Study On Large-scale RAAV1 Production Using The Baculovirus

Gene therapy offers much promise for the treatment of acquired and inherited genetic diseases. While significant progress has been made in identifying therapeutic genetic cargo capable of treating diseases, investigations have revealed that selection of a vector is one of the most critical steps for a success of gene therapy. Many viral vectors derived from adenovirus, adeno-associated virus, lentivirus, retrovirus, herpes simplex virus, etc. have been intensively studied in the past 20 years while each one of them has shown their own unique advantages and disadvantages. For instance, adenoviral vectors have a high transduction efficiency, are capable of containing DNA inserts up to 8 kilobases, have high viral titers, and infect both replicating and differentiated cells while adenoviral vectors-mediated gene expression is transient since the viral DNA does not integrate into the host and systemic delivery may be hampered by the preexisting antibodies. Adeno-associated viral vector (AAV) may overcome some of these limitations.AAV vectors are less immunogenic, not pathogenic, can transduce many types of tissues and cells while leads to a long-lasting expression following gene transfer. Over 100 homologous human and nonhuman serotypes have been isolated; among them AAV1 was found to be specifically targeting muscle cells and has become one of very attractive vectors for gene delivery through muscle cells. However, the current production procedure via plasmid transfection is not only limited by the yield but also time consuming and label intensive. Therefore, my efforts were devoted to the development of a novel methodology to package and produce AAV1.The process I have developed is based on the utilization of baculovirus-insect cells. This production system requires three plasmids that contain all cis- and trans- elements for viral packaging: pFBD-AAV1RepiCapi, pFBD-AAV1RFP, and pFBD-AAV1AT7(Avastin). By transforming these plasmidsl into MAX Efficiency? DH10Bac? competent E. coli cells, site-specific transposition of the expression cassette into a baculovirus vector (bacmid) and transfected into the Sf9 cells to generate recombinant baculoviruses: Bac-AAV1RepiCapi, Bac-AAV1RFP and Bac-AAV1-AT7 (Avastin). Their titers were 5.42×10⁸VG/ml, 2.63×10⁸ VG/ml and 5.95×10¹²VG/ml, respectively. Using the two baculoviral vectors packaging system, rAAV1RFP and rAAV1AT7(Avastin) were consequently created. The yield of rAAV1RFP was 3.7×1013vg in 100ml, was 35 TU/cell and highly infectious, produced significant Red fluorescence while antibody of Avastin was detectable at various time points in cells following infection. Taken together, these results have suggested that the baculovirus-based system I have developed worked well for the packaging and production of AAV1 derived viral vectors, which may meet the need of large quantity of AAV1 vectors required for gene therapy.