Claims for Patent: 9,365,864

Summary for Patent: 9,365,864

Title:	Meganuclease recombination system
Abstract:	The invention relates to a set of genetic constructs which allow the efficient and reproducible introduction of a specific nucleotide sequence at a fixed position in the genome by generating a double strand break at a specific position in the genome using a meganuclease and so stimulating a homologous recombination event at this locus between the genomic site and a transfected donor sequence. The present invention also relates to methods using these constructs and to these materials in the form of a kit.
Inventor(s):	Cabaniols; Jean-Pierre (Saint Leu la Foret, FR), Choulika; Andre (Paris, FR), Delenda; Christophe (Paris, FR)
Assignee:	CELLECTIS (Paris, FR)
Application Number:	13/900,099
Patent Claims:	1. A method for transforming by homologous recombination at least one cell in vitro, comprising: (a) stably transforming at least one cell by inserting construct (i), which is encoded by a nucleic acid molecule, which comprises: A1-A2-A3-A4-A5 (i), wherein A1 is a first promoter, A2 is a first homologous portion, A3 is a meganuclease cleavage site, A4 is a first marker gene, A5 consists of the neomycin resistance gene of SEQ ID NO:7, and wherein construct (i) is configured to be stably integrated into the genome of at least one target cell, into the genome of said at least one cell; (b) cloning a sequence coding for a gene of interest into position B3 of construct (ii), which is encoded by a nucleic acid molecule, which comprises at least the following components: A2' -B1-B2-B3-B4-A5' (ii), wherein A2' comprises a portion of said first homologous portion A2, B1 is a second marker gene different from said first marker gene, B2 is a second promoter, B3 is a multiple cloning site, B4 is a third promoter, A5' consists of the inactive neomycin resistance gene of SEQ ID NO:13; (c) co-transfecting said cell of (a) with said construct (ii) of (b) and construct (iii), (iv), or (v), which are encoded by nucleic acid molecules, which comprise components: C1-C2 (iii), C3 (iv), or an isolated or recombinant protein which comprises component: C4 (v), wherein C1 is a fourth promoter, C2 is the open reading frame (ORF) of a meganuclease, C3 is a messenger RNA (mRNA) encoding said meganuclease, and C4 is an isolated or recombinant protein of said meganuclease, wherein a meganuclease from construct (iii), (iv), or (v) recognizes and cleaves A3, and construct (iii), (iv), or (v) are configured to be co-transfected with construct (ii) into said at least one target cell; (d) following homologous recombination between said construct (ii) and said stably inserted construct (i), selecting at least one cell from (c) based upon: the absence of a first marker gene encoded by component A4 of said construct (i) and the activity of a second marker gene encoded by component B1 and neomycin resistance activity. 2. The method of claim 1, wherein A1 is a promoter selected from an EF1.alpha. promoter, an SV40 promoter, a CMV promoter, and a Ubiqitin subunit c promoter. 3. The method of claim 1, wherein A2 consists of the EF1.alpha. intron 1 sequence of SEQ ID NO:3. 4. The method of claim 1, wherein A3 consists of the meganuclease cleavage site of SEQ ID NO:8. 5. The method of claim 1, wherein A4 is a hygromycin gene. 6. The method of claim 1, wherein A4 is a puromycin gene. 7. The method of claim 1, wherein A2' consists of the EF1 .alpha. intron 1 sequence of SEQ ID NO:29. 8. The method of claim 1, wherein B1 is a hygromycin gene. 9. The method of claim 1, wherein B1 is a puromycin gene. 10. The method of claim 1, wherein B2 is a promoter selected from an EF1.alpha. promoter, an SV40 promoter, a CMV promoter, and a Ubiqitin subunit c promoter. 11. The method of claim 1, wherein B3 consists of the multiple cloning site of SEQ ID NO:23. 12. The method of claim 1, wherein B4 is a promoter selected from an EF1.alpha. promoter, an SV40 promoter, a CMV promoter, and a Ubiqitin subunit c promoter. 13. The method of claim 1, wherein C1 is a promoter selected from an EF1.alpha. promoter, an SV40 promoter, a CMV promoter, and a Ubiqitin subunit c promoter. 14. The method of claim 1, wherein C2 consists of the meganuclease ORF of SEQ ID NO:14. 15. The method of claim 1, wherein C2 consists of the meganuclease ORF of SEQ ID NO:15. 16. The method of claim 1, wherein C3 is an mRNA equivalent of the sequence of SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:35. 17. The method of claim 1, wherein C4 is a meganuclease protein encoded by SEQ ID NO:14. 18. The method of claim 1, wherein C4 is a meganuclease protein encoded by SEQ ID NO:15. 19. The method of claim 1, wherein C4 is the meganuclease protein of SEQ ID NO:58.

Details for Patent 9,365,864

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		2028-10-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-10-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-10-23