Assessment of AAV3B transduction efficiency in a liver normothermic machine perfusion model
摘要
Normothermic machine perfusion (NMP) enables liver preservation under close-to physiological conditions and thus, may serve as a platform to treat livers ex situ. Liver-directed adeno-associated virus (AAV) vectors are a promising candidate for the treatment of liver genetic diseases, but highly specific AAV vectors are lacking. We herein utilized the NMP platform for porcine livers to optimize AAV vector-based gene therapy protocols. Porcine livers (n = 4) were subjected to NMP (OrganOx® Metra) for 48 h and AAV3B encoding eYFP was applied to the perfusate. Serial perfusate samples were analysed for liver function and complement activation. Tissue biopsies were taken pre virus application, after 3 h, 24 h and 48 h for real-time confocal microscopy and immunofluorescent staining to assess eYFP protein or cryopreserved for quantification of AAV3B cDNA and gDNA levels. AAV3B vector administration in the perfusate did not disrupt stable liver NMP nor induce complement activation or an inflammatory response. At a dose of 1 × 1013 vg/kg AAV transduction was successfully detected by eYFP signals in immunofluorescence microscopy and confirmed by quantification of AAV3B vector genomes in tissue biopsies taken after 24 h and 48 h of NMP, but not as early as 3 h after vector application. Administration of a poloxamer (Pluronic®) enhanced transduction efficiency of AAV3B, while a dose reduction to 2 × 1012 vg/kg with Pluronic® resulted in comparable transduction efficiency to the high-dosage protocol without Pluronic®. AAV application during liver NMP is reasonable and does not induce adverse effects, which makes the pre-clinical porcine liver NMP model an excellent platform for the development of AAV-based gene therapies.