Modified MSP-1 nucleic acid sequences and methods for increasing MRNA levels and protein expression in cell systems

• US 6,593,463 B1
• Filed: 10/20/1998
• Issued: 07/15/2003
• Est. Priority Date: 10/20/1997
• Status: Expired due to Fees

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: Genzyme Transgenics Corporation
• Inventors: Chen, Li How, Meade, Harry
• Primary Examiner(s): Priebe, Scott D.
• Assistant Examiner(s): Paras, Jr., Peter
• Application Number:
  US09/175,684
• Time in Patent Office: 1,729 Days
• Field of Search: 424/272.1, 424/185.1, 424/268.1, 424/93.2, 435/320.1, 435/69.1, 435/69.3, 530/300, 530/350, 536/23.1
• US Class Current: 536/23.7
• 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 modified nucleic acid sequence comprising SEQ ID NO 1.

2. A nucleic acid sequence comprising a modified nucleic acid sequence encoding a merozoite surface protein 1 (MSP-1) operably linked to a promoter which directs expression in mammary epithelial cells wherein the AT content of the modified nucleic acid sequence has been reduced by replacing codons from a nucleic acid sequence of SEQ ID NO:

2 with mammary gland preferred codons encoding the same amino acid as the replaced codon such that the AT content of the modified nucleic acid sequence is 50% or less.

9. The nucleic acid of claim 2, wherein the modified nucleic acid comprises at least one additional mammary gland preferred codon other than the mammary gland preferred codons which lowers the AT content.

13. The nucleic acid of claim 9, wherein the promoter is a beta casein promoter.

3. A vector comprising a modified merozoite surface protein 1 (MSP-1) nucleic acid according to clam 2.

4. The vector of claim 3, wherein the promoter is a beta casein promoter.

5. A host cell transformed with the vector according to claim 3.

6. The cell of claim 5, wherein the cell is a mammalian cell.

7. The cell of claim 6, wherein the cell is a COS cell.

8. The cell of claim 6, wherein the cell is a mammary gland epithelial cell.

10. A nucleic acid sequence comprising a modified nucleic acid sequence encoding a merozoite surface protein 1 (MSP-1) operably linked to a promoter which directs expression in mammary epithelial cells wherein the AT content of the modified nucleic acid sequence has been reduced by replacing codons from a nucleic acid sequence of SEQ ID NO:

2 with mammary gland preferred codons such that the AT content of the modified nucleic acid sequence is 50% or less and, wherein at least one glycosylation site of SEQ ID NO;

10 has been altered such that it is not functional.

11. The nucleic acid of claim 10, wherein the glycosylation site which is not functional is at position 182 of SEQ ID NO:

10.

12. The nucleic acid of claim 10, wherein the glycosylation site which is not functional is at position 263 of SEQ ID NO:

10.

14. The nucleic acid of claim 10, wherein the promoter is a beta casein promoter.

15. A nucleic acid comprising a modified nucleic acid encoding merozoite surface protein 1 (MSP-1) operably linked to a promoter which directs expression in mammary epithelial cells, wherein all mRNA instability motifs present in the wild-type nucleic acid sequence encoding MSP-1 have been eliminated by replacing codons of SEQ ID NO:

2 with mammary gland preferred codons encoding the same amino acid as the replaced codon.

16. The nucleic acid of claim 15, wherein the promoter is a beta casein promoter.

17. The nucleic acid of claim 15, wherein the modified nucleic acid comprises at least one additional mammary gland preferred codon other than the codon replaced to eliminate an mRNA instability motif.

18. The nucleic acid of claim 15, wherein all of the codons of the modified nucleic acid are mammary gland preferred codons.

19. A nucleic acid comprising a modified nucleic acid encoding merozoite surface protein 1 (MSP-1) operably linked to a promoter which directs expression in mammary epithelial cells, wherein all mRNA instability motifs present in the wild-type nucleic acid sequence encoding MSP-1 have been eliminated by replacing codons of SEQ ID NO:

2 with mammary gland preferred codons, and wherein at least one glycosylation site of SEQ ID NO;

10 has been altered such that it is not functional.

20. The nucleic acid of claim 19, wherein the glycosylation site which is not functional is at position 182 of SEQ ID NO:

10.

21. The nucleic acid of claim 19, wherein the glucosylation site which is not functional is at position 263 of SEQ ID NO:

10.

22. The nucleic acid of claim 19, wherein the glycosylation sites at positions 182 and 263 of SEQ ID NO:

10 are not functional.