COMPOSITIONS AND METHODS FOR THE DELIVERY OF THERAPEUTIC PEPTIDES

• US 20130259834A1
• Filed: 05/19/2011
• Published: 10/03/2013
• Est. Priority Date: 05/19/2010
• Status: Active Grant

• Current Assignee: University of Virginia Patent Foundation (University of Virginia)
• Original Assignee: North Carolina State University (University of North Carolina System), University of Virginia Patent Foundation (University of Virginia)
• Inventors: Klaenhammer, Todd R., Guerrant, Richard L., Durmaz, Evelyn, Kolling, Glynis L., Timko, Michael P., Warren, Cirle Alcantara
• Granted Patent: US 9,040,302 B2
• Time in Patent Office: Days
• US Class Current: 424/93.2
• CPC Class Codes: A23C 9/1238 using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
• CPC Class Codes: A23L 33/135 Bacteria or derivatives thereof, e.g. probiotics
• CPC Class Codes: A61K 2035/11 Medicinal preparations comprising living procariotic cells
• CPC Class Codes: A61K 35/74 Bacteria therapeutic use of a bacterial protein A61K38/00
• CPC Class Codes: A61K 38/164 from bacteria
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Methods and compositions for targeted delivery of biotherapeutics are provided. The compositions comprise bile-sensitive St. thermophilus bacteria modified to release a biotherapeutic agent following bile exposure. Biotherapeutic agents released by the St. thermophilus bacteria disclosed herein include AQ and AQR rich peptides. Methods of the invention comprise administering to a subject a St. thermophilus bacterium modified to release a biotherapeutic agent following bile exposure. Administration of the St. thermophilus bacterium promotes a desired therapeutic response. The bacterium may be modified to express and release AQ or AQR rich peptides which subsequently inhibit cellular apoptosis or reduce mucosal damage. Thus, methods of the invention find use in treating or preventing a variety of gastrointestinal disorders including C. difficile infection and antibiotic-associated diarrhea.

Claims

1. A Streptococcus thermophilus bacterium that is modified to express a heterologous polypeptide, wherein said heterologous polypeptide is released from said bacterium following exposure to bile.

2. The bacterium of claim 1, wherein said heterologous polypeptide is a therapeutic polypeptide.

3. The bacterium of claim 1, wherein said heterologous polypeptide is an Alanine-Glutamine rich peptide.

4. The bacterium of claim 3, wherein said Alanine-glutamine rich peptide is selected from the group consisting of

a) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

2 or 8;

b) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO;

4 or 10;

c) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO;

6 or 12;

d) a peptide encoded by a polynucleotide comprising a nucleotide sequence as set forth in any SEQ ID NOs;

1, 3, 5, 7, 9, or 11; and

e) a peptide encoded by a polynucleotide comprising a nucleotide sequence having at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to at least one of SEQ ID NOs;

1, 3, 5, 7, 9, or 11; and

f) a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 2, 4, 6. 8, 10, or 12.

6. The bacterium of claim 1, wherein said bacterium does not survive said bile exposure.

7. The bacterium of claim 1, wherein said Streptococcus thermophilus is selected from the group consisting of:

a) Streptococcus thermophilus LMD-9, having been deposited under ATCC Accession Number PTA-11888;

b) Streptococcus thermophilus NCK2071, having been deposited under ATCC Accession Number PTA-11889;

c) Streptococcus thermophilus NCK2072, having been deposited under ATCC Accession Number PTA-11890;

d) Streptococcus thermophilus NCK2073, having been deposited under ATCC Accession Number PTA-11891; and

e) a probiotic Streptococcus thermophilus bacterium.

14. A method of delivering a heterologous polypeptide to a subject, said method comprising orally administering to a subject the bacterium according to claim 1.

15. A method of treating or preventing a disorder in a subject, said method comprising orally administering to a subject a therapeutically effective amount of the bacterium according to claim 1.

16. The method of claim 14, wherein said subject is an animal.

17. The method of claim 16, wherein said subject is a mammal.

21. The method of claim 15, wherein said disorder is a gastrointestinal disorder.

22. The method of claim 15, wherein said disorder is a bacterial infection.

23. The method of claim 22, wherein said bacterial infection is a Clostridium difficile infection.

25. A pharmaceutical composition comprising the bacterium according to claim 1.

26. A food or feed product comprising the bacterium according to claim 1.

5. (canceled)

8. A method of making a Streptococcus thermophilus bacterium, said method comprising genetically modifying said bacterium to express a heterologous polypeptide, wherein said polypeptide is released from said bacterium following exposure to bile.

10. The method of claim 8, wherein said heterologous polypeptide is selected from the group consisting of:

a) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO;

2 or 8;

b) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO;

4 or 10;

c) a peptide comprising the amino acid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO;

6 or 12;

d) a peptide encoded by a polynucleotide comprising a nucleotide sequence as set forth in any SEQ ID NOs;

1, 3, 5, 7, 9, or 11; and

e) a peptide encoded by a polynucleotide comprising a nucleotide sequence having at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to at least one of SEQ ID NOs;

1, 3, 5, 7, 9, or 11; and

f) a peptide comprising an amino acid sequence selected from the group consisting of;

SEQ. ID NO;

2, 4, 6, 8, 10,or 12.

9. (canceled)

11-13. -13. (canceled)

18-20. -20. (canceled)

24. (canceled)

27-37. -37. (canceled)

38. A method of decreasing inflammation in a subject comprising administering to a subject a therapeutically effective amount of an isolated bile-sensitive Streptococcus thermophilus bacterium.

39. The method of claim 38, wherein said subject has an inflammatory gastrointestinal disorder.

40. The method of claim 39, wherein said inflammatory gastrointestinal disorder is a Clostridium difficile infection.

41-43. -43. (canceled)