LACTOCOCCUS LACTIS EXPRESSION SYSTEM FOR DELIVERING PROTEINS EFFICACIOUS FOR THE TREATMENT OF EPITHELIAL BARRIER FUNCTION DISORDERS
The disclosure relates to live biotherapeutic products, probiotics, and therapeutic composition comprising said probiotics having therapeutic proteins, and methods of using them to treat various human diseases. In particular aspects, the disclosure provides such compositions comprising strains of the Lactococcus lacus bacterium within which said therapeutic protein are present. The disclosed pharmaceutical compositions are useful for treating gastrointestinal inflammatory diseases and gastrointestinal conditions associated with decreased epithelial cell barrier function or integrity, especially, for treating or preventing various types of mucositis.
1 . A recombinant host comprising:
a first nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a signal peptide and a protein of interest;
wherein the signal peptide is N-terminal to the protein of interest;
wherein the promoter is selected from the group consisting of usp45 and thyA;
wherein the first nucleic acid is integrated into the genome of the host; and
wherein the host is a thymidylate synthase (thyA) auxotroph, a 4-hydroxy-tetrahydrodipicolinate synthase (dapA) auxotroph, or both.
2 . The host of claim 1 , wherein the host is a bacterium.
3 . The host of claim 2 , wherein the signal peptide is a usp45 signal peptide.
4 . The host of claim 2 , said host further comprising a viability enhancement.
5 . The host of claim 4 , wherein the viability enhancement comprises disruption of an endogenous gene encoding a protein involved in the catabolism or export of lactose, maltose, sucrose, trehalose, or glycine betaine.
6 . The host of claim 5 , wherein the protein involved in the catabolism of lactose, maltose, sucrose, trehalose, or glycine betaine is selected from the group consisting of a sucrose 6-phosphate, a maltose phosphorylase, a beta-galactosidase, a phospho-b-galactosidase, a trehalose 6-phosphate phosphorylase, permease IIC component, and combinations thereof.
7 .- 8 . (canceled)
9 . The host of claim 4 , wherein the viability enhancement comprises an exogenous nucleic acid encoding a protein involved in the import or production of lactose, maltose, sucrose, trehalose, or glycine betaine.
10 . The host of claim 9 , wherein the protein involved in the import of lactose, maltose, sucrose, trehalose, or glycine betaine is selected from the group consisting of a sucrose phosphotransferase, a maltose ABC-transporter permease, a maltose binding protein, a lactose phosphotransferase, a lactose permease, a glycine betaine/proline ABC transporter permease component, a trehalose-6-phosphate synthase, a trehalose-6-phosphate phosphatase and combinations thereof.
11 .- 12 . (canceled)
13 . The host of claim 2 , wherein the host is a non-pathogenic bacterium.
14 . The host of claim 13 , wherein the bacterium is a probiotic bacterium.
15 . The host of claim 14 , wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Eubacterium, Lactobacillus, Lactococcus , and Roseburia.
16 . The host of claim 15 , wherein the host is Lactococcus lactis.
17 . The host of claim 16 , wherein Lactococcus lactis is strain MG1363 or strain NZ9000.
18 . The host of claim 15 , wherein the protein of interest comprises an amino acid sequence with at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19 and/or SEQ ID NO: 34.
19 .- 22 . (canceled)
23 . The host of claim 18 , wherein the protein of interest comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:34.
24 . The host of claim 18 , wherein the protein of interest comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 19; and wherein
(i) the amino acid at position 147 of the protein of interest is valine, and/or
(ii) the amino acid at position 151 of the protein of interest is serine, and/or
(iii) the amino acid at position 84 of the protein of interest is aspartic acid, and/or
(iv) the amino acid at position 83 of the protein of interest is serine, and/or
(v) the amino acid at position 53 of the protein of interest is serine.
25 . The host of claim 18 , wherein the protein of interest comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 19; and wherein
i. the amino acid at position 147 of the protein of interest is valine and the amino acid at position 151 of the protein of interest is serine; or
ii. the amino acid at position 84 of the protein of interest is aspartic acid, the amino acid at position 147 of the protein of interest is valine, and the amino acid at position 151 of the protein of interest is serine: or
iii. the amino acid at position 83 of the protein of interest is serine, the amino acid at position 147 of the protein of interest is valine, and the amino acid at position 151 of the protein of interest is serine; or
iv. the amino acid at position 53 of the protein of interest is serine, the amino acid at position 84 of the protein of interest is aspartic acid, the amino acid at position 147 of the protein of interest is valine, and the amino acid at position 151 of the protein of interest is serine; or
v. the amino acid at position 53 of the protein of interest is serine, the amino acid at position 83 of the protein of interest is serine, the amino acid at position 147 of the protein of interest is valine, and the amino acid at position 151 of the protein of interest is serine; or
vi. the amino acid at position 147 of the protein of interest is not cysteine, the amino acid at position 151 of the protein of interest is not cysteine, the amino acid at position 83 of the protein of interest is not asparagine, and/or the amino acid at position 53 of the protein of interest is not asparagine.
26 .- 30 . (canceled)
31 . The host of claim 18 , wherein the protein of interest comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 34; and wherein
(i) the amino acid at position 76 of the protein of interest is valine, and/or
(ii) the amino acid at position 80 of the protein of interest is serine; and/or
(iii) the amino acid at position 13 of the protein of interest is aspartic acid; and/or
(iv) the amino acid at position 12 of the protein of interest is serine.
32 . The host of claim 18 , wherein the protein of interest comprises an amino acid sequence having at least about 90/a sequence identity to SEQ ID NO: 34; and wherein
i. the amino acid at position 76 of the protein of interest is valine, and the amino acid at position 80 of the protein of interest is serine; or
ii. the amino acid at position 13 of the protein of interest is aspartic acid, the amino acid at position 76 of the protein of interest is valine, and the amino acid at position 80 of the protein of interest is serine; or
iii. the amino acid at position 12 of the protein of interest is serine, the amino acid at position 76 of the protein of interest is valine, and the amino acid at position 80 of the protein of interest is serine; or
iv. the amino acid at position 76 of the protein of interest is not cysteine, the amino acid at position 80 of the protein of interest is not cysteine, and the amino acid at position 12 of the protein of interest is not asparagine.
33 .- 35 . (canceled)
36 . The host of claim 15 , wherein the protein of interest comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49.
37 .- 38 . (canceled)
39 . A method of treating a gastrointestinal epithelial cell barrier function disorder, comprising: administering to a subject in need thereof a pharmaceutical composition comprising:
i. a therapeutically effective amount of the recombinant host of claim 2 ;
ii. a pharmaceutically acceptable carrier.
40 . The method of claim 39 , wherein the composition comprises viable recombinant hosts.
41 . The method of claim 39 , wherein the composition comprises non-viable recombinant hosts.
42 . The method of claim 39 , wherein the gastrointestinal epithelial cell barrier function disorder is a disease associated with decreased gastrointestinal mucosal epithelium integrity.
43 . The method of claim 39 , wherein the disorder is selected from the group consisting of: inflammatory bowel disease, ulcerative colitis, Crohn's disease, short bowel syndrome, GI mucositis, oral mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, a metabolic disease, celiac disease, inflammatory bowel syndrome, and chemotherapy associated steatohepatitis (CASH).
44 . (canceled)
45 . The method of claim 39 , wherein the composition is formulated for oral ingestion.
46 . The method of claim 39 , wherein the composition is an edible product or the composition is formulated as a pill, a tablet, a capsule, a suppository, a liquid, or a liquid suspension.
47 .- 101 . (canceled)