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Bacteria engineered to treat diseases associated with hyperammonemia

A bacterial, genetically engineered technology, applied in the field of genetically engineered bacteria, capable of solving problems such as effective, reliable, and/or long-term unmet treatment

Active Publication Date: 2021-08-13
SYNLOGIC OPERATING CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, there is a significant unmet need for effective, reliable and / or long-term treatments for diseases associated with hyperammonemia, including urea cycle disorders

Method used

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  • Bacteria engineered to treat diseases associated with hyperammonemia
  • Bacteria engineered to treat diseases associated with hyperammonemia
  • Bacteria engineered to treat diseases associated with hyperammonemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0495] Example 1. ARG box mutations

[0496] The wild-type genome sequence containing the ArgR binding site for each arginine biosynthesis operon in E. coli Nissle is shown in Figure 6 middle. Modifications to these sequences were designed according to the following parameters. For each wild-type sequence, the ARG box is shown in italics. The ARG box of the arginine regulon overlaps with the promoter region of each operon. quilt underlined Sequences represent RNA polymerase binding sites, and those sequences were not altered. Bases that are protected from DNA methylation during ArgR binding are highlighted, and bases that are protected from hydroxyl radical attack during ArgR binding are bolded. Highlighted and bolded bases are the primary targets of mutations for disrupting ArgR binding.

Embodiment 2

[0497] Embodiment 2.λred recombination

[0498] λred recombination is used to make chromosomal modifications such as ARG box mutations. λred is a program that uses the recombinase from bacteriophage λ to insert a piece of custom DNA into the E. coli chromosome. The pKD46 plasmid was transformed into E. coli Nissle host strain. E. coli Nissle cells were grown overnight in LB medium. Dilute the overnight culture 1:100 in 5 mL LB media and grow until it reaches OD 600 0.4-0.6. Pre-cool all tubes, solutions and cuvettes to 4°C. The E. coli cells were centrifuged at 2,000 rpm for 5 min at 4°C, the supernatant was removed, and the cells were resuspended in 1 mL of 4°C water. Escherichia coli was centrifuged at 2,000 rpm at 4°C for 5 min, the supernatant was removed, and the cells were resuspended in 0.5 mL of 4°C water. Escherichia coli was centrifuged at 2,000 rpm at 4°C for 5 min, the supernatant was removed, and the cells were resuspended in 0.1 mL of 4°C water. The electr...

Embodiment 3

[0504] Example 3. Transformation of Escherichia coli Nissle

[0505] The mutated ARG cassette construct was transformed into E. coli Nissle containing pKD46. Pre-cool all tubes, solutions and cuvettes to 4°C. Dilute the overnight culture 1:100 in 5 mL of LB medium containing ampicillin and grow until it reaches OD 600 is 0.1. Add 0.05 mL of 100X L-arabinose stock solution to induce pKD46 λred expression. Grow the culture until it reaches OD 600 0.4-0.6. The E. coli cells were centrifuged at 2,000 rpm for 5 min at 4°C, the supernatant was removed, and the cells were resuspended in 1 mL of 4°C water. Escherichia coli was centrifuged at 2,000 rpm at 4°C for 5 min, the supernatant was removed, and the cells were resuspended in 0.5 mL of 4°C water. Escherichia coli was centrifuged at 2,000 rpm at 4°C for 5 min, the supernatant was removed, and the cells were resuspended in 0.1 mL of 4°C water. Set the electroporator to 2.5 kV. 0.5 μg of the mutated ARG cassette construct wa...

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PUM

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Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with hyperammonemia are disclosed.

Description

[0001] This application claims the benefit of: U.S. Provisional Application No. 62 / 087,854, filed December 5, 2014; U.S. Provisional Application No. 62 / 173,706, filed June 10, 2015; U.S. Provisional Application No. 62 / 173,706, filed November 16, 2015 No. 62 / 256,041; U.S. Provisional Application No. 62 / 103,513, filed January 14, 2015; U.S. Provisional Application No. 62 / 150,508, filed April 21, 2015; U.S. Provisional Application No. 62, filed June 10, 2015 / 173,710; U.S. Provisional Application No. 62 / 256,039, filed November 16, 2015; U.S. Provisional Application No. 62 / 184,811, filed June 25, 2015; U.S. Provisional Application No. 62 / 183,935, filed June 24, 2015 and U.S. Provisional Application No. 62 / 263,329, filed December 4, 2015, the entire contents of which are incorporated herein by reference to provide continuity of disclosure. technical field [0002] The present disclosure relates to compositions and methods of treatment for reducing excess ammonia and converting ammo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21A61K35/745A61K35/747A61K35/744A61K35/741A61P1/16C12P13/10
CPCA61K35/741A61K35/744A61K35/745A61K35/747C12N9/1029C12N15/52C12N15/70C12P13/10C07K14/245C12R2001/19C12N1/205C12N9/1025C12Y203/01001C12R2001/01
Inventor 迪安·法尔勃文森特·M·伊莎贝拉乔纳森·W·科图拉保罗·F·米勒
Owner SYNLOGIC OPERATING CO INC
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