TARGETED TRANSGENE INSERTION INTO THE AAVS1 LOCUS VIA ZINC FINGER NUCLEASE TECHNOLOGY FOR CANCER IMMUNOTHERAPY

Abstract

IN THIS STUDY, WE DEMONSTRATE HOW ZINC FINGER NUCLEASE (ZFNS) CAN BE UTILIZED FOR THE ADVANCEMENT IN CANCER IMMUNOTHERAPY. FIRSTLY, A BACULOVIRAL VECTOR-MEDIATED ZINC FINGER NUCLEASE (BV-ZFN) TECHNOLOGY WAS DEVELOPED. IT DEMONSTRATED A HIGH TARGETING EFFICIENCY AND MAINTAINED EXPRESSION STABILITY OF THE TRANSGENE INSERTED INTO THE AAVS1 LOCUS OF HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS). NEXT, USING THE BV-ZFN TECHNOLOGY, HUMAN FIBROBLASTS WERE EFFICIENTLY REPROGRAMMED INTO HIPSCS BY INTEGRATING 4 REPROGRAMMING GENES INTO THE AAVS1 LOCUS. THE SAME REPROGRAMMING TECHNIQUE WAS USED TO REPROGRAM HEPATOCELLULAR CARCINOMA (HCC) CELLS INTO CANCER STEM CELL (CSC)-LIKE CELLS THAT EXHIBITED CLASSICAL CSC CHARACTERISTICS. THESE CSC-LIKE CELLS COULD BE USED AS AN INFINITE SOURCE OF CSC-ASSOCIATED ANTIGENS FOR THE GENERATION OF DENDRITIC CELL (DC)-BASED VACCINE AGAINST HCC CSC. LASTLY, IMPROVEMENTS IN THE EXPANSION OF NATURAL KILLER (NK) CELL WERE ACHIEVED BY UTILIZING ZFNS TO INTRODUCE MEMBRA