Claims for Patent: 8,790,928

Summary for Patent: 8,790,928

Title:	Utilisation of constructs comprising recombination sequence motifs for enhancing gene expression in moss
Abstract:	A method of amplifying gene expression in a moss plant cell or moss tissue, DNA constructs therefor, moss plant cells and uses thereof for the production of protein.
Inventor(s):	Gorr; Gilbert (Freiburg, DE)
Assignee:	Greenovation Biotech GmbH (Freiburg, DE)
Application Number:	10/566,496
Patent Claims:	1. A method of amplifying gene expression in a moss plant cell by increasing the copy number of integrated transforming DNA constructs, the method comprising: 1) providing at least a first heterologous nucleic acid construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by a first recombination sequence and is flanked at the 3' end of said construct by a second recombination sequence; 2) providing at least a second heterologous nucleic acid construct different from the first, said second construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by said second recombination sequence and is flanked at the 3' end of said construct by said first recombination sequence; 3) transforming into the moss plant cell at least said first and said second heterologous nucleic acid constructs; and 4) regenerating the transformed moss plant cell into moss protonema comprising a plurality of copies of said at least one heterologous nucleotide sequence; wherein said first recombination sequence differs from said second recombination sequence and wherein recombination occurs between said different constructs at said first recombination sequences and at said second recombination sequences after transformation. 2. A method according to claim 1 wherein said at least first construct and said at least second construct are co-transformed into a moss protoplast. 3. A method according to claim 1 wherein the recombination sequences are selected from the group consisting of genomic DNA and cDNA. 4. A method according to claim 3 wherein the recombination sequence is selected from an intron or non-coding region. 5. A method according to claim 3 wherein the length of the recombination sequences is from 25 to 1000 nucleotides long. 6. A method according to claim 5 wherein the length of the recombination sequences is from 50-650 nucleotides long. 7. A method according to claim 6 wherein the length of the recombination sequences is from 100-400 nucleotides long. 8. A set of nucleic acid vectors suitable for amplifying gene expression in a moss plant cell, wherein said set of nucleic acid vectors comprises: 1) at least a first heterologous nucleic acid construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by a first recombination sequence and is flanked at the 3' end of said construct by a second recombination sequence; and 2) at least a second heterologous nucleic acid construct different from the first, said second construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by said second recombination sequence and is flanked at the 3' end of said construct by said first recombination sequence; wherein said first recombination sequence differs from said second recombination sequence, wherein said first and said second recombination sequences permit recombination in vivo between said different constructs at said first recombination sequences and at said second recombination sequences, and wherein said first and second recombination sequences are homologous to sequences in the moss plant cell's genome thereby permitting integration of heterologous sequences obtained from said recombination into the moss plant cell's genome. 9. A set of nucleic acid vectors according to claim 8, wherein the constructs are linear DNA constructs. 10. A moss cell transformed with a set of nucleic acid vectors suitable for amplifying gene expression in a moss plant cell, wherein said set of nucleic acid vectors comprises: 1) at least a first heterologous nucleic acid construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by a first recombination sequence and is flanked at the 3.degree. end of said construct by a second recombination sequence; and 2) at least a second heterologous nucleic acid construct different from the first, said second construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by said second recombination sequence and is flanked at the 3' end of said construct by said first recombination sequence; wherein said first recombination sequence differs from said second recombination sequence and wherein said first and said second recombination sequences permit recombination in vivo between said different constructs at said first recombination sequences and at said second recombination sequences. 11. A moss cell according to claim 10 which is a moss protoplast or a moss protonema cell. 12. A moss cell according to claim 11 which is Physcomitrella patens. 13. Moss protonema tissue comprised of cells transformed with a set of nucleic acid vectors suitable for amplifying gene expression in a moss plant cell, wherein said set of nucleic acid vectors comprises: 1) at least a first heterologous nucleic acid construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by a first recombination sequence and is flanked at the 3' end of said construct by a second recombination sequence; and 2) at least a second heterologous nucleic acid construct different from the first, said second construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5.degree. end thereof by said second recombination sequence and is flanked at the 3.degree. end of said construct by said first recombination sequence; wherein said first recombination sequence differs from said second recombination sequence and wherein said first and said second recombination sequences permit recombination in vivo between said different constructs at said first recombination sequences and at said second recombination sequences. 14. Use of moss protonema cells transformed with a set of nucleic acid vectors in the production of protein therefrom, comprising 1) providing moss protonema cells transformed with the set of DNA vectors suitable for amplifying gene expression in a moss plant cell, wherein said set of nucleic acid vectors comprises: a) at least a first heterologous nucleic acid construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by a first recombination sequence and is flanked at the 3' end of said construct by a second recombination sequence, and b) at least a second heterologous nucleic acid construct different from the first, said second construct comprising at least one heterologous nucleotide sequence operably linked to a promoter, wherein said construct is flanked at the 5' end thereof by said second recombination sequence and is flanked at the 3' end of said construct by said first recombination sequence; wherein said first recombination sequence differs from said second recombination sequence and wherein said first and said second recombination sequences permit recombination in vivo between said different constructs at said first recombination sequences and at said second recombination sequences; and 2) inducing expression of protein encoded in said DNA constructs. 15. Use according to claim 14 wherein said moss protonema cells are Physcomitrella patens. 16. A method according to claim 1 further wherein said recombination results in heterologous sequences, the method further comprising integrating said heterologous sequences into the moss plant cell's genome. 17. A set of nucleic acid vectors according to claim 8 wherein said recombination sequences are selected from the group consisting of a genomic DNA sequence and a cDNA sequence. 18. A method according to claim 1 wherein the recombination sequences are selected from the group consisting of an intron, a non-coding region, an exon, and a combination thereof. 19. A set of nucleic acid vectors according to claim 8 wherein said recombination sequences are selected from the group consisting of an intron sequence, a non-coding region sequence, an exon sequence, and a combination thereof.

Details for Patent 8,790,928

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		2023-07-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2023-07-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2023-07-31