Chromosome-based platforms

• US 20060246586A1
• Filed: 06/29/2006
• Published: 11/02/2006
• Est. Priority Date: 05/30/2001
• Status: Abandoned Application

• Current Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Original Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Inventors: Stewart, Sandra, Fleming, Elena, Leung, Josephine, Shellard, Joan, Lindenbaum, Michael, Perez, Carl, Perkins, Edward, Greene, Amy
• Application Number:
  US11/480,175
• Publication Number:
  US 20060246586A1
• Time in Patent Office: Days
• US Class Current: 435/455
• 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 provide tractable, efficient and rational engineering of the chromosome for a variety of applications.

Claims

1. A platform artificial chromosome expression system (ACes) comprising one or a plurality of sites that participate in recombinase catalyzed recombination.

2. The ACes of claim 1 that contains a plurality of sites.

3. The ACes of claim 1 that is predominantly heterochromatin.

4. The ACes of claim 1 that is an animal ACes.

5. The ACes of claim 4 that is a mammalian Aces.

6. The artificial chromosome expression system (ACes) of claim 1, wherein the recombinase and site(s) are from among the Cre/lox system of bacteriophage P1, the int/att system of lambda phage, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Cin recombinase system, the Pin recombinase system of E. coli and the R/RS system of the pSR1 plasmid and any combination thereof.

7. An isolated cell comprising, the chromosome of claim 1.

8. The cell of claim 7 that is an animal cell.

9. The cell of claim 7 that is a stem cell.

10. The cell of claim 7 that is a nuclear donor cell.

11. The stem cell of claim 9 that is human and is selected from among a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell and an embryonic stem cell.

19. A combination, comprising, the chromosome of claim 1 and a first vector comprising the cognate recombination site, wherein the cognate recombination site is a site that recombines with the site engineered into the chromosome.

20. The combination of claim 19, further comprising a nucleic acid molecule encoding a recombinase, wherein the nucleic acid molecule encoding the recombinase is provided on a second vector or on the first vector, or on the chromosome and its expression is under the control of an inducible promoter.

21. The combination of claim 20, wherein a vector is the plasmid pCXLamIntR.

22. The combination of claim 20, wherein a vector is the plasmid pDsRedN1-attB.

23. A kit, comprising:

the combination of claim 19;

and

optionally, instructions for introducing heterologous nucleic acid into the chromosome.

12. An animal artificial chromosomes comprising one or a plurality of att site(s), wherein:

an att site is heterologous to the chromosome; and

an att site permits site-directed integration in the presence of lambda integrase.

13. The artificial chromosome of claim 12 that is an artificial chromosome expression system (ACes).

14. The chromosome of claim 12, wherein the att sites are selected from the group consisting of attP and attB or attL and attR, or variants thereof.

15. The chromosome of claim 13 that is an artificial chromosome that is predominantly heterochromatin.

16. The chromosome of claim 12 that is a mammalian artificial chromosome.

17. The chromosome of claim 13 that is a mammalian artificial chromosome.

18. The chromosome of claim 12, wherein the integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR

24. An isolated cell comprising, the chromosome of claim 12.

25. The cell of claim 24, wherein the cell is a stem cell.

26. The cell of claim 24, wherein the cell is an animal cell.

27. The chromosome of claim 12, wherein the integrase is a lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.