Claims for Patent: 6,413,776

Summary for Patent: 6,413,776

Title:	High throughput screening of gene function using adenoviral libraries for functional genomics applications
Abstract:	Novel adenovirus vectors and methods for their use are provided to determine the function of the product(s) of one or more sample nucleic acids. The sample nucleic acids are synthetic oligonucleotides, DNA, or cDNA and encode polypeptides, antisense nucleic acids or GSEs. The sample nucleic acids are expressed in a host by recombinant adenovirus vectors to alter at least one phenotype of the host. The altered phenotype(s) is identified as a means to assign a biological function to the product(s) encoded by the sample nucleic acid(s).
Inventor(s):	Vogels; Ronald (Linschoten, NL), Bout; Abraham (Moerkapelle, NL), van Es; Helmuth H. G. (Hoofddorp, NL), Shouten; Govert (Leiden, NL)
Assignee:	Galapagos Geonomics N.V. (Mechelen, BE)
Application Number:	09/097,239
Patent Claims:	1. A method of producing a recombinant adenoviral vector library consisting of distinct recombinant adenoviral vectors, wherein each distinct vector contains an unique nucleic acid, by: growing a plurality of cell cultures, each said cell culture containing at least one cell comprising adenoviral nucleic acid sequences consisting essentially of adenoviral E1-complementing sequences, and transfecting, into said at least one cell of each said cell culture, an adapter plasmid and a recombinant nucleic acid, under conditions such that said recombinant adenoviral vector library is produced, wherein (A) i) said adapter plasmid comprises adenoviral nucleic acid sequence but does not comprise E1 region sequences that overlap with E1 region sequences in said at least one cell and E1 region sequences that overlap with E1 region sequences in said recombinant nucleic acid, wherein said overlap would otherwise result in generation of replication competent adenovirus in said at least one cell, (A) ii) said adapter plasmid further does not comprise E2B region sequences other than essential E2B sequences, E2A region sequences, E3 region sequences and E4 region sequences, (A) iii) said adapter plasmid further comprises, in operable configuration, a nucleic acid sequence coding for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sufficient adenoviral sequences to allow for homologous recombination with said recombinant nucleic acid, and a promoter and a unique nucleic acid sequence operatively linked to said promoter; and wherein (B) said recombinant nucleic acid comprises, in operable configuration, a sequence for a functional Inverted Terminal Repeat and adenoviral sequence sufficient for replication, wherein said recombinant nucleic acid sequence partially overlaps with sequence of said adapter plasmid to allow for homologous recombination resulting in replication-defective, recombinant adenoviral vector. 2. The method according to claim 1 wherein at least one of said adapter plasmids and said recombinant nucleic acid is heat denatured prior to transfecting said plurality of cells or ancestors of said plurality of cells. 3. The method according to claim 1 wherein said adenoviral E1-complementing sequences, said adapter plasmid and said recombinant nucleic acid contain no overlapping sequence which allows for homologous recombination resulting in replication competent adenovirus in a cell transfected therewith. 4. A method of producing a recombinant adenovirus vector library consisting essentially of a plurality of cell cultures containing distinct recombinant adenoviral vectors each containing a unique nucleic acid, by: growing a plurality of cell cultures, each said cell culture containing at least one cell, said at least one cell comprising adenoviral E1-complementing sequences; and transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one cell with i) a first recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for one functional Inverted Terminal Repeat, and sequence coding for a promoter and a unique nucleic acid operatively linked to said promoter; and ii) a second recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for one functional Inverted Terminal Repeat and sufficient for replication, wherein said second recombinant nucleic acid does not comprise the adenoviral E1 region, wherein one of said first or second recombinant nucleic acids comprises a functional encapsidation signal. 5. The method according to claim 4, wherein at least one of said plurality of cell cultures is in a multiwell format. 6. The method according to claim 4 wherein said adenovirus E1-complementing sequences, said first recombinant nucleic acid and said second recombinant nucleic acid contain no overlapping sequence which allows for homologous recombination resulting in replication competent adenovirus in a cell transfected therewith. 7. The method according to claim 4 wherein at least one of said plurality of cell cultures is a PER.C6 cell culture. 8. The method according to claim 4 wherein at least one of said plurality of cell cultures is grown in a medium containing sodium butyrate in an amount sufficient to enhance production of said recombinant adenovirus vector. 9. The method according to claim 4 wherein said at least one cell further comprises at least one of an adenovirus preterminal protein and a polymerase complementing sequence. 10. The method according claim 4 wherein said at least one cell further comprises an adenovirus E2 complementing sequence. 11. The method according to claim 10 wherein said E2 complementing sequence is selected from the group consisting of an E2A complementing sequence and an E2B complementing sequence. 12. The method according to claim 1 wherein said at least one cell further comprises a recombinase protein, whereby said homologous recombination resulting in replication-defective, recombinant adenovirus is enhanced. 13. The method according to claim 12 wherein said recombinase protein is a Kluyveromyces waltii recombinase. 14. The method according to claim 1 wherein said at least one cell further comprises a nucleotide sequence coding for a recombinase protein. 15. The method according to claim 14 wherein said recombinase protein is Kluyveromyces waltii recombinase. 16. The method according to claim 4 wherein each cell culture consists essentially of cells containing said recombinant adenovirus vectors, comprising said unique nucleic acids that are identical. 17. The method according claim 4 wherein said promoter is an inducible promoter. 18. The method according to claim 4 wherein said promoter is repressed or down modulated by an adenovirus E1 gene product. 19. The method according to claim 18 wherein said promoter comprises an AP1 dependent promoter. 20. The method according to claim 19 wherein said AP1 dependent promoter is derived from a collagenase, a c-myc, a monocyte chemoattractant protein or a stromelysin gene. 21. The method according to claim 4 wherein said unique nucleic acid sequence encodes a product of unknown function. 22. The method according to claim 4 wherein said nucleic acid sequence is selected from the group consisting of synthetic oligonucleotides, DNAs, cDNAs, genes, ESTs, antisense nucleic acids and genetic suppressor elements. 23. The method according to claim 4 wherein said method is automated. 24. A method of producing a recombinant adenovirus vector library consisting essentially of distinct recombinant adenoviral vectors each distinct vector containing a unique nucleic acid, said method comprising: growing a plurality of cell cultures containing at least one cell, said one cell expressing adenoviral sequence consisting essentially of E1-region sequences and expressing one or more functional gene products encoded by at least one adenoviral region selected from an E2A region and an E4 region; and transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one cell in each of said plurality of cell cultures with i) an adapter plasmid comprising adenoviral sequence coding, in operable configuration, for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sequences sufficient to allow for homologous recombination with a first recombinant nucleic acid, and not coding for E1 region sequences which overlap with E1 region sequences in said at least one cell, for E1 region sequences which overlap with E1 region sequences in a first recombinant nucleic acid, for E2B region sequences other than essential E2B sequences, for E2A region sequences, for E3 region sequences and for E4 region sequences, and further comprises a unique nucleic acid sequence and promoter operatively linked to said unique nucleic acid sequence; and ii) a first recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for a functional adenoviral Inverted Terminal Repeat and for sequences sufficient for replication in said at least one cell, but not comprising adenoviral E1 region sequences which overlap with E1 sequences in said at least one cell, and not comprising E2A region sequences or E4 region sequences expressed in said plurality of cells which would otherwise lead to production of replication competent adenovirus wherein said first recombinant nucleic acid has sufficient overlap with said adapter plasmid to provide for homologous recombination resulting in production of recombinant adenovirus in said at least one cell. 25. The method according to claim 24 wherein said first recombinant nucleic acid further comprises no E3 region sequences. 26. The method according to claim 24 wherein said at least one cell expresses at least one functional E2A gene product. 27. The method according to claim 26 wherein said at least one functional E2A gene product is a mutated gene product. 28. The method according to claim 27 wherein said mutated gene product is temperature sensitive. 29. The method according to claim 24 wherein at least one of said adapter plasmids and said first recombinant nucleic acids is heat denatured prior to transfecting said at least one cell or ancestor of said at least one cell. 30. The method according to claim 24 wherein said at least one cell expresses one or more functional gene product encoded by E2B region sequences and wherein said first recombinant nucleic acid comprises E2B region sequences required for adenoviral generation but does not comprise E2B region sequences coding for said functional E2B region gene products. 31. The method according to claim 30 wherein said at least one cell expresses all gene products encoded by E2B region sequences, and said adapter plasmid does not comprise E2B gene region sequences. 32. The method according to claim 24 wherein said at least one cell is a PER.C6 cell culture. 33. The method according to claim 24 wherein said promoter is an inducible promoter. 34. A plurality of cell cultures containing a recombinant replication-defective adenoviral vector library consisting essentially of distinct recombinant adenoviral vectors each distinct vector containing a unique nucleic acid, wherein said recombinant replication-defective adenoviral vector library is produced according to the method of claim 24. 35. The plurality of cultures of claim 34 wherein said at least one cell is a PER.C6 cell. 36. The method according to claim 24 wherein said method is automated.

Details for Patent 6,413,776

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-02-26