Claims for Patent: 8,158,414

Summary for Patent: 8,158,414

Title:	Gene silencing
Abstract:	The present invention relates to unique strategies and constructs for producing a nucleic acid product that down-regulates or prevents expression of a desired target polynucleotide. Such construct may include an expressing cassette having two convergently-oriented promoters each operably linked to a polynucleotide, but not to a terminator. The two polynucleotides are fully identical in sequence over at least 23 nucleotides.
Inventor(s):	Rommens; Caius (Boise, ID), Yan; Hua (Boise, ID), Bougri; Oleg (Boise, ID), Swords; Kathy M. M. (Boise, ID)
Assignee:	J.R. Simplot Company (Boise, ID)
Application Number:	11/662,872
Patent Claims:	1. A construct, comprising an expression cassette which comprises convergently-oriented first and second promoters, wherein (i) the first promoter is operably linked to a first polynucleotide and (ii) the second promoter is operably linked to a second polynucleotide, wherein (a) neither the first nor the second polynucleotide is operably linked to a terminator, and (b) the second polynucleotide and the first polynucleotide are fully identical in sequence over at least 23 nucleotides, wherein the second polynucleotide is positioned within the cassette in a different orientation to the first polynucleotide, and wherein the direction of transcription initiated from the first promoter is toward the second promoter and the direction of transcription initiated from the second promoter is toward the first promoter. 2. The construct of claim 1, wherein at least part of the second polynucleotide is oriented as perfect or imperfect an inverse complement copy of at least part of the first polynucleotide. 3. The construct of claim 1, wherein neither the first nor the second polynucleotide is operably linked to (i) a nos gene terminator, (ii) the 3' untranslated sequence of T-DNA gene 7, (iii) the 3' untranslated sequences of the major inclusion body protein gene of cauliflower mosaic virus, (iv) the 3' untranslated sequences of the pea ribulose 1,5-bisphosphate carboxylase small subunit, (v) the 3' untranslated sequences of the potato ubiquitin-3 gene, (vi) the 3' untranslated sequences of the potato proteinase inhibitor II gene, (vii) the 3' untranslated sequences of opine genes, or (viii) the 3' untranslated sequences of endogenous genes. 4. The construct of claim 1, wherein the first polynucleotide comprises a sequence that shares sequence identity with a target gene or at least one of a regulatory element that is associated with the target gene, an exon of the target gene, an intron of the target gene, the 5'-untranslated region of the target gene, or the 3'-untranslated region of the target gene. 5. The construct of claim 4, wherein the target gene is a COMT gene involved in lignin biosynthesis, a CCOMT gene involved in lignin biosynthesis, any other gene involved in lignin biosynthesis, an R1 gene involved in starch phosphorylation, a phosphorylase gene involved in starch phosphorylation, a PPO gene involved in oxidation of polyphenols, a polygalacturonase gene involved in pectin degradation, a gene involved in the production of allergens, or a gene involved in fatty acid biosynthesis. 6. The construct of claim 1, wherein the first and second promoters are functional in a plant and wherein the expression cassette is located between transfer-DNA border sequences of a plasmid that is suitable for bacterium-mediated plant transformation, wherein the bacterium is a strain of Agrobacterium, Rhizobium, or Phyllobacterium. 7. The construct of claim 1, further comprising a spacer polynucleotide positioned between the first and second polynucleotides, wherein the spacer polynucleotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or more than 500 nucleotides long. 8. The construct of claim 1, wherein the first promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter and wherein the second promoter is a constitutive promoter, a near-constitutive promoter, a tissue-specific promoter, or an inducible promoter. 9. A transformation plasmid, comprising an expression cassette, which comprises in the 5' to 3' orientation (1) a first promoter that is operably linked to (2) a first desired polynucleotide, which abuts (3) at least one optional spacer polynucleotide, where the 3'-end of one of the spacer polynucleotides abuts a (4) a second desired polynucleotide, which is operably linked to (5) a second promoter, wherein neither desired polynucleotide in the expression cassette is operably linked to any known transcription terminator, and wherein the second desired polynucleotide and the first desired polynucleotide are fully identical in sequence over at least 23 nucleotides. 10. The transformation plasmid of claim 9, wherein (a) at least part of the first desired polynucleotide is in the antisense orientation and wherein at least part of the second desired polynucleotide is oriented as the perfect or imperfect inverse complement of the first desired polynucleotide; or (b) at least part of the first desired polynucleotide is in the sense orientation and wherein at least part of the second desired polynucleotide is oriented as the perfect or imperfect inverse complement of the first desired polynucleotide; or (c) at least part of the first desired polynucleotide of (a) or (b) is a promoter sequence or shares sequence identity with a promoter sequence. 11. The transformation plasmid of claim 10, wherein the sequence of (c) shares sequence identity with a promoter that is associated with an endogenous gene and selected from the group consisting of (1) a starch-associated R1 gene promoter, (2) a polyphenol oxidase gene promoter, (3) a fatty acid desaturase 12 gene promoter, (4) a microsomal omega-6 fatty acid desaturase gene promoter, (5) a cotton stearoyl-acyl-carrier protein delta 9-desaturase gene promoter, (6) an oleoyl-phosphatidylcholine omega 6-desaturase gene promoter, (7) a Medicago truncatula caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene promoter, (8) a Medicago sativa (alfalfa) caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene promoter, (9) a Medicago truncatula caffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (10) a Medicago sativa (alfalfa) caffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (11) a Zea mays (maize) COMT gene, (12) a major apple allergen Mal d 1 gene promoter, (13) a major peanut allergen Ara h 2 gene promoter, (14) a major soybean allergen Gly m Bd 30 K gene promoter, (15) a polygalacturonase gene promoter, (16) any other endogenous promoter. 12. The transformation plasmid of claim 10, wherein (1) (i) at least one of the first and second promoters is a GBSS promoter and (ii) the first desired polynucleotide is a sequence that shares sequence identity with at least a part of a promoter associated with a polyphenol oxidase gene; (2) both the first and second promoters are GBSS promoters; or (3) the first promoter is a GBSS promoter and the second promoter is an AGP promoter. 13. A method of reducing expression of a gene normally capable of being expressed in a plant cell, comprising exposing a plant cell to the construct of claim 1, wherein the construct is maintained in a bacterium strain, selected from the group consisting of Agrobacterium tumefaciens, Rhizobium trifolii, Rhizobium leguminosarum, Phyllobacterium myrsinacearum, SinoRhizobium meliloti, and MesoRhizobium loti, wherein the desired polynucleotide comprises a sequence that shares sequence identity to a target sequence in the plant cell genome. 14. A method for enhancing tolerance to black spot bruising in a tuber, comprising expressing the construct of claim 1 in a cell of a tuber, wherein (a) the first polynucleotide comprises the sequence that shares sequence identity with at least a part of a tuber-expressed polyphenol oxidase gene a tuber-expressed polyphenol oxidase gene promoter, (b) the second polynucleotide is the inverse complement of the first polynucleotide or an imperfect inverse complement of the first polynucleotide, (c) one or both of the first and second promoters are GBSS or AGP, and (d) expression of the construct in the cell reduces transcription and/or translation of a polyphenol oxidase gene in the tuber cell genome, thereby enhancing the tolerance of the tuber to black spot bruising. 15. The method of claim 14, wherein the first polynucleotide comprises the sequence of SEQ ID NO: 26 or 27. 16. The construct of claim 5, wherein the gene involved in fatty acid biosynthesis is FAD2.

Details for Patent 8,158,414

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		2024-09-24
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2024-09-24
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2024-09-24
Aimmune Therapeutics, Inc.	PALFORZIA	peanut (arachis hypogaea) allergen powder	Powder	125696	01/31/2020		2024-09-24