US 12,359,210 B2
Genetically modified bacteria stably expressing IL-10 and insulin
Pieter Rottiers, De Pinte (BE); and Lothar Steidler, Lokeren (BE)
Assigned to INTREXON ACTOBIOTICS N.V., Zwijnaarde (BE)
Filed by INTREXON ACTOBIOTICS NV, Zwijnaarde (BE)
Filed on Nov. 29, 2022, as Appl. No. 18/059,689.
Application 17/092,624 is a division of application No. 16/329,321, granted, now 10,858,663, issued on Dec. 8, 2020, previously published as PCT/IB2017/055287, filed on Sep. 2, 2017.
Application 18/059,689 is a continuation of application No. 17/092,624, filed on Nov. 9, 2020, granted, now 11,549,118.
Claims priority of provisional application 62/383,079, filed on Sep. 2, 2016.
Prior Publication US 2023/0348918 A1, Nov. 2, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/74 (2006.01); A61K 35/744 (2015.01); A61K 39/39 (2006.01); A61K 39/395 (2006.01); A61P 3/10 (2006.01); C07K 14/54 (2006.01); C07K 14/62 (2006.01); C07K 16/28 (2006.01)
CPC C12N 15/746 (2013.01) [A61K 35/744 (2013.01); A61K 39/3955 (2013.01); A61P 3/10 (2018.01); C07K 14/5428 (2013.01); C07K 14/62 (2013.01); C07K 16/2809 (2013.01); C12N 15/74 (2013.01)] 14 Claims
 
1. A recombinant Lactococcus lactis comprising two chromosomally integrated exogenous nucleic acids,
the first exogenous nucleic acid encoding a human interleukin-10 (hIL-10) comprising (i) an amino acid sequence at least 95% identical to SEQ ID NO: 1 and (ii) a proline (Pro) to alanine (Ala) substitution at position 2 of a mature hIL-10, and
the second exogenous nucleic acid encoding a human proinsulin (hPINS) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 3;
wherein the recombinant L. lactis (i) constitutively expresses and secretes the hIL-10 and the hPINS, (ii) expresses an OtsB from Escherichia coli, (iii) constitutively overexpresses a gene encoding a trehalose transporter encoded by an ptsI gene and an ptsII gene, (iv) lacks trehalose-6-phosphate phosphorylase (TrePP) activity; and (v) lacks cellobiose-specific PTS system IIC component (PtcC) activity; and
wherein the recombinant L. lactis comprises:
a) an expression cassette chromosomally integrated at the thyA locus, wherein said expression cassette comprises an hl1A promoter (PhllA) that transcribes a nucleic acid sequence encoding a Usp45 secretion leader (SSusp45) and said first exogenous nucleic acid, and wherein said hIL-10 is expressed as a fusion protein comprising said SSusp45;
b) a first chromosomally integrated polycistronic expression cassette comprising, in 5′ to 3′ order, a gapB promoter, a gapB gene, a nucleic acid sequence encoding a Usp45 secretion leader, and said second exogenous nucleic acid, wherein said hPINS is expressed as a fusion protein comprising said SSusp45;
c) a second chromosomally integrated polycistronic expression cassette comprising, in 5′ to 3′ order, a usp45 promoter, a usp45 gene, an intergenic region that is immediately 5′ to an rpmD gene, and a third exogenous nucleic acid encoding the OtsB from Escherichia coli;
d) a third polycistronic expression cassette comprising, in 5′ to 3′ order, an hllA promoter (PhllA) and a nucleic acid comprising the ptsI gene and the ptsII gene;
e) an inactivated endogenous trePP gene, wherein said trePP gene is inactivated by gene deletion; and
f) an inactivated endogenous ptcC gene, wherein said ptcC gene is inactivated by insertion of a premature stop codon.