Periodic Reporting for period 2 - NEUROPA (Non-invasive dynamic neural control by laser-based technology)
Período documentado: 2021-01-01 hasta 2022-07-31
1) Development of compact ultrashort pulse lasers in 1000-1700nm wavelength range for two-photon phytochrome activation and deactivation. Non-invasive phytoptogenetic brain excitation and cerebral blood flowmetry is one of the key goals of the project. Existing phytochromes are excited with visible light which can penetrate only a few hundred m into tissues, and certainly cannot go through a skull. Objectives:
2) Development and characterisation of phytochromes suitable for 2P activation. A key goal is the development of a new class of light activated proteins that will enable the control necessary to realise the vision. Because of their properties and attributes, we have chosen phytochromes and will use these to activate relevant gene expression and enzymes that may potentially alleviate symptoms in mouse models. Objectives:
3) Non-invasive gene delivery for cell-type specific expression of phytochromes. We will engineer new AAVs that can be delivered intranasally to provide access to the brain in a non-invasive manner. We will follow a previously established directed evolution method to engineer an AAV capsid with desired properties. We will then adjust the transgene cargo, and viral dose appropriate for the experimental aims. Objectives:
4) Development of non-invasive monitoring technique for brain hemodynamic changes. Increased network activity by phytochrome activation will be monitored in-vivo by through-skull observation of brain blood flow changes with diffusing-wave spectroscopy method with resolution down to 1 micron. Objectives:
5) Non-invasive recovery of mouse motor and cognitive deficits by 2P laser activation of expressed phytochromes in specific brain circuits in disease mouse models. Expression of phytochromes in specific neuronal types will be achieved by coupling the generated phytochromes with promoters specific for the chosen cells. The use of 2P stimulation will engender the stimulation with volume specificity so that only cells in the targeted region will be modulated. As a proof of concept, we will express our developed phytochromes in cortical neurons from mouse models of HD and AD to evaluate motor and cognitive recovery. Objectives:
• Website – 388 total site sessions and 317 unique visitors
• 46 different communication/dissemination outputs at events across 12 different countries
• LinkedIn site achieves 7000+ views during the period
• Highest number views for piece on leading edge development of AAVs using directed evolution
• 7 journal publications
• All Deliverables due in the Period submitted.
Specific networks in the brain are comprised of cortical and subcortical loops. We aim to target the cortical component of loops which is accessible through 2P absorption and use this to activate and in turn deactivate phytochromes. Long-term modulation will be achieved by modifying gene expression which will result in the long-term desired change in loop network activity. The non-invasive delivery and stimulation of Phytochromes to modify gene expression and so long-term activity in dysfunctional cortico-subcortical networks surpasses current technological paradigms. Achieving our vision requires, and will achieve, major advances in the technologies and biotechnologies of phytochromes, AAV- mediated gene delivery, laser systems and DWS for non-invasive monitoring of brain activity. We will develop a new class of phytochrome actuators that will be activated by pulsed laser wavelengths and selectively control gene expression for long term neuronal network modulation. The bi-stable nature of phytochromes will enable control of the degree and duration of activation.
To target specific cortical cell types, we will develop AAV based delivery via non-invasive injection routes. We will first deliver engineered AAVs encoding phytochromes via intravenous infusion to obtain wide-spread phytochrome expression in the brain. In the perspective of human application, we will also design a new set of AAVs to target the cortex via intranasal instillation. We will develop an ultra-short pulse system utilising advanced compact lasers in the near-infrared (NIR) spectral range specifically for brain activation. The skull is semi-transparent in the wavelength range of 1000-1700nm therefore, we aim to be able to excite phytochromes non-invasively in situ. Changes in local cerebral blood flow induced by the resultant neuronal activity will be monitored non- invasively using NIR DWS technique which will provide a measure of the phytochrome stimulating effect and enable control of brain activity.