Modelos celulares avançados para remodelar a resposta imune inata a vectores de terapia génica

Adeno-associated viral vector (rAAV)-mediated gene therapy is promising for curing monogenic central nervous system (CNS) disorders, such as mucopolysaccharidoses (MPSs). Despite promising preclinical data, successful translation of gene therapy for MPSs is still pending due to poor long-term efficacy in clinical trials (6). Building upon preclinical and clinical data (3, 6) , we hypothesize that the disease-associated pre-activation of the host innate immune system (i.e., neuroinflammation (7, 9) may exacerbate responses to therapeutic vectors and impair the survival of corrected cells. Direct administration into the CNS can activate resident immune-competent cells, leading to direct toxicity towards neurons. Subsequent secretion of neuroinflammatory cytokine/chemokines establishes immunomodulatory loops between the different cell players, amplifying inflammatory effects. The role of the different immune-competent cell types in this early tissue response and their crosstalk mechanisms remain elusive in humans. To address this gap, we set out 4 specific objectives: 1. To recapitulate in vitro the molecular crosstalk between the main cellular components implicated in the innate immune response in the CNS. For this, we will craft an innate immunocompetent 3D cell model of the human CNS, using hiPSC as a scalable supply of neural, immune, and vascular cells (iiNSoids, Fig.1). 2. To depict disease-associated neuroinflammation, we will establish an MPSVII-iiNSoid model and deconvolute inflammatory pathways specific for each of the immune-competent cell types. 3. To dissect the cellular responders and the molecular effectors of the innate immune response to rAAV9, in the MPS VII-iiNSoid model. 4. To propose novel targeted approaches for amelioration of the innate immune responses to rAAVs Fluorescent reporters of NF-kB activity and biotin ligase-based proximity binding approaches will leverage the interrogation of the iiNSoid models and enable a deeper understanding of the innate immune response against rAAV therapies. This knowledge has the potential to improve the efficacy of gene therapies. These assays are also modular and can therefore be adapted to other cell types enabling a broad characterization for different therapies and target cells.