Hereditary spastic paraplegia type 52 (SPG52) is an ultra-rare neurological disorder characterized by weakness and spasticity in the lower limbs, developmental delay, intellectual disability, seizures, and thinning of the corpus callosum (CC). SPG52 is caused by biallelic loss-of-function mutations in the AP4S1 gene, which encodes the sigma subunit of the adaptor protein complex 4 (AP-4). Disruptions in any subunit of the AP-4 complex lead to its destabilization and degradation, resulting in a shared pathophysiology and symptomatology, known as the AP-4 complex associated hereditary spastic paraplegia. Although the exact role of the AP-4 complex remains undefined, it is hypothesized to facilitate the biogenesis of autophagosomes through the sorting and trafficking of ATG9A. At present, no cure or disease-modifying treatment is available. Therefore, a gene therapy approach aimed at restoring AP4S1 gene expression represents a rational therapeutic strategy for SPG52.
This doctoral thesis presents the development of two models to assess the efficacy and safety of the proposed gene therapy approach employing an adeno-associated viral vector. Specifically, an in vivo model has been established, namely the first mouse model for SPG52 featuring a complete knockout of the AP4S1 gene (AP4S1 KO). Furthermore, an in vitro model has been constructed through the generation of induced pluripotent stem cells (iPSC) derived from skin biopsies of a patient and two controls, with the purpose of producing iPSC-derived cortical neurons to create a human disease-relevant model.
Following the successful generation and characterization of the iPSC-derived cortical neurons, patient-derived neurons demonstrated an accumulation of the primary cargo of the AP-4 complex, ATG9A, within the Trans-Golgi network. Additionally, abnormalities in neurite morphology and reduced neuronal network activity were observed. The overexpression of the AP4S1 gene successfully reestablished complex formation, thereby ameliorating the neuropathological phenotype.
In parallel, the generation, establishment, and characterization of the AP4S1 KO mouse model were undertaken. As observed in 94% of SPG52 patients, this animal model effectively recapitulated the thinning of the CC. In addition to this phenotype, KO mice exhibited reductions in body weight, muscle strength, motor coordination, and lifespan in comparison to their wild-type (WT) littermates. Neuromuscular deficits identified in the hindlimb musculature of KO mice were substantiated by neuromuscular function assessments, which demonstrated decreased compound muscle action potential (CMAP). Furthermore, KO mice showed behavioral differences relative to WT mice, such as increased exploratory behavior without signs of anxiety, as observed in patients. To evaluate the efficacy of the proposed gene therapy approach, two cohorts of animals were administered an AAV9P31 adeno-associated viral vector carrying a correct copy of the human AP4S1 gene. One cohort received treatment during the neonatal stage, while the second cohort was treated during young adulthood. The phenotype rescue was assessed in both cohorts, demonstrating a complete restoration of CMAP and CC integrity, a partial rescue of CC thinning, motor coordination, and muscle strength; however, no effect was observed on the body weight of the animals. These promising results provide a foundation for safety trials, advancing the prospects for initiating clinical trials for SPG52 therapy.
| Date of Award | 7 Nov 2025 |
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| Original language | English |
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| Awarding Institution | - Universitat Autònoma de Barcelona (UAB)
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| Supervisor | Miguel Chillón Rodriguez (Director) |
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