Periodic Reporting for period 3 - PRIME (A Personalised Living Cell Synthetic Computing Circuit for Sensing and Treating Neurodegenerative Disorders)
Période du rapport: 2023-08-01 au 2025-07-31
There remain urgent, unmet needs for the treatment of neurological diseases. Epilepsy is a serious, chronic brain disease characterized by recurrent seizures - one of the most common, serious neurological conditions. PRIME aimed to develop an innovative diagnostic-therapeutic treatment by implanting engineered biological cells in the brain that will act as biocomputers to treat + suppress impending seizures. The use of engineered biological cells in the revolutionary technology means the implants to be developed by PRIME can also be personalised, using artificial intelligence, to accommodate the different types + levels of seizures experienced by individual epilepsy patients.
Obj1: Developing molecular communication simulation + modeling design tool.
Obj2: Engineering cells to sense, perform logic computing + release GDNF.
Obj3: Developing an encapsulated implantable device that integrates three-dimensional (3D) constructs of the cells from Objective 2 grown in hybrid biomaterial scaffolds.
Obj4: Experimental testing + validation of device in vivo.
During the 54-month period, we made significant progress relative to our objectives. Each WP has undertaken its tasks without significant variation from the Description of Work. Despite COVID-19, the project completed experimental activities. Given the multidisciplinary nature of the project, strong cross-collaboration emerged that were vital to driving it forward + delivering objectives. This is evident through a range of smaller synergetic subexperiments + studies that naturally emerged between the partners. An ambitious plan for dissemination + communication activities was executed.
Obj1: Developing molecular communication simulation + modeling design tool.
Obj2: Engineering cells to sense, perform logic computing + release GDNF.
Obj3: Developing an encapsulated implantable device that integrates three-dimensional (3D) constructs of the cells from Objective 2 grown in hybrid biomaterial scaffolds.
Obj4: Experimental testing + validation of device in vivo.
During the 54-month period, we made significant progress relative to our objectives. Each WP has undertaken its tasks without significant variation from the Description of Work. Despite COVID-19, the project completed experimental activities. Given the multidisciplinary nature of the project, strong cross-collaboration emerged that were vital to driving it forward + delivering objectives. This is evident through a range of smaller synergetic subexperiments + studies that naturally emerged between the partners. An ambitious plan for dissemination + communication activities was executed.
WP1: We obtained a 6 month extension + ensured deliverables + milestones were achieved in coherence with the updated timelines, extending the GANTT span from 48 to 54 months.
WP2: We submitted a major review on modeling microRNA regulation of the TGF-β/SMAD signaling pathways for seizure control in temporal lobe epilepsy, had one research article on microRNA-based modeling of TGF-β/SMAD signaling for seizure control in temporal lobe epilepsy accepted for publication, published a paper in IEEE Transactions on Molecular, Biological, and Multi-Scale Communications focusing on molecular communication frameworks for brain signal analysis + have 2 additional manuscripts in progress on molecular communication models + loss budget estimation for tsRNA detection in the brain. We integrated AGO2-immunoprecipitated small RNA sequencing with matched transcriptomic and proteomic datasets across seven stages of epileptogenesis in the perforant pathway stimulation (PPS) rat model of temporal lobe epilepsy. This work provides the first integrative multi-omics view of tsRNA activity in epilepsy and highlights novel RNA-mediated mechanisms with potential relevance for disease progression.
WP3: We determined the expression and secretion of GDNF with both a constitutive and a regulated pathway in human ARPE-19 cells and rat rMC-1 cells and also analysed the contribution of intracellular Ca2+ in promoting GDNF secretion. We achieved high expression and secretion of GDNF in engineered ARPE-19 cells that overexpress GDNF alone, and the group also produced lentiviral vectors that will be used to generate cells over-expressing both P2X7 (the ATP-gated purinergic P2X7 receptor) and GDNF (according to the fail-safe strategy). We have established assays for the detection of extracellular 5’-tRFs by quantitative PCR and extracellular GDNF by ELISA, enabling the testing of the engineered cells. 5’tRFs were also quantified in the CSF (cerebrospinal fluid) and serum of epileptic rats, in relation to seizure occurrence and inter-ictal activity.
WP4: We further optimized hybrid scaffolds of collagen hydrogels and laminin-functionalized electrospun polymer nanorods as a biomimetic habitant for engineered ARPE-19 cells mimicking the brain extracellular microenvironment with the implementation of finite element simulations + further introduced innovative design approaches for nano-or micro-fiber tubes via coaxial electrospinning and via additive manufacturing. We successfully developed a sealed porous encapsulating membrane using commercially available polyethersulfone (PES) tubes.We studied the permeability of the PES tubes to naked mRNA, lipid nanoparticle-encapsulated mRNA, exosomes, and GDNF using ELISA. The results confirmed effective bidirectional transport of these biomolecules across the device membrane.
WP5: We measured the release of GDNF from the devices into the local brain environment and studied the effects of this on animal behaviours. We also tested the effects of encapsulated cell devices that release GDNF on seizures in the RCSI mouse model of epilepsy. We completed an extensive gene expression profiling of the RNA landscape in the tissue surrounding the explanted encapsulated device. The findings represent a comprehensive characterisation of the fuctionality of the encapsulated device + the impact of GDNF on brain excitability + the underlying gene expression programmes.
WP6: Dissemination - we successfully promoted the project via visible online platforms including an active website + social media channels. The website shows public deliverables, publications, news + events, reports of communication activities, results, opinions; operating as the supporting pillar of the project’s online image. It was updated during the project’s lifetime providing a permanent reference for PRIME work. D6.4 provides collective data of the impact metrics + statistical figures for key milestones across the project. Exploitation - despite the low TRL of the project a number of exploitable deliverables have already been identified + subjected to a long-term Technology Transfer Strategy. In addition to the IPR Management + Patent Filing in D6.4 we also out a Sustainability Plan.
WP2: We submitted a major review on modeling microRNA regulation of the TGF-β/SMAD signaling pathways for seizure control in temporal lobe epilepsy, had one research article on microRNA-based modeling of TGF-β/SMAD signaling for seizure control in temporal lobe epilepsy accepted for publication, published a paper in IEEE Transactions on Molecular, Biological, and Multi-Scale Communications focusing on molecular communication frameworks for brain signal analysis + have 2 additional manuscripts in progress on molecular communication models + loss budget estimation for tsRNA detection in the brain. We integrated AGO2-immunoprecipitated small RNA sequencing with matched transcriptomic and proteomic datasets across seven stages of epileptogenesis in the perforant pathway stimulation (PPS) rat model of temporal lobe epilepsy. This work provides the first integrative multi-omics view of tsRNA activity in epilepsy and highlights novel RNA-mediated mechanisms with potential relevance for disease progression.
WP3: We determined the expression and secretion of GDNF with both a constitutive and a regulated pathway in human ARPE-19 cells and rat rMC-1 cells and also analysed the contribution of intracellular Ca2+ in promoting GDNF secretion. We achieved high expression and secretion of GDNF in engineered ARPE-19 cells that overexpress GDNF alone, and the group also produced lentiviral vectors that will be used to generate cells over-expressing both P2X7 (the ATP-gated purinergic P2X7 receptor) and GDNF (according to the fail-safe strategy). We have established assays for the detection of extracellular 5’-tRFs by quantitative PCR and extracellular GDNF by ELISA, enabling the testing of the engineered cells. 5’tRFs were also quantified in the CSF (cerebrospinal fluid) and serum of epileptic rats, in relation to seizure occurrence and inter-ictal activity.
WP4: We further optimized hybrid scaffolds of collagen hydrogels and laminin-functionalized electrospun polymer nanorods as a biomimetic habitant for engineered ARPE-19 cells mimicking the brain extracellular microenvironment with the implementation of finite element simulations + further introduced innovative design approaches for nano-or micro-fiber tubes via coaxial electrospinning and via additive manufacturing. We successfully developed a sealed porous encapsulating membrane using commercially available polyethersulfone (PES) tubes.We studied the permeability of the PES tubes to naked mRNA, lipid nanoparticle-encapsulated mRNA, exosomes, and GDNF using ELISA. The results confirmed effective bidirectional transport of these biomolecules across the device membrane.
WP5: We measured the release of GDNF from the devices into the local brain environment and studied the effects of this on animal behaviours. We also tested the effects of encapsulated cell devices that release GDNF on seizures in the RCSI mouse model of epilepsy. We completed an extensive gene expression profiling of the RNA landscape in the tissue surrounding the explanted encapsulated device. The findings represent a comprehensive characterisation of the fuctionality of the encapsulated device + the impact of GDNF on brain excitability + the underlying gene expression programmes.
WP6: Dissemination - we successfully promoted the project via visible online platforms including an active website + social media channels. The website shows public deliverables, publications, news + events, reports of communication activities, results, opinions; operating as the supporting pillar of the project’s online image. It was updated during the project’s lifetime providing a permanent reference for PRIME work. D6.4 provides collective data of the impact metrics + statistical figures for key milestones across the project. Exploitation - despite the low TRL of the project a number of exploitable deliverables have already been identified + subjected to a long-term Technology Transfer Strategy. In addition to the IPR Management + Patent Filing in D6.4 we also out a Sustainability Plan.
Significant progress has been achieved, with key developments spanning molecular communication simulation, cell engineering + implantable device design. Advanced modelling tools have been established to enhance the understanding and design of interactions within the biocomputer system. Partners successfully engineered mammalian ARPE-19 cells with molecular computing functions capable of detecting seizure-related signals (tsRNAs) and triggering the release of a therapeutic molecule (GDNF). This includes the exploration of innovative cell circuit designs utilizing L7Ae/kink-turn technology to optimize gene regulation + signal amplification.
A focus was on developing an encapsulated, implantable device to house these engineered cells. Research was conducted into biocompatible materials that support the diffusion of critical molecules (tsRNAs and GDNF), incorporating hybrid biomaterial scaffolds designed to replicate the brain’s extracellular matrix. Porous membrane structures, including PDMS hollow tubes and coaxial electrospun nanofibers were developed to facilitate cell encapsulation. In vitro epilepsy models were established to assess the PRIME concept prior to in vivo studies. Progress was made in identifying + characterising tsRNAs as potential biomarkers + sensors for seizure detection, reinforcing the personalised + responsive nature of the proposed therapy. These advancements in cell engineering, biomaterial science + molecular sensing represent a substantial step forward beyond current state-of-the-art treatments for neurodegenerative disorders.
A focus was on developing an encapsulated, implantable device to house these engineered cells. Research was conducted into biocompatible materials that support the diffusion of critical molecules (tsRNAs and GDNF), incorporating hybrid biomaterial scaffolds designed to replicate the brain’s extracellular matrix. Porous membrane structures, including PDMS hollow tubes and coaxial electrospun nanofibers were developed to facilitate cell encapsulation. In vitro epilepsy models were established to assess the PRIME concept prior to in vivo studies. Progress was made in identifying + characterising tsRNAs as potential biomarkers + sensors for seizure detection, reinforcing the personalised + responsive nature of the proposed therapy. These advancements in cell engineering, biomaterial science + molecular sensing represent a substantial step forward beyond current state-of-the-art treatments for neurodegenerative disorders.