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US 12,721,882 B2Granted
Enumeration of genetically engineered microorganisms by live cell counting techniques
AssigneeSynlogic Operating Company, Inc.
InventorEugene Antipov
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Abstract
Genetically engineered microorganisms, e.g., genetically engineered bacteria, compositions and formulations thereof, as well as methods for characterizing, dosing, and determining the activity of the bacteria, compositions, and formulations, e.g., using a live cell counting method are disclosed.
Claims12
A method for selecting a pharmaceutical composition for reducing hyperphenylalaninemia, comprising a genetically engineered bacteria comprising one or more gene(s) for producing a phenylalanine ammonia lyase (PAL), wherein the method comprises:
- determining the live cell count of the genetically engineered bacteria in a first composition, wherein the live cell count provides a more accurate measure of therapeutic activity than colony-forming units (CFU) by determining a percent of live cells in the first composition, wherein the percent of live cells is the number of living cells divided by the total number of cells multiplied by 100;
- wherein when the first composition comprises between 1×108 to 1×1013 live cells as determined by live cell counting, and wherein when the percent live cells in the first composition is at least 60% of the total number of cells, selecting the first composition as a second composition;
- determining the activity of the second composition by measuring:
- (1) a transcinnamic acid (TCA) production rate of the second composition, and selecting the second composition as the pharmaceutical composition wherein it is capable of producing transcinnamic acid (TCA) at a rate of at least approximately 0.5 μmol/hour/109 live cells,
- (2) a phenyl pyruvic acid (PPA) production rate of the second composition, and selecting the second composition as the pharmaceutical composition wherein it is capable of producing PPA at a rate of at least approximately 1.0 μmol/hour/109 live cells, and/or
- (3) an increased hippurate (HA) or labeled D5-HA of the second composition relative to a control, wherein the control comprises the unmodified bacterium, and selecting the second composition as the pharmaceutical composition wherein it demonstrates an increased HA or labeled D5-HA level relative to the control;
- and
- administering the selected pharmaceutical composition to a subject in need thereof for reducing hyperphenylalaninemia.
5 more elements
Dependencies are inferred from the claim text.