Periodic Reporting for period 3 - NEUROPA (Non-invasive dynamic neural control by laser-based technology)
Reporting period: 2022-08-01 to 2023-07-31
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 set out to develop a non-invasive system, which modulates long-term activity in specific cortico-sub-cortical networks implicated in specific brain disorders, with Huntington’s (HD) and cognitive impairment in Alzheimer’s (AD) disease the targets in the first instance. The means of achieving the main aim is by two-photon (2P) activation of novel phytochrome actuators that control the expression of genes involved in synaptic plasticity. Existing phytochromes are excited with visible light which can penetrate only a few hundred microns into tissues, and certainly cannot go through a skull. With activation of new phytochromes, the resulting increase in cortical and subcortical network activity will lead to the long term alleviation of dysfunction. The main objectives of the project were as follows:
1) Development of compact ultrashort pulse lasers in 1000-1700nm wavelength range for two-photon phytochrome activation and deactivation.
2) Development of phytochromes suitable for 2-photon (non-linear) activation.
3) Engineering new Adeno-Associated Viruses (AAVs) that can be delivered intranasally to provide access to the brain in a non-invasive manner.
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.
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.
NEUROPA has partially achieved the objectives for the project. Significant progress has been made in key areas including phytochrome development, and AAVs to deliver the phytochromes to the brain. And crucially, the monitoring of changes in brain plasticity were observed in a mouse model following non-invasive activation with a laser. Specifically NEUROPA has demonstrated that activation of phytochromes in the M2 cortex of a mouse model can result in astrocytic modulation of synaptic plasticity and improvements in the motor learning of Huntington's Disease (HD) mice using single-photon laser activation. Challenges that are still to be overcome include being able to demonstrate non-linear activation which is important for deeper penetration which enables the ability to modulate brain circuits in a wide range of neurological disorders. We fully expect to see further public and private investment in NEUROPA technologies in the future.
1) Development of compact ultrashort pulse lasers in 1000-1700nm wavelength range for two-photon phytochrome activation and deactivation.
2) Development of phytochromes suitable for 2-photon (non-linear) activation.
3) Engineering new Adeno-Associated Viruses (AAVs) that can be delivered intranasally to provide access to the brain in a non-invasive manner.
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.
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.
NEUROPA has partially achieved the objectives for the project. Significant progress has been made in key areas including phytochrome development, and AAVs to deliver the phytochromes to the brain. And crucially, the monitoring of changes in brain plasticity were observed in a mouse model following non-invasive activation with a laser. Specifically NEUROPA has demonstrated that activation of phytochromes in the M2 cortex of a mouse model can result in astrocytic modulation of synaptic plasticity and improvements in the motor learning of Huntington's Disease (HD) mice using single-photon laser activation. Challenges that are still to be overcome include being able to demonstrate non-linear activation which is important for deeper penetration which enables the ability to modulate brain circuits in a wide range of neurological disorders. We fully expect to see further public and private investment in NEUROPA technologies in the future.
Proof of Concept (POC) has been achieved in key constituent technologies of NEUROPA. The efficacy of the Phytochromes as effective photoreceptors capable of activation and deactivation in brain tissue has been demonstrated. This result also demonstrated that the use of Adeno Associated Virus (AAV) as a vector to transport Phytochromes to the brain, and together the system has been capitalised upon successfully in demonstrating phytochrome effects on synaptic plasticity in the mouse brain. As a consequence, there is now a foundation for further development efforts to expand the validation of the optogenetic platform created by NEUROPA partners. In addition, a POC has been achieved in the development of the Mouse Phantom to effectively mimic the small vessel structure of the mouse brain thereby reducing the number of animal models required . The Diffusing Wave Spectroscopy (DWS) system has also achieved a POC as a means of non-invasively monitoring changes in brain hemodynamics, which coupled with the ability to modulate circuits in the brain means a feedback system of initiation, modulation, and observation of changes has been established. At the conclusion of NEUROPA, two areas have not got the results they desired. The non-linear laser has not yet achieved a POC in demonstrating non-invasive activation of Phytochromes in-vivo. Further experiments are planned which it is hoped will achieve the targeted outcomes in the coming months. Similarly the Directed Evolution efforts to develop an Adeno-associated virus (AAV) for intranasal delivery of the AAV-Phytochrome constructs has not yet concluded, although again it is hoped that the coming months will produce the required result.
Dissemination activity has produced 19 Journal publications in total, 4 publications in Conference Proceedings, and a batch of further Journal publications in preparation or submitted. Market research data has been collected in support of the Key Exploitable Results (KERs). Routes to market have been investigated and user information collected through the networks of industrial contacts held by consortium members. Pathways to eventual Regulatory compliance have been investigated and challenges identified. A methodology for the calculation of Return on Investment (ROI) has been devised and used to estimate the ROI of each KER. A patent application has been submitted to the UK patent office for the Mouse Phantom.
Dissemination activity has produced 19 Journal publications in total, 4 publications in Conference Proceedings, and a batch of further Journal publications in preparation or submitted. Market research data has been collected in support of the Key Exploitable Results (KERs). Routes to market have been investigated and user information collected through the networks of industrial contacts held by consortium members. Pathways to eventual Regulatory compliance have been investigated and challenges identified. A methodology for the calculation of Return on Investment (ROI) has been devised and used to estimate the ROI of each KER. A patent application has been submitted to the UK patent office for the Mouse Phantom.
The NEUROPA consortium was assembled to develop a conceptually novel non-invasive theranostic approach. 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.
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.