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.