1047. Neural Progenator Cells Can Be Genetically-Modified by a Novel Double-Stranded Adeno-Associated Viral (AAV) Vectors
Authors: Chi-Hsieh Wang, Ga-Chen Yin, Xiao Xiao, Hsin-I. Ma, Yung-Hsiao Chiang
Journal: Molecular Therapy (2006)

Neural stem cells (NSCs) may be used either for cell replacement or for gene delivery vehicles in neurodegenerative diseases, stroke and malignant brain tumor. The expression of therapeutic proteins by NSCs can be enhanced by viral-mediated gene transfer, though the effects of several common recombinant viruses on primary progenitor cell populations have not been widely tested.AAV is a promising gene vector based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a slow process responsible for the delayed and the occasional inefficient transduction. By mutating the inverted terminal repeat (ITR), novel AAV vectors that predominantly package the dsDNA genome were made. In addition to circumventing, the requirement for DNA synthesis, the dsAAV exhibited higher DNA stability in vivo and more effective circularization than the ssAAV, suggesting potential molecular mechanisms for the faster, stronger and longer transgene expression. These findings suggest that a new approach for gene therapy of neurodegenerative diseases, stroke and malignant brain tumors, based on the grafting of NSCs producing therapeutic molecules, is very possible.The overall goal of this study is to identify and develop a safe and effective NSCs-based gene therapy protocol and examine which serum type of the novel double-stranded recombinant adeno- associated viral (AAV) vector can efficiently deliver reporter gene (eGFP) into the rat NSCs. Meanwhile, we will investigate the gene product delivered by the genetically engineered NSCs in the adult normal brain. Several serum types of AAV vector (carrying the eGFP reporter gene) were applied in vitro and different buffers and transduction conditions were also test. Different density of neural progenitor cells in flask also played a critical role in the transduction efficiency. Totally 8 serum types included type 1, type 2, type 3, type 4, type 5, type 6, type 7 and type 8 were used. Novel transduction procedures (SOPs) were developed. The transduction efficiency of type 2 ds AAV-eGFP was almost the same as type 5 but superior to other types in the in vitro transduction efficiency, but the duration of expression in type 2 was longer than the type 5. The green neurospheres can be found over one month after AAV vector (type 2) infection, but gradually fade away around three weeks in the type 5. In the type 2 AAV-eGFP, the transduction efficiency was almost 80 – 90 % in the whole neurosphere, and around 80 % in the type 5. After the immunocytology study, the ds type 2 AAV can transducer MAP-2 (+), Nestin (+), and NF (+) cells. Our study proved that the neural progenitor cells can be genetically modified by the double-stranded type 2 and type 5 AAV vectors. Neural stem cells (NSCs) may be used either for cell replacement or for gene delivery vehicles in neurodegenerative diseases, stroke and malignant brain tumor. The expression of therapeutic proteins by NSCs can be enhanced by viral-mediated gene transfer, though the effects of several common recombinant viruses on primary progenitor cell populations have not been widely tested. AAV is a promising gene vector based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a slow process responsible for the delayed and the occasional inefficient transduction. By mutating the inverted terminal repeat (ITR), novel AAV vectors that predominantly package the dsDNA genome were made. In addition to circumventing, the requirement for DNA synthesis, the dsAAV exhibited higher DNA stability in vivo and more effective circularization than the ssAAV, suggesting potential molecular mechanisms for the faster, stronger and longer transgene expression. These findings suggest that a new approach for gene therapy of neurodegenerative diseases, stroke and malignant brain tumors, based on the grafting of NSCs producing therapeutic molecules, is very possible. The overall goal of this study is to identify and develop a safe and effective NSCs-based gene therapy protocol and examine which serum type of the novel double-stranded recombinant adeno- associated viral (AAV) vector can efficiently deliver reporter gene (eGFP) into the rat NSCs. Meanwhile, we will investigate the gene product delivered by the genetically engineered NSCs in the adult normal brain. Several serum types of AAV vector (carrying the eGFP reporter gene) were applied in vitro and different buffers and transduction conditions were also test. Different density of neural progenitor cells in flask also played a critical role in the transduction efficiency. Totally 8 serum types included type 1, type 2, type 3, type 4, type 5, type 6, type 7 and type 8 were used. Novel transduction procedures (SOPs) were developed. The transduction efficiency of type 2 ds AAV-eGFP was almost the same as type 5 but superior to other types in the in vitro transduction efficiency, but the duration of expression in type 2 was longer than the type 5. The green neurospheres can be found over one month after AAV vector (type 2) infection, but gradually fade away around three weeks in the type 5. In the type 2 AAV-eGFP, the transduction efficiency was almost 80 – 90 % in the whole neurosphere, and around 80 % in the type 5. After the immunocytology study, the ds type 2 AAV can transducer MAP-2 (+), Nestin (+), and NF (+) cells. Our study proved that the neural progenitor cells can be genetically modified by the double-stranded type 2 and type 5 AAV vectors.