Chromosome-based platforms

• US 7,521,240 B2
• Filed: 12/06/2004
• Issued: 04/21/2009
• Est. Priority Date: 05/30/2001
• Status: Expired due to Term

• Current Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Original Assignee: Smithkline Beecham Corporation (Smithkline Beecham PLC)
• Inventors: Lindenbaum, Michael, Perez, Carl, Stewart, Sandra, Fleming, Elena, Perkins, Edward, Greene, Amy, Leung, Josephine
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US11/006,076
• Publication Number:
  US 20050181506A1
• Time in Patent Office: 1,597 Days
• Field of Search: None
• US Class Current: 435/462
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2795/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/208 of mammalian origin, e.g. minichromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/40 being an insulator
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome for a variety of applications.

Claims

1. A method of introducing heterologous nucleic acid into an artificial chromosome, comprising:

contacting an animal or plant artificial chromosome comprising one or a plurality of att site(s) with a nucleic acid molecule comprising an insect artificial chromosome (IAC) and comprising both the heterologous nucleic acid and an att recombination site, in the presence of a recombinase that promotes recombination between the sites in the chromosome and in the nucleic acid molecule, wherein;

the one or a plurality of att site(s) are heterologous to the chromosome;

the recombinase is lambda integrase and the att site is a substrate for lambda integrase; and

the one or a plurality of att site(s) directs site-directed integration in the presence of lambda integrase.

2. The method of claim 1, wherein the nucleic acid molecule encodes a therapeutic protein, antisense nucleic acid, or comprises an artificial chromosome.

3. The method of claim 1, wherein the nucleic acid molecule with a recombination site is a PCR product.

4. The method of claim 1, wherein the recombinase is a protein and the recombination event occurs in vitro.

5. A method for introducing heterologous nucleic acid into a human mesenchymal cell, comprising:

(a) introducing into the human mesenchymal cell a platform-ACes, wherein the platform-ACes has a first recombination site;

(b) introducing into the resulting cell a vector comprising at least a second recombination site and the heterologous nucleic acid; and

(c) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the platform-ACes within the mesenchymal cells.

6. The method of claim 5, wherein the at least one recombination protein is encoded by a bacteriophage selected from the group consisting of bacteriophage lambda integrase (Int), phi 80, P22, P2, 186, P4 and P1.

7. The method of claim 6, wherein the at least one recombination protein is encoded by bacteriophage lambda integrase (Int), or a mutant of bacteriophage lambda integrase (Int).

8. The method of claim 5, wherein the at least one recombination protein is selected from the group consisting of Int, IHF, Xis, Fis, Cre, γ

δ

, Tn3 resolvase, Hin, Gin, Cin and Flp.

9. The method of claim 5, wherein the recombination sites are selected from the group consisting of att and lox P sites.

10. The method of claim 5, wherein the first and/or second recombination site contains at least one mutation in the sequence of nucleic acids encoding the recombination site that removes one or more stop codons introduced by virtue of the recombination site.

11. The method of claim 5, wherein the first and/or second recombination site contains at least one mutation in the sequence of nucleic acids encoding the recombination site that avoids hairpin formation introduced by virtue of the recombination site.

12. The method of claim 5, wherein the first and/or second recombination sites comprises at least a first nucleic acid sequence selected from among:

13. The method of claim 5, wherein the first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of:

a mutated att recombination site containing at least one mutation that enhances recombinational specificity relative to the absence of the mutation; and

a nucleic acid molecule whose sequence is complementary to the mutated att recombination site sequence.

14. The method of claim 5, wherein the vector comprising the second recombination site further encodes at least one selectable marker.

15. The method of claim 14, wherein the marker is a promoterless marker, which, upon recombination is under the control of a prometer and is thereby expressed.

16. The method of claim 15, where the first recombination site is attP and is in the sense orientation relative to a promoter on the platform ACes prior to recombination.

17. The method of claim 15, wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic, fluorescent, or capable of being bound by an antibody and FACs sorted.

18. The method of claim 17, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase (hisD).

19. The method of claim 15. wherein the promoterless marker is transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and

wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein.

20. The method of claim 19, wherein the selectable marker and the heterologous nucleic acid are transcriptionally linked by the presence of an IRES between them.

21. The method of claim 20, wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic or fluorescent.

22. The method of claim 21, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase.

23. The method of claim 19, further comprising expressing the heterologous protein and isolating the heterologous protein.

24. The method of claim 5, wherein the vector is a PCR product comprising a second recombination site.