Renal CLCN5 gene therapy for Dent disease type 1: local rapamycin immune modulation and age-dependent outcomes with lentiviral and AAV9 vectors

Dent disease type 1 is a rare X-linked renal tubulopathy caused by loss of the Cl⁻/H⁺ exchanger ClC-5, leading to defective proximal tubular endocytosis and low-molecular-weight proteinuria. Viral gene therapy represents a promising disease-modifying strategy; however, the relative performance of integrating versus non-integrating vectors, the impact of immune responses, and the influence of renal developmental stage on therapeutic durability remain unresolved. In this study, we used a ClC-5-null mouse model to systematically address three key questions relevant to Dent disease type 1 gene therapy: (i) whether local immune suppression can prolong viral vector-mediated therapeutic effects, (ii) whether adeno-associated virus (AAV) is suitable for renal delivery of CLCN5 , and (iii) how age at treatment influences outcomes. We first show that lentiviral delivery of human CLCN5 to the kidney of mutant mice, when combined with local co-delivery of rapamycin-loaded biodegradable microspheres, resulted in enhanced ClC-5 expression and reduction of urinary β2-microglobulin for up to 10 months, supporting a role for localized immune modulation in extending gene therapy efficacy. We then evaluated AAV serotype 9-mediated retrograde ureteral delivery of codon-optimized human CLCN5 . In neonatal mice, AAV treatment produced marked but transient reductions in urinary protein markers despite persistent ClC-5 expression, coinciding with postnatal tubular growth and spatial redistribution of transgene expression. In adult mutant mice, AAV9- CLCN5 delivery achieved long-term transgene expression lasting up to 8–10 months; however, this expression did not result in consistent reduction of urinary β2-microglobulin excretion, even with rapamycin co-delivery. Together, these findings demonstrate that Dent disease type 1 gene therapy outcomes are strongly influenced by vector type, immune modulation, and developmental stage, and that sustained transgene expression alone is insufficient for durable phenotypic correction. This work provides a comparative framework for optimizing vector selection, treatment timing, and immune management strategies for gene therapy of Dent disease type 1.