRNPs comprise a U1 snRNP, a U2 snRNP, a U4 snRNP, a U5 snRNP, and a U6 snRNP. 29. The method of claim 21, wherein said exonic sequence comprises a polypeptide encoding sequence. 30. The nucleic acid construct of claim 29, wherein said 3' and 5' intron fragments comprise nuclear pre-mRNA intron fragments. 31. The method of claim 21, wherein said exonic sequence comprises a ribozyme sequence. 32. A method for generating a chimeric ribonucleic acid by trans-splicing, comprising admixing a two or more splicing constructs under trans-splicing reaction conditions, which splicing constructs comprise a ribonucleic acid represented by the general formula (3' IVS)-EX-(5' IVS), wherein EX represents an ribonucleic acid encoding at least a portion of a mammalian polypetide, said exon having a 5' exon end and a 3' exon end, (3' IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5' IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least one of (3' IVS) and (5' IVS) is present on each splicing construct, wherein said exons of said set of splicing constructs comprise a variegated population of polypeptide-encoding sequences, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid. 33. A library of splicing constructs comprising a variegated population of nucleic acids each represented by the general formula (3' IVS)-EX-(5' IVS), wherein EX represents an exonic ribonucleic acid which is intended to be present in a chimeric ribonucleic acid, said exon having a 5' exon end and a 3' exon end, (3' IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5' IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least one of (3' IVS) and (5' IVS) is present on each splicing construct; and the intron fragments for at least a portion of said splicing constructs are not from intronic sequences which naturally flank the corresponding covalently attached exon; wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid. 34. A splicing construct comprising a nucleic acid represented by the general formula (3' IVS)-EX-(5' IVS), wherein EX represents an ribonucleic acid encoding at least a portion of a mammalian polypetide, said exon having a 5' exon end and a 3' exon end, (3' IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5' IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least one of (3' IVS) and (5' IVS) is present on each splicing construct, wherein said exon sequence is discontinuous with any nucleic acid sequences other than said flanking intron sequences, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs. 35. The nucleic acid construct of claim 34, wherein said 3' and 5' intron fragments comprise group II intron fragments. 36. The nucleic acid construct of claim 34, wherein said 3' and 5' intron fragments comprise group I intron fragments. 37. A method for generating a circular ribonucleic acid by intron-mediated splicing comprising, providing a ribonucleic acid, represented by the general formula (3' IVS)-EX-(5' IVS), wherein EX represents an exonic nucleic acid which is intended to be present in a circular nucleic acid, said exon having a 5' exon end and a 3' exon end, (3' IVS) represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and (5' IVS) represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, under trans-splicing conditions which cause reconstitution of a functional intron by intramolecular complementation of said 3' and 5' intron fragments, which functional intron, under said trans-splicing conditions, ligatates said 3' and 5' end of said exon to generate a circular ribonucleic acid. 38. The method of claim 37, wherein said trans-splicing conditions include providing a bridging oligonucleotide with said ribonucleic acid to facilitate intramolecular complementation of said 3' and 5' intron fragments. 39. The method of claim 37, wherein the ribonucleic acid includes a structural element which non-covalently links the (3' IVS) and (5' IVS). 40. The method of claim 39, wherein (3' IVS) and (5' IVS) each contain complementary sequences which anneal to non-covalently link the (3' IVS) and (5' IVS). 41. The method of claim 37, wherein at least one of the (3' IVS) or (5' IVS) represents a fragment of an intron not normally associated with the exonic nucleic acid. 42. The method of claims 37, wherein (3' IVS) or (5' IVS) represent fragments of an autocatalytic intron, and combine through intramolecular complementation to form a functional intron. 43. The method of claim 42, wherein (3' IVS) or (5' IVS) represent fragments of a group II autocatalytic intron. 44. The method of any of claims 37, 39-43, wherein the ribonucleic acid is produced by transcription of an expression construct in a host cell, and the trans-splicing which generates the circular ribonucleic acid occurs in the host cell. 45. The method of claim 44, wherein the host cell is a mammalian cell.

Details for Patent 5,780,272

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		2015-07-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2015-07-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2015-07-14