3Dbig AAV – Exploring hiPSC-derived 3D Models to Tackle the Brain Innate Immune Response to Gene Therapy rAAV

Financiado por / Funded by:

Designação do Projeto | Project Name 3Dbig-AAV – Exploring hiPSC-derived 3D models to tackle the brain innate immune response to gene therapy rAAV

Código do Projeto | Project Code 2023.16122.ICDT

Entidade beneficiária | Beneficiary Entity ITQB NOVA – Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa and iBET – Instituto de Biologia Experimental e Tecnológica

Data de início | Starting Date 01/07/2025

Data de conclusão | Conclusion Date 30/06/2028

Breve Descrição do Projeto | Brief Project Description

The 3Dbig AAV project addresses a critical bottleneck in the clinical translation of adeno-associated viral vector (rAAV) gene therapies for central nervous system (CNS) disorders: the poorly understood innate immune response triggered within the human brain microenvironment. Although rAAV-based therapies have shown promise in treating monogenic neurological diseases, including mucopolysaccharidoses (MPS), their long-term efficacy and safety remain limited by neuroinflammatory reactions that are not adequately predicted by current animal or simplified in vitro models.

This project proposes the development of an innovative, fully human, innate immunocompetent 3D in vitro model of the CNS (iiNSoids), derived from human induced pluripotent stem cells (hiPSCs). These advanced models will integrate neurons, astrocytes, oligodendrocytes, microglia, endothelial cells, and pericytes in a scalable and reproducible 3D architecture generated using bioreactor technology. By recreating the cellular complexity and molecular crosstalk of the brain’s innate immune compartment, iiNSoids will enable unprecedented investigation of early neuroinflammatory events following rAAV administration.

The project combines state-of-the-art cellular engineering with novel molecular tools, including cell-type-specific NF‑κB fluorescent reporters and proximity-dependent biotinylation approaches, to monitor inflammatory signalling and secretome dynamics at high spatial and temporal resolution. These tools will be applied to model disease-associated neuroinflammation using hiPSCs derived from patients with mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disorder characterised by chronic CNS inflammation.

Using this human disease context, the project will dissect the cellular responders and molecular effectors of the innate immune response to rAAV9-mediated gene delivery. Integrated single-cell transcriptomics and proteomics will map immune activation pathways across distinct CNS cell populations. Finally, the project will test targeted genetic and pharmacological strategies to mitigate rAAV-induced neuroinflammation, aiming to improve vector efficacy and safety.

Overall, 3Dbig AAV will deliver advanced human preclinical models, mechanistic insight into CNS innate immunity, and actionable strategies to optimise gene therapies, with broad impact for neurological disease research, biotechnology innovation, and personalised medicine.