Protease sensitive gvpc and related gas vesicle gene clusters, expression systems, constructs, vectors, genetic circuits, cells, compositions, methods and systems for contrast-enhanced imaging
AssigneeCalifornia Institute of Technology
InventorsAnupama LAKSHMANAN, Mikhail SHAPIRO, Suchita P. NETY, Zhiyang JIN
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Abstract
Provided herein are engineered protease sensitive gas vesicles and related engineered protease genetically GvpC constructs, vectors, gas vesicles gene clusters, genetic circuits, cells, compositions, methods and systems, which in several embodiments can be used together with contrast-enhanced imaging technique, to detect and report protease activity and related biological events in an imaging target site.
Claims39
A method to provide a protease sensitive gas vesicle, the method comprising
- i) providing one or more engineered gas vesicles in which a gas is enclosed by a protein shell, the engineered gas vesicles comprising a gas vesicle, a GvpA/B protein and an engineered GvpC protein,
- the engineered GvpC protein comprising:
- multiple repeat regions within a central portion of the GvpC flanked by an N-terminal region having an N-terminus and a C-terminal region having a C-terminus, and
- at least one protease recognition site inserted within the central portion and/or attached to at least one of the N-terminus and the C-terminus of the GvpC,
- each of the one or more engineered gas vesicles exhibiting an initial collapse pressure and an initial ultrasound response up to collapse, the initial ultrasound response having a baseline nonlinearity,
- ii) contacting the one or more engineered gas vesicles with a protease capable of binding the at least one protease recognition site to allow cleavage of the protease recognition site,
- iii) following the contacting, detecting a protease induced collapse pressure and/or a protease induced ultrasound response of the one or more engineered gas vesicles;
- and
- iv) following the detecting, selecting engineered gas vesicles having a detected protease induced collapse pressure lower than the initial collapse pressure and/or a protease induced ultrasound response having a nonlinearity enhanced with respect to the baseline nonlinearity, the selecting performed to provide the protease sensitive gas vesicles.
A method for producing and screening protease sensitive gas vesicles (GVs), the method comprising:
- designing a plurality of protease sensitive GvpC;
- cloning the plurality of protease sensitive GvpC;
- producing GVs with the plurality of protease sensitive GvpC, creating GV and GvpC combinations;
- measuring the mechanical stiffness of the GVs over a range of pressures for each of the plurality of GvpCs;
- and determining which GV and GvpC combination provides a largest shift in collapse pressure based on the measuring.
A method for producing and screening protease sensitive GVs comprising:
- designing a plurality of protease sensitive GvpC;
- cloning the plurality of protease sensitive GvpC;
- producing GVs with the plurality of protease sensitive GvpC, creating GV and GvpC combinations;
- measuring the nonlinear ultrasound response over a range of pressures for each of the plurality of GvpCs;
- and determining which GV and GvpC combination provides the maximum nonlinear ultrasound imaging contrast to noise ratio before and after exposure to the protease.
Dependencies are inferred from the claim text.