Merozoite surface protein 1 lacking glycosylation sites

• US 7,354,594 B2
• Filed: 05/27/2005
• Issued: 04/08/2008
• Est. Priority Date: 10/20/1997
• Status: Expired due to Fees

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Meade, Harry M., Chen, Li How
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US11/140,676
• Publication Number:
  US 20050235371A1
• Time in Patent Office: 1,047 Days
• Field of Search: 530/395, 424/191.1, 424/268.1, 800/7, 800 14- 18, 514/12
• US Class Current: 424/268.1
• 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 composition comprising a merozoite surface protein 1 (MSP-1) or fragment thereof, wherein at least one glycosylation site of the merozoite surface protein 1 (MSP-1) or fragment thereof has been eliminated.

3. The composition of claim 1 or 2, wherein the sequence of the MSP-1 is as set forth in SEQ ID NO:

9, but wherein the glycosylation site at position 182 has been eliminated by an amino acid substitution at position 182.

4. The composition of claim 1 or 2, wherein the sequence of the MSP-1 is as set forth in SEQ ID NO:

9, but wherein the glycosylation site at position 263 has been eliminated by an amino acid substitution at position 263.

5. The composition of claim 1 or 2, wherein the sequence of the MSP-1 is as set forth in SEQ ID NO:

9, but wherein the glycosylation sites at positions 182 and 263 have been eliminated by amino acid substitutions at positions 182 and 263.

2. A composition comprising a merozoite surface protein 1 (MSP-1) or fragment thereof, wherein all of the glycosylation sites of the merozoite surface protein 1 (MSP-1) or fragment thereof have been eliminated.

6. A composition comprising a merozoite surface protein 1 (MSP-1) or fragment thereof produced by:

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

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

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

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

2, thereby producing MSP-1 in its milk, wherein the MSP-1 or fragment thereof lacks at least one glycosylation site.

7. The composition of claim 6, wherein the MSP-1 or fragment thereof lacks all glycosylation sites.

8. The composition of claim 6, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 182.

9. The composition of claim 6, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 263.

10. The composition of claim 6, wherein the modified SEQ ID NO:

2 codes for amino acid substitutions at positions 182 and 263.

11. The composition of claim 6, wherein the promoter is a β

-casein promoter.

12. A composition comprising a merozoite surface protein 1 (MSP-1) or fragment thereof produced by:

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

2 encoding the MSP-1 or fragment thereof 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 each encode the same amino acid as the replaced codon; and

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

2, thereby producing MSP-1 in its milk,

wherein the MSP-1 or fragment thereof lacks at least one glycosylation site.

13. The composition of claim 12, wherein the MSP-1 or fragment thereof lacks all glycosylation sites.

14. The composition of claim 12, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 182.

15. The composition of claim 12, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 263.

16. The composition of claim 12, wherein the modified SEQ ID NO:

2 codes for amino acid substitutions at positions 182 and 263.

17. The composition of claim 12, wherein the promoter is a β

-casein promoter.

18. A composition comprising a merozoite surface protein 1 (MSP-1) or fragment thereof produced by:

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

2 encoding the MSP-1 or fragment thereof 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 the AT content of SEQ ID NO;

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

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

2, thereby producing MSP-1 in its milk,

wherein the MSP-1 or fragment thereof lacks at least one glycosylation site.

19. The composition of claim 18, wherein the MSP-1 or fragment thereof lacks all glycosylation sites.

20. The composition of claim 18, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 182.

21. The composition of claim 18, wherein the modified SEQ ID NO:

2 codes for an amino acid substitution at position 263.

22. The composition of claim 18, wherein the modified SEQ ID NO:

2 codes for amino acid substitutions at positions 182 and 263.

23. The composition of claim 18, 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.

24. The composition of claim 23, 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.

25. The composition of claim 24, 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.

26. The composition of claim 18, wherein all protozoan codons are replaced with codons preferred by mammalian cells.

27. The composition of claim 18, wherein the promoter is a β

-casein promoter.