• creatine transporter
• creatine transporter deficiency
• gene therapy

Creatine (Cr) transporter deficiency (CTD) is an X-linked metabolic disorder caused by mutations in the SLC6A8 gene, encoding for the creatine transporter (CrT). The disruption of the SLC6A8 gene leads to the loss-of-function of the CrT protein and consequently to brain Cr deficiency. The main hallmarks of CTD include intellectual disability (ID), autistic behavior, and epilepsy.
Although some symptoms can be managed through pharmacological intervention, effective treatments for CTD are sorely missing yet. This is an important problem, because CTD, despite rare, has a great impact on the society and the healthcare system, due to the lifelong care needed by patients. We hypothesized that adeno-associates virus (AAV)-mediated transfer of a functional SLC6A8 transgene might succeed in reinstating proper and long-lasting Cr transporter expression. CTD is an ideal target for gene replacement therapy for at least three reasons: i) it is a monogenic condition; ii) the replenishment of brain Cr ameliorates the symptoms of the two syndromes caused by the disruption of Cr endogenous synthesis, proving the reversibility of the pathological state due to Cr deficiency; iii) CTD phenotype exhibits a dose-dependent relationship with the expression of a functional SLC6A8 gene. The key goal of this project is to develop a gene therapy strategy for CTD and to test it in a mouse model of CTD carrying the deletion of Slc6a8 gene.
Intracerebroventricular injection in male neonatal mice (postnatal day 1, PND1) of an AAV9 vector carrying a plasmid with a functional copy of the human SLC6A8 gene under the control of the CAG promoter resulted, 2 weeks later, in the expression of the CrT protein in the brain. The exogenous protein was functional since Cr brain levels significantly increased. However, the overexpression of CrT protein led to neurotoxic effects, including neuronal degeneration, neuroinflammation, and death of animals. We replaced the CAG promoter with the weaker JeT promoter. Again, injection of AAV9-JeT-hCRT (6x1013vg/ml) resulted in increased brain Cr levels and neurotoxic effects, although the mortality of animals diminished in parallel with the reduction of the viral dose. Since animals injected with a low-titer AAV (1.5x1012 vg/ml) displayed a survival rate comparable to mice injected with the AAV carrying the GFP protein, we conducted behavioral tests in PND40 and PND100 mice. We found a significant increase in the body weight, but no improvement in cognitive deficits in KO mice and a deterioration of the performance in WT mice. These results indicate that there is a delicate balance between the expression levels of Slc6a8, and the fitness of cerebral cells and circuits, prompting us to investigate the neurotoxicity mechanisms of CrT overexpression and alternative strategies to promote the success of AAV-based gene therapy in CTD.
We found that the primary cause of neurotoxicity is not related to inflammation caused by the injection of an exogenous antigen nor to the heterogeneity of SLC6A8 gene expressions across brain circuits. In contrast, our gene therapy strategy activates the stress of endoplasmic reticulum and likely induces cellular overload of Cr. Based on these results, we focused on identifying possible strategies to modulate transgene expression. We modified our plasmid in the following ways: i) we replaced the JeT promoter with the human endogenous SLC6A8 promoter (hu::hSLC6A8); ii) we removed the WPRE sequence (JeT::hSLC6A8-noWPRE); iii) we added a ligand-dependent destabilizing domain at the 3’ end of the gene (JeT::hSLC6A8-DD). Using HEK293T cells as testbed, we found that the expression level of the exogenous CrT is not reduced in the cells transfected with the JeT::hSLC6A8-noWPRE plasmid, while it is significantly decreased with the hu::hSLC6A8 and the JeT::hSLC6A8-DD plasmids. However, the destabilizing domain seemed to induce to efficient degradation of the exogenous protein, preventing the possible restoration of intracellular Cr levels. Thus, we identified the human endogenous promoter as the most promising strategy. The next step will be to validate the hu::SLC6A8 plasmid in other cell models such as fibroblast and neurons derived from KO mice before testing its effects in vivo.