Restoring blood-brain barrier extrusion activity with endothelial-targeting gene therapy to manage Alzheimer’s disease: viral versus non-viral approaches

• Applicant: Amira Hanafy, Ph.D.
• Subject Area: Pharmacy
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 560699507

Dysregulated ß-amyloid (Aß) homeostasis is evident in Alzheimer’s disease (AD). The blood-brain barrier (BBB) plays a critical role in Aß homeostasis via a group of transporters, among which is the luminal ATP-binding cassette (ABC) transporter ABCC1 in cerebral endothelial cells (Ecs), which contributes to Aß clearance to the blood stream. Using ISMICAP, a BBB model based on in situ micro-perfusion of capillaries in brain slices, I identified, for the first time, the significant decrease in ABCC1 activity in the Tg2576 mouse model of AD. This project addresses the restoration of ABCC1 activity in Ecs using gene therapy as a novel therapeutic approach to manage AD via enhancing Aß clearance. While gene therapy can be delivered to Ecs by viral and non-viral vectors, it is not known yet whether the delivery approach may influence the therapeutic outcomes. Here we propose CRISPR activation (CRISPRa) to promote the transcription of abcc1 in cerebral Ecs. To deliver the CRISPRa tool, we will use viral and non-viral approaches. An all-in-one dCas9-VP64 abcc1-sgRNA construct will be selectively delivered to cerebral Ecs in an adeno-associated virus (AAV)-based vector, AAV-BI30, by intravenous administration. Our pilot studies showed that AAV-BI30 transduces cerebral Ecs with high specificity and efficiency without crossing the BBB. As the non-viral approach, dCas9-VP64 abcc1-sgRNA plasmids will be packaged in polyethylenimine (PEI)-based polyplexes (nanoplexes). To achieve cell targeting specificity, the short peptide CFAG will be used as a molecular tag due to ist selective surface retention to cerebral Ecs. In addition, we will examine whether the subcellular compartmentalization of endothelial ABCC1 is altered in Tg2576 mice and in response to treatment using focused ion beam-scanning electron microscopy. With the outlook of early AD management, the optimized delivery approach will be administered to young Tg2576 mice and the disease progression will be monitored compared to untreated mice. Using ISMICAP, we will establish the restoration of endothelial ABCC1 activity as an unprecedented approach to slow down AD progression. The generated all-in-one viral particles to promote the transcription of abcc1 have the potential to be used as a proof-of-principle viral platform to target other genes in different diseases involving BBB disruption. Moreover, we will establish nanoplexes packaging dCas9-VP64 abcc1-sgRNA plasmids with superior retention and endocytosis in cerebral Ecs and minimal BBB crossing. Similarly, the nanoplexes can be used as a platform for the delivery of different cargos to the cerebral Ecs with high specificity. This project will ultimately provide essential insights into the use of viral and non-viral approaches to address the same cellular target. There is the intrinsic potential to uncover their respective advantages and disadvantages, not only in the context of AD, but also as drug delivery platforms in the general sense.

• DFG Programme: Research Grants