Recombinant DNA molecules and expression vectors for erythropoietin

• US 5,888,774 A
• Filed: 12/19/1994
• Issued: 03/30/1999
• Est. Priority Date: 12/19/1994
• Status: Expired due to Fees

• Current Assignee: Rh Nanopharmaceuticals LLC
• Original Assignee: Cangene Corporation (Emergent Biosolutions Inc.)
• Inventors: Delcuve, Genevieve
• Primary Examiner(s): Chambers, Jasemine C.
• Application Number:
  US08/358,918
• Time in Patent Office: 1,562 Days
• Field of Search: 536/23.1, 536/24.1, 435/172.3, 435/240.2, 435/320.1, 435/69.6, 935/11, 935/24, 935/34, 935/70, 935/71
• US Class Current: 435/69.6
• 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, 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 in vitro at levels of at least 1,500 u/10⁶ cells in 24 hours. The invention further relates to a method of expressing recombinant mammalian erythropoietin 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 erythropoietin, comprising a nucleic acid molecule encoding mammalian erythropoietin, an expression control sequence operatively linked thereto and at least one human apolipoprotein B SAR element.

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

3. A recombinant DNA molecule as claimed in claim 2 wherein the nucleic acid molecule encodes erythropoietin having the amino acid sequence shown in SEQ ID NO 34.

4. A recombinant DNA molecule as claimed in claim 3 wherein the nucleic acid molecule has the sequence as shown in SEQ ID NO. 35.

5. A recombinant DNA molecule as claimed in claim 1 wherein the SAR element comprises the sequence as shown in SEQ ID NO. 36 or 37.

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

7. An expression vector comprising a recombinant DNA molecule as claimed in claim 1.

10. A mammalian cell transformed with an expression vector as claimed in claim 7.

11. A mammalian cell as claimed in claim 10 lacking a selectable gene capable of being amplified, said cell being capable of expressing recombinant erythropoietin in vitro at levels of at least about 2,000 u/10⁶ cells in 24 hours.

12. A method of expressing recombinant mammalian erythropoietin comprising the steps of culturing a mammalian cell as claimed in claim 11 in a suitable medium until sufficient amounts of erythropoietin are produced by the cell and separating the erythropoietin produced.

13. A method of preparing recombinant erythropoietin comprising transfecting a mammalian cell with an expression vector comprising a recombinant DNA molecule as claimed in claim 1, and culturing the transfected cell in a suitable medium until sufficient amounts of erythropoietin are produced by the cell and separating the erythropoietin 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 14 wherein the selectable marker gene is neo.

16. A method as claimed in claim 12 comprising the additional step of identifying and selecting cells producing high levels of erythropoietin;

subcloning the selected cells;

establishing long term cell lines from the selected cells and;

culturing the selected cells in a suitable medium until sufficient amounts of erythropoietin are produced by the cell and separating the erythropoietin produced.

17. A method as claimed in claim 12 wherein erythropoietin is produced at levels of at least 1,500 u/10⁶ cells in 24 hours in the absence of gene amplification.

8. An expression vector comprising a nucleic acid molecule encoding erythropoietin and having the sequence shown in SEQ ID NO.35 under the control of a promoter, flanked by 5'"'"' and 3'"'"' human apolipoprotein B SAR elements.

9. An expression vector as claimed in claim 8 wherein the promoter is a human cytomegalovirus IE enhancer and promoter, or an elongation factor-1 alpha promoter.