Optimization of and new insights into a novel and empowered TARGETed gene addition approach at a relevant microglia locus for the treatment of inherited NeuroMetabolic Diseases

Inherited metabolic disorders belong to a class of monogenic diseases caused by defects in lipids, proteins or organelle metabolism. Among these pathologies, peroxisomal and lysosomal storage disorders are recurrently associated with severe neurological involvement (NeuroMetabolic Diseases - NMDs) resulting in the most frequent cause of pediatric neurodegenerative conditions. In the therapeutic options spectrum, allogeneic hematopoietic stem cell transplantation and enzyme replacement therapy can modify disease course when applied early, yet they are constrained by donor availability, conditioning toxicity, and limited central nervous system (CNS) penetration. Autologous lentiviral (LV) hematopoietic stem and progenitor cell (HSPC) gene therapy has established clinical benefit, but concerns persist regarding vectors semi-random genomic integration profile, delayed CNS repopulation by HSPCs progeny, and non-physiological, ubiquitous transgenic cassettes expression. To address these limitations, this thesis validated and advanced a targeted gene addition strategy that couples CRISPR/Cas9 editing with AAV6 donor delivery to insert therapeutic cassettes at a microglia-biology informed locus in clinically relevant, mobilized peripheral-blood HSPCs. By positioning ABCD1 (for cerebral X-linked adrenoleukodystrophy, cc X-ALD) and ARSA (for metachromatic leukodystrophy, MLD) cDNAs at the prototypical microglial marker locus CX3CR1, the approach is designed to minimize genotoxic risk, accelerate functional myeloid output to the CNS, and achieve lineage-appropriate, regulated expression in disease-relevant compartments. In-vitro studies demonstrated efficient, site-specific integration while partially preserving HSPC phenotype and function, supporting the feasibility of durable metabolic correction through edited progeny. Recognizing delivery modalities as a central bottleneck for genome editing in primitive compartment of HSPCs, we further developed an innovative lipid-nanoparticle (LNP) platform for intracellular delivery of Cas9/sgRNA ribonucleoprotein (RNP). This non-viral modality provides a conceptual path to reduce electroporation-associated stress while maintaining editing competence, thereby broadening the manufacturability and safety envelope of ex vivo editing workflows. Finally, to interrogate the contribution of CX3CR1 dosage to engraftment and myeloid differentiation, we’ve generated an allele-dosage engineered iPSC lines and deploy them with brain organoids to delineate mechanisms that may enhance microglia-like cell generation post-transplantation and inform HSPC gene-therapy design. Collectively, these results outline a next-generation HSPC gene therapy paradigm, precise, regulated, and CNS-oriented, that directly addresses key constraints of LV platforms and potentially advances the translational prospects for patients with pediatric NMDs.