Gene delivery of AGAT and GAMT boosts creatine levels in creatine transporter deficiency patient fibroblasts

Abstract: Creatine is a critical metabolite used to buffer cellular energy demands in highly energetic tissues such as the brain and muscle. Genetic defects in endogenous creatine synthesis or transport across cellular membranes lead to a common set of phenotypes referred to as Cerebral Creatine Deficiency Syndrome (CCDS). The most common form of CCDS is Creatine Transporter 1 (CT1) Deficiency (CTD). It accounts for ~ 70% of cases and results from loss-of-function mutations in the X-linked gene SLC6A8. Affected individuals suffer from intellectual disability, autistic-like behaviors, and epilepsy. There are currently no effective therapies for this disorder, but gene therapy has emerged as a potential approach. The two enzymes which comprise the endogenous creatine synthetic pathway (AGAT and GAMT) are selectively expressed by specific cell types throughout the body. However, after synthesized, creatine uptake relies on the protein product of SLC6A8, CT1, to transport creatine into target cell types. We hypothesized that gene delivery of GATM (encoding AGAT) and GAMT into end-user cell types would bypass the need for CT1, allowing for intracellular synthesis of creatine. We tested this strategy in two human cell types: HEK293T cells and primary fibroblasts. Co-delivery of GATM and GAMT increased internal creatine concentrations by 7.6-fold in HEK293T cells and 12.3-fold in healthy control fibroblasts. We then employed this approach to primary fibroblasts from patients with CTD. This resulted in an up to 11.6-fold increase in intracellular creatine concentrations, far exceeding the intracellular concentration of creatine in healthy control fibroblasts. Importantly, overexpression of AGAT and GAMT resulted in proper targeting of these enzymes to their natural cellular compartment and did not impair the growth of patient fibroblasts. These findings establish gene therapy with GATM and GAMT as a potential strategy for patients with CTD.

Parent Summary: This study explores a new gene therapy approach for Creatine Transporter Deficiency (CTD). Rather than using the gene encoding for the dysfunctional creatine transporter, the researchers tested whether delivering the two enzymes needed to make creatine, AGAT and GAMT, directly into cells could allow the cells to make their own creatine. In lab experiments, they showed that this strategy greatly increased creatine levels in both normal and CTD patient cells without harming cell growth or function. This study provides early evidence that gene therapy targeting creatine synthesis could be a promising treatment option for CTD.

Link to article: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0319350

PubMed:  https://pubmed.ncbi.nlm.nih.gov/40338959/

Authors: Chloe Wells, Jon Sorgentrei, Sadie L. Johnson, Devin Albertson, Jared Rutter, Steven Andrew Baker

Key Terms: Multiple CCDS, Gene Therapy, In vitro, Basic Science, Therapeutics, GAMT, AGAT, CTD