Creatine transporter deficiency: a journey on the road to gene therapy

Creatine Transporter Deficiency (CTD) is a rare X-linked neurodevelopmental disorder resulting from loss-of-function mutations in the SLC6A8 gene, which encodes the creatine transporter (CrT). The condition is characterized by cerebral creatine deficiency, early-onset intellectual disability, epilepsy, and autistic-like behavior. To date, no effective treatment is available. To explore the feasibility of gene therapy as a disease-modifying treatment for CTD, I developed an adeno-associated viral vector serotype 9 (AAV9) carrying the human SLC6A8 gene under the control of the synthetic ubiquitous promoter JeT. A single intracerebroventricular (ICV) injection of AAV-JeT::hSLC6A8 in male neonatal SLC6A8 knockout (KO) mice led to widespread transgene expression and a significant increase in brain creatine levels, demonstrating that the exogenous CrT was functionally active. This metabolic rescue was accompanied by the prevention of early brain hypoconnectivity and significant improvements in autistic-like stereotyped behavior and declarative memory in adult KO animals. However, working memory deficits persisted in KO mice, and treated WT animals showed impaired mnemonic performance. Moreover, administration of higher AAV-JeT::hSLC6A8 titers resulted in neurotoxicity, characterized by neuronal loss and neuroinflammation. Mechanistic analyses suggested that these adverse effects were not primarily mediated by endoplasmic reticulum stress or immune activation, but likely stemmed from excessive or ectopic transgene expression. These results highlight both the potential and challenges of gene therapy for CTD, highlighting the critical need for precise regulation of transgene expression. To address this issue, I optimized the gene cassette using multiple strategies aimed at fine-tuning SLC6A8 transgene expression. Among these new genetic constructs, FF351 displayed the most favorable expression profile, producing lower and more physiological CrT levels. For these reasons, it was selected for the development of a next-generation viral vector (AAV-FF351). Following ICV administration, AAV-FF351 effectively restored brain creatine levels and rescued cognitive deficits in KO mice, demonstrating a more promising outcome compared to the JeT strategy, and without detectable adverse effects. Overall, these findings provide robust proof-of-concept evidence that gene therapy is a promising strategy for CTD and emphasize the critical importance of finely tuning transgene expression to ensure both therapeutic efficacy and safety.