Non-human transgenic mammal comprising a modified MSP-1 nucleic acid

• US 7,501,553 B2
• Filed: 02/20/2002
• Issued: 03/10/2009
• Est. Priority Date: 10/20/1997
• Status: Expired due to Fees

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Chen, Li-How, Meade, Harry M.
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US10/082,018
• Publication Number:
  US 20020144299A1
• Time in Patent Office: 2,575 Days
• Field of Search: 800/4, 800/14, 800/7, 800/16, 800/17, 800/18
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 2039/51 comprising whole cells, viruses or DNA/RNA
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/445 Plasmodium
• CPC Class Codes: C07K 14/4732 Casein in foodstuffs A23J
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• 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
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides modified recombinant nucleic acid sequences (preferably DNA) and methods for increasing the mRNA levels and protein expression of malarial surface protein MSP-1 which is known to be difficult to express in cell culture systems, mammalian cell culture systems, or in transgenic animals. The preferred protein candidates for expression using the recombinant techniques of the invention are MSP-1 proteins expressed from DNA coding sequences comprising reduced overall AT content or AT rich regions and/or mRNA instability motifs and/or rare codons relative to the native MSP-1 gene.

Claims

1. A non-human transgenic mammal whose genome comprises a modified SEQ ID NO:

2 encoding wild-type merozoite surface protein (MSP-1) operably linked to a mammary gland promoter,

wherein the modification reduces the AT content of SEQ ID NO;

2 by 50% or less by replacement of protozoan codons with codons preferred by mammalian cells,

wherein the replacement codons encode the same amino acid as the replaced codon, and

wherein the transgenic mammal expresses said modified SEQ ID NO;

2, thereby to produce MSP-1 in its milk.

2. The mammal of claim 1, wherein the promoter is a β

-casein promoter.

3. The mammal of claim 1, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks at least one glycosylation site.

4. The mammal of claim 3, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks all glycosylation sites.

5. The mammal of claim 1, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

6. The mammal of claim 1, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

7. The mammal of claim 1, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising amino acid substitutions at positions 181 and 263.

8. A method of producing a merozoite surface protein 1 (MSP-1) in the milk of a non-human transgenic mammal, comprising:

providing a non-human transgenic mammal whose genome comprises a modified SEQ ID NO;

2 encoding wild-type MSP-1 operably linked to a mammary gland promoter, wherein the modification reduces the AT content of SEQ ID NO;

2 by 50% or less by replacement of protozoan codons with codons preferred by mammalian cells, wherein the replacement codons encode the same amino acid as the replaced codon; and

allowing the transgenic mammal to express said modified SEQ ID NO;

2, thereby to produce MSP-1 in its milk.

9. The method of claim 8, wherein the promoter is a β

-casein promoter.

10. The method of claim 8, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks at least one glycosylation site.

11. The method of claim 10, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks all glycosylation sites.

12. The method of claim 8, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

13. The method of claim 8, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

14. The method of claim 8, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising amino acid substitutions at positions 181 and 263.

15. A non-human transgenic mammal whose genome comprises a modified SEQ ID NO:

2 encoding a wild-type MSP-1 operably linked to a mammary gland promoter,

wherein the modification eliminates all the mRNA instability motifs in said SEQ ID NO;

2 by replacement of protozoan codons with codons preferred by mammalian cells,

wherein the replacement codons encode the same amino acid as the replaced codon, and

wherein the transgenic mammal expresses said modified SEQ ID NO;

2, to thereby produce MSP-1 in its milk.

16. The mammal of claim 15, wherein the promoter is a β

-casein promoter.

17. The mammal of claim 15, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks at least one glycosylation site.

18. The mammal of claim 17, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks all glycosylation sites.

19. The mammal of claim 15, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

20. The mammal of claim 15, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

21. The mammal of claim 15, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising amino acid substitutions at positions 182 and 263.

22. A method of producing a merozoite surface protein 1 (MSP-1) sequence in the milk of a non-human transgenic mammal, comprising:

providing a non-human transgenic mammal whose genome comprises a modified SEQ ID NO;

2 encoding a wild-type MSP-1 operably linked to a mammary gland promoter,

wherein the modification eliminates all the mRNA instability motifs in said SEQ ID NO;

2 by replacement of protozoan codons with codons preferred by mammalian cells, and

wherein the replacement codons encode the same amino acid as the replaced codon; and

,

allowing the transgenic mammal to express said modified SEQ ID NO;

2, to thereby produce MSP-1 in its milk.

23. The method of claim 22, wherein the promoter is a β

-casein promoter.

24. The method of claim 22, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks at least one glycosylation site.

25. The method of claim 24, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid sequence that lacks all glycosylation sites.

26. The method of claim 22, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

27. The method of claim 22, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

28. A transgenic non-human mammal whose genome comprises a modified SEQ ID NO:

2 encoding a wild-type MSP-1 operably linked to a mammary gland specific promoter, wherein the modification eliminates all the mRNA instability motifs of said SEQ ID NO;

2 by replacement of one or more protozoan codons with codons preferred by mammalian cells and the modification reduces the AT content of said SEQ ID NO;

2 by 50% or less by replacement of protozoan codons with codons preferred by mammalian cells, wherein the replacement codons encode the same amino acid as the replaced codon and wherein the transgenic mammal expresses said modified SEQ ID NO;

2, thereby to produce MSP-1 in its milk.

29. The mammal of claim 28, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 25% more than the wild-type sequence is expressed under the same conditions.

30. The mammal of claim 28, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 50% more than the wild-type nucleic acid sequence is expressed under the same conditions.

31. The mammal of claim 28, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 100% more than the wild-type nucleic acid sequence is expressed under the same conditions.

32. The mammal of claim 28, wherein all protozoan codons are replaced with codons preferred by mammalian cells.

33. The mammal of claim 28, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

34. The mammal of claim 28, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

35. The mammal of claim 28, wherein the promoter is a β

-casein promoter.

36. A method for producing a merozoite surface protein 1 (MSP-1) sequence in the milk of a non-human transgenic mammal, comprising:

providing a non-human transgenic mammal whose genome comprises a modified SEQ ID NO;

2 encoding a wild-type MSP-1 operably linked to a mammary gland promoter, wherein the nucleic acid has been modified by

a) elimination of mRNA instability motifs by the replacement of protozoan codons in SEQ ID NO;

2 with codons preferred by mammalian cells; and

b) reduction of AT content by 50% a or less by the replacement of one or more AT-containing protozoan codons of SEQ ID NO;

2 with codons preferred by mammalian cells,

wherein the replacement codons encode the same amino acid as the replaced codon; and

allowing the transgenic mammal to express said modified SEQ ID NO;

2, to thereby produce MSP-1 in its milk.

37. The method of claim 36, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 25% more than the wild-type sequence is expressed under the same conditions.

38. The method of claim 36, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 50% more than the wild-type nucleic acid sequence is expressed under the same conditions.

39. The method of claim 36, wherein the modified SEQ ID NO:

2 is expressed in milk at a level which is at least 100% more than the wild-type nucleic acid sequence is expressed under the same conditions.

40. The method of claim 36, wherein all protozoan codons are replaced with codons preferred by mammalian cells.

41. The method of claim 36, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 182.

42. The method of claim 36, wherein the modified SEQ ID NO:

2 encodes an MSP-1 comprising an amino acid substitution at position 263.

43. The method of claim 36, wherein the promoter is a beta casein promoter.