Quantitative regulation of a g protein signalling pathway

A method of quantitative regulation of G protein activation in a cell, wherein the method comprises i) transforming the cell with a vector encoding a bistable opsin wherein the bistable opsin has an inactive and an active state, and transition in both directions between inactive and active states are driven by light absorption;

• and wherein the bistable opsin in the inactive state is sensitive to a first wavelength of light for transition to an active state, and the bistable opsin in the active state is sensitive to a second wavelength of light for transition to an inactive state, the first and second wavelengths being different;
• ii) expressing the bistable opsin in the transformed cell;
• iii) illuminating the transformed cell expressing the bistable opsin with light comprising the first and/or second wavelength, wherein the ratio of the two wavelengths of light regulates the amplitude of a G protein signalling cascade.

A method for quantitative regulation of the quantity of bistable opsin in an inactive and active state in a cell, wherein transition in both directions between inactive and active states are driven by light absorption;

• and wherein the bistable opsin in the inactive state is sensitive to a first wavelength of light for transition to an active state, and the bistable opsin in the active state is sensitive to a second wavelength of light for transition to an inactive state, the first and second wavelengths being different;
• wherein the method comprises illuminating the cell expressing the bistable opsin with light of a first wavelength and/or light of a second wavelength, and wherein the ratio of the two wavelengths of light regulates the amount of bistable opsin in the active state.

A method of restoring vision in a subject by restoring a visual signal transduction response in a retinal cell in a subject, the method comprising i) transforming a retinal cell in the subject with a vector encoding a bistable opsin wherein the bistable opsin has an inactive and an active state, and transition in both directions between inactive and active states are driven by light absorption;

• and wherein the bistable opsin in the inactive state is sensitive to a first wavelength of light for transition to an active state, and the bistable opsin in the active state is sensitive to a second wavelength of light for transition to an inactive state, the first and second wavelengths being different;
• ii) expressing the bistable opsin in the transformed cell;
• iii) illuminating the transformed cell expressing the bistable opsin with light of a first wavelength and/or light of a second wavelength, wherein the ratio of the two wavelengths of light regulates the amplitude of the visual signal transduction response in the cell by regulating the amplitude of G protein activation.

A method of treatment of retinal degeneration by introducing a visual signal transduction response in a retinal cell in a subject, the method comprising i) transforming a retinal cell in the subject with a vector encoding a bistable opsin wherein the bistable opsin has an inactive and an active state, and transition in both directions between inactive and active states are driven by light absorption;

• and wherein the bistable opsin in the inactive state is sensitive to a first wavelength of light for transition to an active state, and the bistable opsin in the active state is sensitive to a second wavelength of light for transition to an inactive state, the first and second wavelengths being different;
• ii) expressing the bistable opsin in the transformed cell;
• iii) illuminating the transformed cell expressing the bistable opsin with light of a first wavelength and/or light of a second wavelength, wherein the ratio of the two wavelengths of light regulates the amplitude of the visual signal transduction response in the cell by regulating the amplitude of G protein activation.

A method according to any one of the preceding claims wherein the first and second wavelength differ by at least 10 nm;

• and wherein where the light of the first and/or second wavelength is not a single wavelength, it is the wavelengths having the maximum output which differ from each other by at least 10 nm.

A method according to any one of the preceding claims wherein the light of a first wavelength is from 380-450 nm for transition from the inactive state to active state;

• and/or the light of the second wavelength is from 450-650 nm for transition from an active to inactive state.

A method according to any one of the preceding claims wherein the method comprises illuminating the cell with light of a first wavelength or light of a second wavelength for a time period from one or more milli-seconds, up to one or more hours, or during waking hours;

• preferably wherein the ratio of light of the first wavelength to light of the second wavelength may change one or more times during the time period.

A nucleic acid vector comprising a nucleic acid encoding i) a bistable opsin which has an inactive and an active state, and transition in both directions between inactive and active states are driven by light absorption;

• and wherein the bistable opsin in the inactive state is sensitive to a first wavelength of light for transition to an active state, and the bistable opsin in the active state is sensitive to a second wavelength of light for transition to an inactive state, the first and second wavelengths being different;
• and ii) a promoter which is specific for expression in an inner retinal cell.

A method, vector or kit according to any one of the preceding claims wherein the bistable opsin is a parapinopsin, for example lamprey parapinopsin, preferably encoded by the sequence of Genbank AB116380.1 or a variant or derivative thereof.

A method, vector or kit according to any one of the preceding claims wherein the sequence encoding a bistable opsin is provided in a viral vector, preferably an AAV vector.