Since the first enzyme replacement therapy (ERT) for Gaucher disease was approved in 1991, ERT has become standard of care for several lysosomal storage diseases (LSD), collectively characterized by dysfunctional lysosomal enzymes leading to pathological substrate accumulation. Sanfilippo Syndrome type A (MPSIIIA) is a monogenetic disease caused by >150 different loss-of-function mutations in the lysosomal enzyme N-Sulfoglucosamine Sulfohydrolase (SGSH), resulting in accumulation of heparan sulfate (HS). This accumulation disrupts cellular homeostasis with consequences for autophagy and mitochondrial and synaptic functions, leading to neuroinflammation and neurodegeneration. Clinically, MPSIIIA is characterized by mild peripheral symptoms and severe CNS degeneration resulting in symptoms such as dementia, hyperactivity, seizures, and loss of motor skills. There is currently no curative treatment.As a pre-requisite for developing an efficient ERT approach also targeting cells in the CNS, it is essential to understand how SGSH is internalized by- and distributed between different brain cell types. We have established an ex vivo model of mouse organotypic hippocampal slice cultures (OHSCs) in which enzyme uptake into relevant brain cell types (microglia, astrocytes, neurons), its subcellular localization and its distribution among cell types can be studied. Furthermore, the model enables the assessment of cell type specific ERT efficacy ex vivo, measured as reduction of HS accumulation, paving the way for efficient drug development for patients living with MPS IIIA. Results from the model will be benchmarked to similar data from human iPSC-derived microglia, astrocytes, and neurons, to provide further insight about suitable models that could benefit ERT drug development.