Periodic Reporting for period 2 - NEUROPA (Non-invasive dynamic neural control by laser-based technology) Reporting period: 2021-01-01 to 2022-07-31 Summary of the context and overall objectives of the project There is an enormous human toll of brain disorders in Europe, with an estimated 83 million people affected and an estimated total cost of €798 billion. This is a great healthcare and societal challenge for the countries of the EU and indeed, the world, which will be exacerbated by an increasingly ageing population. NEUROPA will develop a non-invasive system, which modulates long-term activity in specific cortico-sub-cortical networks implicated in specific brain disorders, Huntington’s (HD) and cognitive impairment in Alzheimer’s (AD) disease in the first instance. This will be achieved by two-photon (2P) activation of novel phytochrome actuators that control the expression of genes involved in synaptic plasticity. The resulting increase in cortical and subcortical network activity will lead to the long term alleviation of dysfunction. The vision is to drive forward the state of the art in key areas and combine them to form a new phytochrome based (phytoptogenetics) technology.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: Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Overview – Significant results have been achieved in progressing towards the target of a new non-invasive means of stimulating brain circuits. Each of the constituent technologies has progressed in this period. Measurements of laser energy penetration into the mouse brain, important for safety and efficacy of the NEUROPA solution have been taken which suggest that safe non-invasive activation is feasible. Optimisation of two-photon conversion of bacteriophytochromes (photoreceptors) is nearing completion, AAV development for nasal administration is well advanced, and the mouse phantom development which helps to minimise animal experiments is also progressing well. And crucially, the project is near to finalising the test beds needed for reporting the end-of-project evaluation of the extent to which the NEUROPA system can modulate cells in the brain. Overall progress is back on target with good prospects of achieving targeted results in the extended 43 month project. • 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The NEUROPA consortium has assembled to develop a conceptually novel non-invasive theranostic approach. We will develop a non-invasive system, which modulates long-term activity in specific cortico-sub-cortical networks implicated in specific brain disorders, Huntington’s (HD) and cognitive impairment in Alzheimer’s (AD) disease in the first instance. This will be achieved by two-photon (2P) activation of novel phytochrome actuators that control the expression of genes involved in synaptic plasticity. The resulting increase in cortical and subcortical network activity will lead to the long-term alleviation of dysfunction. The vision is to drive forward the state of the art in key areas and combine them to form a new phytochrome based (phytoptogenetics) technology.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. alzheimers-disease-optogenetics.jpeg
