Claims for Patent: 6,150,141

Summary for Patent: 6,150,141

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. (Lincoln, MA)
Assignee:	Trustees of Boston University (Boston, MA)
Application Number:	08/814,412
Patent Claims:	1. A method of producing a recombinant DNA molecule, the method comprising steps of: providing a first DNA/RNA hybrid molecule comprising a first DNA linked to a first splicing component; providing a second DNA/RNA hybrid molecule comprising a second DNA linked to a second splicing component, which second splicing component is selected so that, when the first and second DNA/RNA hybrid molecules are admixed together, trans-splicing between the first and second splicing components covalently links the first DNA with the second DNA to form a single, recombinant DNA molecule; and admixing the first and second DNA/RNA hybrid molecules together so that the recombinant DNA molecule is produced by trans-splicing. 2. The method of claim 1 wherein the first DNA is single-stranded. 3. The method of claim 2 wherein the second DNA is single-stranded. 4. The method of claim 2 wherein the second DNA is double-stranded. 5. The method of claim 1 wherein the first DNA is double-stranded. 6. The method of claim 5 wherein the second DNA is single-stranded. 7. The method of claim 5 wherein the second DNA is double-stranded. 8. The method of claim 7 wherein the step of providing a second DNA/RNA hybrid molecule comprises: integrating a third splicing ribozyme into a third target site in a second DNA molecule; integrating a fourth splicing ribozyme into a fourth target site in the second DNA molecule, the fourth target site being removed from the third target site, so that DNA between the third and fourth target sites constitutes the second DNA and is flanked by splicing components as a result of integration by the third and fourth ribozymes. 9. The method of claim 1 wherein the step of providing a first DNA/RNA hybrid molecule comprises: providing a first DNA/RNA hybrid molecule comprising a first DNA flanked by first and third splicing components. 10. The method of claim 1 or claim 9 wherein the step of providing a second DNA/RNA hybrid molecule comprises; providing a second DNA/RNA hybrid molecule comprising a second DNA flanked by second and first splicing components. 11. The method of claim 10 wherein the step of providing a second DNA/RNA hybrid comprises; ligating at least one of the second and fourth splicing components the second DNA by oligonucleotide-bridge-mediated DNA/RNA ligation. 12. The method of claim 9 wherein the step of providing a first DNA/RNA hybrid molecule comprises: integrating a first splicing ribozyme into a first target site in a DNA molecule; integrating a second splicing ribozyme into a second target site in the DNA molecule, the second target site being located at a distance removed from the first target site, so that DNA between the first and second target sites constitutes the first DNA and is flanked by splicing components as a result of integration by the first and second ribozyme. 13. The method of claim 12 wherein the first splicing ribozyme constitutes a lariat intron. 14. The method of claim 13 wherein the first splicing ribozyme constitutes a lariat group II intron. 15. The method of claim 14 wherein the group II intron is selected from the group consisting of aI5.gamma., bI1, aI1 and aI2. 16. The method of claim 14 wherein the first splicing ribozyme constitutes a lariat group II intron whose EBS1 site has been engineered to recognize the first target site, which first target site is distinct from the target site into which the intron would naturally integrate without having been so engineered. 17. The method of claim 12 wherein the first splicing ribozyme constitutes a Y-branched RNA molecule. 18. The method of claim 17 wherein the Y-branched RNA molecule comprises: a first arm comprising at least domains 1-3 of a group II intron; and a second arm comprising at least domains 5-6 of the group II intron, the first and second arms being covalently linked to one another via a 2'-5' phosphodiester linkage to the group II intron branch-point residue in domain 6. 19. The method of claim 18 wherein the group II intron EBS1 site has been engineered to recognize the first target site, which first target site is distinct from the target site into which the intron would naturally integrate had the EBS1 site not been so engineered.

Details for Patent 6,150,141

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		2013-09-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2013-09-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2013-09-10