Recombinant DNA molecules and expression vectors for tissue plasminogen activator

• US 5,985,607 A
• Filed: 06/27/1997
• Issued: 11/16/1999
• Est. Priority Date: 12/19/1994
• Status: Expired due to Term

• Current Assignee: Cangene Corporation (Emergent Biosolutions Inc.)
• Original Assignee: Cangene Corporation (Emergent Biosolutions Inc.)
• Inventors: Delcuve, Genevieve, Awang, Gregor
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): SCHWARTZMAN, ROBERT A
• Application Number:
  US08/883,795
• Time in Patent Office: 872 Days
• Field of Search: 435/69.1, 435/70.1, 435/70.3, 435/320.1, 435/325, 435/358, 435/359, 536/23.1, 536/23.2, 536/23.5, 536/24.1
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/0375 Animal model for cardiovascular diseases
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

A recombinant DNA molecule adapted for transfection of a host cell comprising a nucleic acid molecule encoding mammalian erythropoietin or tissue plasminogen activator, an expression control sequence operatively linked thereto and at least one SAR element. The invention also relates to expression vectors having the recombinant DNA molecule and to mammalian cells transformed with the expression vector. The mammalian cells lack multiple copies of an amplified amplification gene and are capable of expressing recombinant EPO or tPA in vitro at levels of at least 1,500 u or 500 u/10⁶ cells in 24 hours respectively. The invention further relates to a method of expressing recombinant mammalian EPO or tPA using the expression vectors and to a transgenic non-human animal or embryo whose germ cells and somatic cells contain a DNA construct having the recombinant DNA molecule of the invention.

Claims

1. A recombinant DNA molecule adapted for expression of tissue plasminogen activator, comprising a nucleic acid molecule encoding mammalian tissue plasminogen activator, an expression control sequence operatively linked thereto and at least one human apolipoprotein B scaffold attachment region (SAR) element wherein the SAR element is other than a 5'"'"' proximal apolipoprotein B SAR element.

2. A recombinant DNA molecule as claimed in claim 1 wherein the nucleic acid molecule encodes human tissue plasminogen activator.

3. A recombinant DNA molecule as claimed in claim 1 wherein the SAR element comprises the sequence as shown in SEQ ID NO:

36 or the sequence represented by nucleotides at positions 1 to approximately 1443 in SEQ ID NO. 37.

4. A recombinant DNA molecule as claimed in claim 1 wherein the SAR element is a SAR element which co-maps with a chromatin domain boundary.

5. A recombinant DNA molecule as claimed in claim 1 wherein the nucleic acid molecule encoding mammalian tissue plasminogen activator and the expression control sequence operatively linked thereto are flanked by SAR elements.

6. An expression vector which integrates into a host cell and comprising a recombinant DNA molecule as claimed in claim 1.

7. A method of expressing recombinant tissue plasminogen activator comprising transfecting a mammalian cell with an expression vector comprising a recombinant DNA molecule as claimed in claim 1 thereby generating a transfected cell, and culturing the transfected cell in a medium suitable for expression of tissue plasminogen activator until tissue plasminogen activator is produced by the cell and separating the tissue plasminogen activator produced.

8. A recombinant DNA molecule as claimed in claim 2 wherein the nucleic acid molecule encodes tissue plasminogen activator having the amino acid sequence shown in SEQ ID NO:

38.

9. A mammalian cell transformed with an expression vector as claimed in claim 6.

10. A recombinant DNA molecule as claimed in claim 8 wherein the nucleic acid molecule has the sequence as shown in SEQ ID NO:

39.

11. A mammalian cell as claimed in claim 9 which has not undergone gene amplification, wherein said cell expresses recombinant tissue plasminogen activator in vitro at levels of at least about 500 u/10⁶ cells in 24 hours.

12. A method of expressing recombinant mammalian tissue plasminogen activator comprising the steps of culturing a mammalian cell as claimed in claim 9 in a suitable medium to produce tissue plasminogen activator by the cell and separating the tissue plasminogen activator produced.

13. A method of expressing recombinant mammalian tissue plasminogen activator comprising the steps of culturing a mammalian cell as claimed in claim 9 in a medium suitable for the expression of tissue plasminogen activator in vitro at levels of at least about 500 u/10⁶ cells in 24 hours and separating the tissue plasminogen activator produced.

14. A method as claimed in claim 12 wherein the mammalian cell is further transfected with a selectable marker gene and wherein transfected cells are selected by means of the selectable marker gene.

15. A method as claimed in claim 12 comprising the additional step of identifying and selecting cells producing the highest levels of tissue plasminogen activator thereby generating selected cells;

subcloning the selected cells;

establishing long term cell lines from the selected cells and;

culturing the cell lines in a medium suitable for expression of tissue plasminogen activator until levels of tissue plasminogen activator of at least 500 u/10⁶ cells/day are produced by the cells and separating the tissue plasminogen activator produced.

16. A method as claimed in claim 12 wherein tissue plasminogen activator is produced at levels of at least 1,000 u/10⁶ cells in 24 hours in the absence of gene amplification.

17. A method as claimed in claim 14 wherein the selectable marker gene is neo.

18. An expression vector comprising a nucleic acid molecule encoding tissue plasminogen activator having the sequence shown in SEQ ID NO:

39 under the control of a promoter wherein said nucleic acid molecule and promoter are flanked by 5'"'"' distal and 3'"'"' human apolipoprotein B scaffold attachment region (SAR) elements.

19. An expression vector as claimed in claim 18 wherein the promoter is a human cytomegalovirus IE enhancer and promoter, or an elongation factor-1 alpha promoter.