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Last Updated: October 18, 2019

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Claims for Patent: 5,770,371

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Summary for Patent: 5,770,371
Title: Modification of cryptic splice sites in heterologous genes expressed in fungi
Abstract:The present invention relates to methods for obtaining a fungal host cell comprising a nucleic acid sequence encoding a heterologous polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence. The present invention also relates to a nucleic acid sequence(s) with a modified cryptic splice site(s) as well as nucleic acid constructs, vectors, and host cells comprising said nucleic acid sequence(s). The present invention further relates to methods for recombinant production of a polypeptide encoded by said nucleic acid sequence.
Inventor(s): Thompson; Sheryl (Davis, CA)
Assignee: Novo Nordisk Biotech, Inc. (Davis, CA)
Application Number:08/672,158
Patent Claims:1. A method for obtaining a recombinant fungal host cell, which produces a biologically active heterologous polypeptide comprising introducing into a fungal host cell a nucleic acid sequence encoding said heterologous polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%, wherein a recombinant fugal host cell which produces a biologically active heterologous polypeptide is obtained.

2. The method according to claim 1, wherein at least one cryptic splice site is modified by replacing a first region comprising at least one cryptic intron or portion thereof with a second region which has a percent G+C content in the range of about 40% to about 70%.

3. The method according to claim 1, wherein at least one cryptic splice site is modified both by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence and by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%.

4. The method according to claim 1, wherein at least one cryptic splice site is modified by replacing at least one cryptic consensus sequence of at lest one cryptic splice site with a non-consensus sequence.

5. The method according to claim 4, wherein the cryptic consensus sequence is a 5' cryptic consensus sequence.

6. The method according to claim 5, wherein the 5' cryptic consensus sequence is GT, GC, or CT.

7. The method according to claim 6, wherein the 5' cryptic consensus sequence is GTANGT, GCANGT, or CTANGT, wherein N is A, C, G, or T.

8. The method according to claim 4, wherein the cryptic consensus sequence is a 3' cryptic consensus sequence.

9. The method according to claim 8, wherein the 3' cryptic consensus sequence is AG.

10. The method according to claim 8, wherein the 3' cryptic consensus sequence is CAG, TAG, or AAG.

11. The method according to claim 1, wherein the nucleic acid sequence encodes a hormone, an enzyme, a receptor, or a reporter.

12. The method according to claim 11, wherein the nucleic acid sequence encodes a reporter.

13. The method according to claim 12, wherein the reporter is an Aequorea victoria green fluorescent protein.

14. The method according to claim 1, wherein the fungal cell is a filamentous fungal cell.

15. The method according to claim 14, wherein the filamentous fungal cell is a cell of a species of Acremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

16. A method for producing a biologically active polypeptide comprising

(a) introducing into a fungal host cell a nucleic acid sequence encoding said biologically active polypeptide, wherein at least one cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about70%;

(b) cultivating the fungal host cell of step (a) in a nutrient medium; and

(c) recovering said biologically active polypeptide from the nutrient medium of step (b).

17. The method accordig to claim 16, in which said cryptic splice site is modified in the nucleic acid sequence by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence and by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%.

18. An isolated nucleic acid sequence with at least one cryptic splice site modified by replacing at least one cryptic consensus sequence of at least one cryptic splice site with a non-consensus sequence and/or by replacing a first region of a cryptic intron with a second region which has a percent G+C content in the range of about 40% to about 70%, said nucleic acid sequence encoding a biologically active polypeptide.

19. A nucleic acid construct comprising the nucleic acid sequence of claim 18.

20. A recombinant fungal host cell comprising the nucleic acid construct of claim 19.

21. A recombinant expression vector comprising the nucleic acid construct of claim 19.

22. The vector according to claim 21, wherein the nucleic acid sequence is operably linked to a promoter sequence.

23. The vector according to claim 21, wherein the nucleic acid sequence is operably linked to a transcription termination signal.

24. The vector according to claim 21, further comprising a selectable marker.

25. A recombinant fungal host cell comprising the recombinant vector of claim 21.

Details for Patent 5,770,371

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Start Trial Novo Nordisk Biotech, Inc. (Davis, CA) 2037-11-20 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Start Trial Novo Nordisk Biotech, Inc. (Davis, CA) 2037-11-20 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Start Trial Novo Nordisk Biotech, Inc. (Davis, CA) 2037-11-20 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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