Genetically modified probiotics for oral delivery of renin-angiotensin related therapeutic proteins and peptides

Patent metadata

Field	Value
Publication number	US-11945853-B2
Application number	US-202217839815-A
Country	US
Kind code	B2
Filing date	Jun 14, 2022
Priority date	Sep 13, 2016
Publication date	Apr 2, 2024
Grant date	Apr 2, 2024

Abstract

Provided herein are polynucleic acids and expression vectors for the expression and secretion of angiotensin peptide fragments (e.g., angiotensin-(1-7)) in probiotic bacteria. Provided herein are also probiotic compositions that enable efficient, cost-effective and patient friendly oral therapeutics for treating diverse pathological conditions that involve the renin-angiotensin system (RAS), e.g., pulmonary hypertension, diabetes, diabetic complications, cardiovascular diseases, and ocular inflammatory and neurodegenerative diseases.

First claim

What is claimed is:

1. An expression vector comprising a polynucleic acid for expressing ACE2 in a bacterium, the polynucleic acid comprising:
a promoter,
a nucleic acid encoding a secretion signal peptide,
a nucleic acid encoding a carrier protein, wherein the nucleic acid encoding the carrier protein has:
(i) a polynucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 3 or
(ii) a polynucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 18,

a nucleic acid encoding ACE2, and;
wherein the nucleic acids encoding the secretion signal peptide, carrier protein and ACE2 encode a fusion protein and are operably linked to the promoter.

2. The expression vector of claim 1 , further comprising a nucleic acid encoding a cleavage site that lies in between the nucleic acids encoding the carrier protein and ACE2.

3. The expression vector of claim 1 , further comprising a nucleic acid encoding a hinge that lies 3′ to the nucleic acid encoding the carrier protein.

4. The expression vector of claim 1 , wherein the promoter is a lactose dehydrogenase (ldh) promoter from Lactococcus lactis.

5. The expression vector of claim 4 , wherein sequence of the ldh promoter is at least 95% identical to SEQ ID NO: 1.

6. The expression vector of claim 5 , wherein sequence of the ldh promoter is SEQ ID NO: 1.

7. The expression vector of claim 1 , wherein the secretion signal peptide is from the usp45 gene of Lactococcus lactis.

8. The expression vector of claim 7 , wherein sequence of the nucleic acid encoding the secretion signal peptide from usp45 gene of Lactococcus lactis is at least 95% identical to SEQ ID NO: 2.

9. The expression vector of claim 8 , wherein sequence of the nucleic acid encoding the secretion signal peptide from usp45 gene of Lactococcus lacti is SEQ ID NO: 2.

10. The expression vector of claim 1 , wherein the nucleic acid encoding the secretion signal peptide lies adjacent and 5′ to the nucleic acid encoding the carrier protein.

11. The expression vector of claim 1 , wherein the carrier protein is cholera toxin B (CTB), and wherein the sequence of the nucleic acid encoding CTB is at least 95% identical to SEQ ID NO: 3.

12. The expression vector of claim 1 , wherein the sequence of the nucleic acid encoding CTB is SEQ ID NO: 3.

13. The expression vector of claim 1 , wherein the carrier protein is a cell-penetrating peptide (CPP) derived from Pancreatic And Duodenal Homeobox I (PDX-1), and wherein the nucleic acid encoding the CPP is as set forth in SEQ ID NO: 18.

14. The expression vector of claim 1 , wherein the sequence of the nucleic acid encoding ACE2 is at least 95% identical to SEQ ID NO: 7.

15. The expression vector of claim 1 , wherein the sequence of the nucleic acid encoding ACE2 is SEQ ID NO: 7.

16. The expression vector of claim 2 , wherein the cleavage site is a furin cleavage site.

17. A genetically engineered bacterium comprising the expression vector of claim 1 .

18. The genetically engineered bacterium of claim 17 , wherein the bacterium is a Lactobacillus.

19. The genetically engineered bacterium of claim 18 , wherein the Lactobacillus is L. paracasei, L. plantarum or L. gasseri.

20. A probiotic composition comprising a plurality of the genetically engineered bacterium of claim 17 , wherein the bacteria are concentrated and live, frozen, in lyophilized powder form or lyophilized tablet form.

21. A method of treating a disease or condition involving the renin-angiotensin system (RAS), the method comprising administering orally to a subject in need thereof a therapeutically effective dose of the probiotic composition of claim 20 .

Assignees

• Univ Florida

Inventors

• Li Qiuhong
• Raizada Mohan

Classifications

• C07K14/575 Primary

  Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695) · CPC title

• A61K35/74

  Bacteria (therapeutic use of a bacterial protein A61K38/00) · CPC title

• A61K35/741

  Probiotics (probiotic yeast, e.g. saccharomyces A61K36/06) · CPC title

• A61K35/747

  Lactobacilli, e.g. L. acidophilus or L. brevis · CPC title

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title