Cholesterolated Rapamycin Prodrug Liposomes Induce Antigen-Specific Tolerance and Enable AAV Redosing

Antidrug antibodies (ADAs) compromise the pharmacokinetics and efficacy of biologics and can trigger adverse reactions. We engineer a tolerogenic liposomal platform in which rapamycin is covalently conjugated to cholesterol (RAPA-chol) and formulated as nanoliposomes (RA-c@L) to induce antigen-specific immune tolerance to coadministered proteins. Covalent anchoring enables high drug encapsulation (>95%) and improves colloidal stability. In a three-dose weekly tolerization regimen with uricase, followed by a high-dose challenge, RA-c@L markedly suppresses ADA formation: by day 42, antiuricase IgG titers are reduced by 2.15-fold compared to uricase alone and 2.17-fold compared to free rapamycin. Responses to an irrelevant antigen (KLH) remain unchanged, indicating antigen specificity. Importantly, coadministration of RA-c@L with rAAV8-SEAP enables vector readministration, yielding approximately 2-fold higher sustained serum SEAP expression after the second dose compared to rAAV alone. Mechanistically, intravenously delivered RA-c@L preferentially accumulates in the liver and reshapes systemic immunity, with reduced splenic T follicular helper cells and germinal-center B cells and an expansion of CD4⁺Foxp3⁺ regulatory T cells. Together, these data show that RA-c@L establishes durable, antigen-specific tolerance to therapeutic proteins and facilitates AAV redosing, offering a practical strategy to mitigate ADA-mediated loss of efficacy in repeated biotherapeutic treatments.