Claims for Patent: 5,498,531

Summary for Patent: 5,498,531

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. (Arlington, MA)
Assignee:	President and Fellows of Harvard College (Cambridge, MA)
Application Number:	08/119,512
Patent Claims:	1. A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon 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 a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; 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 a variegated population of ribonucleic acids encoding chimeric gene products. 2. The method of claim 1, wherein said exons are randomly trans-spliced to each other to generate a library of combinatorial genes comprising, for every N different internal exons, N.sup.y different genes having y internal exons. 3. The method of claim 1, wherein at least a portion of said exons are spliced to each other in predetermined order. 4. The method of claim 1, 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 exon. 5. The method of claim 4, wherein said group II intron fragments further comprise at least a portion of a group II domain V sufficient to reconstitute said functional intron. 6. The method of claim 4, wherein said trans-splicing reaction conditions further comprise 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. 7. The method of claim 1, 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 exon. 8. The method of claim 1, 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). 9. The method of claim 8, wherein said snRNPs comprise a U1 snRNP, a U2 snRNP, a U4 snRNP, a U5 snRNP, and a U6 snRNP. 10. The method of claim 1 further comprising the step of reverse transcribing said variegated population of ribonucleic acids encoding chimeric gene products. 11. A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) is absent or represents a 3' fragment of a group II intron, which 3' intron fragment includes a branch acceptor site and at least a portion of a domain V, and 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 a group II intron, which 5' intron fragment includes an exon binding site, and is covalently attached to the 3' exon end of said exon by a phosphodiester bond, with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation and trans-splice said exons to generate a variegated library of chimeric ribonucleic acids. 12. The method of claim 11, wherein said branch acceptor site comprises a stem loop structure of a group II domain VI having a branch nucleotide disposed in said 3' intron fragment in the range of 5 to 10 nucleotides from said 5' exon end of said exon and oriented to facilitate nucleophilic attack of its 2'-hydroxyl on a phosphodiester at a 3' exon end of another of said exons. 13. The method of claim 12, wherein said branch nucleotide is an unbase-paired adenosine residue bulging from said stem loop structure of said domain VI. 14. The method of claim 11, wherein said exon binding site comprises from 3 to 8 consecutive nucleotides complementary in sequence to an internal binding sequence located within said exon and proximate to said 3' exon end. 15. The method of claim 11, wherein said 5' intron fragment comprises intron domains V and VI, and said 3' intron fragment comprises intron domains I-III. 16. A method of isolating a gene for chimeric gene product having a desired activity, comprising the steps of: (i) admixing a variegated population of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, 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 a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments; wherein said trans-splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation, which functional intron, under said trans-splicing conditions, causes ligation of said exons to generate a combinatorial library of chimeric ribonucleic acids; (ii) generating, from said library of chimeric ribonucleic acids, a library of replicable expression vectors; (iii) transforming suitable host cells with said library of replicable expression vectors; (iv) culturing said transformed host cells under conditions causing expression of said library of expression vectors to generate a library of gene products; (v) detecting, in said library of gene products, individual gene products having a desired activity; and (vi) isolating, from said library of expression vectors, genes for the gene products detected in step (v). 17. The method of claim 16, wherein said 3' and 5' intron fragments comprise group II intron fragments including an exon binding site, a branch acceptor site, and at least a potion of a domain V. 18. The method of claim 16, wherein said 3' and 5' intron fragments comprise group I intron fragments including an internal guide sequence, a GTP-binding site, and a guanosine nucleotide located in said 3' intron fragment immediately adjacent said 5' exon end. 19. The method of claim 16, wherein (i) said 3' and 5' intron fragments comprise nuclear pre-mRNA intron fragments including a 5' splice junction sequence, a 3' splice junction sequence, and a branchpoint sequence; and (ii) said trans-splicing reaction conditions include admixing, with said splicing constructs, adenosine triphosphate (ATP) and small nuclear ribonucleoproteins (snRNPs). 20. The method of claim 16, wherein said gene product is a polypeptide. 21. The method of claim 16, wherein said gene product is a ribozyme. 22. The method of claim 16, wherein said desired activity is an enzymatic activity and detecting said desired activity comprises detecting an enzymatic alteration of a substrate by individual gene products of said library of gene products. 23. The method of claim 16, wherein said desired activity is a binding affinity for a target molecule and detecting said desired activity comprises detecting binding to said target molecule by individual gene products of said library of gene products. 24. The method of claim 23, wherein said gene product is an antibody or functional binding fragment thereof. 25. The method of claim 16, wherein said library of replicable expression vectors comprises a phage display library. 26. A splicing construct comprising a nucleic acid represented by the general Formula (3'IVS)-EX-(5'IVS), wherein EX represents an exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, 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 not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said exon is discontinuous with any nucleic acid sequences other than said 3' and 5' intron fragments, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs. 27. The nucleic acid construct of claim 26, wherein said exon comprises an open reading frame encoding at least a portion of a protein. 28. The nucleic acid construct of claim 27, wherein said protein is naturally encoded by genomic DNA of a eukaryotic cell. 29. The nucleic acid construct of claim 26, wherein said exon comprises at least a portion of a ribozyme nucleic acid sequence. 30. The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise group II intron fragments. 31. The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise group I intron fragments. 32. The nucleic acid construct of claim 26, wherein said 3' and 5' intron fragments comprise nuclear pre-mRNA intron fragments. 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 exon, said exon having a 5' exon end and a 3' exon end, (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, 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 not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond, wherein said 3' and 5' intron fragments can, through intermolecular complementation, cause trans-splicing of two or more exons to each other, and said variegated population of nucleic acids is heterogeneous with respect to nucleotide sequences of said exon. 34. The library of splicing constructs of claim 33, wherein exons of said variegated population of nucleic acids encode portions of at least one thrombolytic protein. 35. The library of splicing constructs of claim 33, wherein exons of said variegated population of nucleic acids encode portions of at least one antibody. 36. A kit for humanizing an antibody by combinatorial intron-mediated ligation, comprising a library of human framework region (FR) constructs each represented by the general formula (3'IVS)-FR-(5'IVS), wherein FR represents an exon encoding a human framework region selected from the group consisting of FR1, FR2, FR3 and FR4, 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 one or both of said 3' and 5' intron fragments are present in each FR construct; wherein said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing between said exon and a complementarity determining region (CDR) of a construct comprising CDR-encoding nucleic acid having 3' and 5' intron fragments attached thereto. 37. The kit of claim 36, wherein said FR constructs are selected for sequential ligation of said exons with said CDR-encoding nucleic acids to form a ribonucleic acid represented by a general formula 5'-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-3'.

Details for Patent 5,498,531

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		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26