Using Bombyx Mori Bioreactor To Produce Recombinant Adeno-associated Virus (rAAV) Vectors

Adeno-associated virus(AAV)has broad application prospects in the field of gene therapy.It has many advantages,such as non-pathogenicity,low immunogenicity,wide tissue tropism,and long-term stable expression of target genes.However,the yield and production cost of recombinant adeno-associated virus(r AAV)vectors limit its further development in gene therapy.At present,several production methods of r AAV have been developed,including adenovirus production system,herpes virus production system,and insect cell baculovirus production system.Using a recombinant baculovirus expression system to produce r AAV in insect cells is an efficient and flexible method to produce r AAV.Currently,the r AAV vectors are mainly produced in Sf9 cells by the expression system of Autographa californica multiple nuclear polyhedrosis virus(Ac MNPV).To establish a more efficient and lower-cost production system,we used the silkworm bioreactor as a platform to produce a recombinant chimeric r AAV2/HBo V1vector using the Bombyx mori nuclear polyhedrosis virus(Bm NPV)expression system.Both AAV2 and HBo V1(Human Bocavirus type-I)belong to the Parvoviridae and have very similar topological structures of the capsid surface.Therefore,packaging the AAV2 genome with the capsid protein of HBo V1 can form a pseudo-packaged chimeric virus r AAV2/HBo V1,which not only has the tissue tropism of HBo V1 but also has the gene loading capacity of HBo V1 capsid(?5.4kb).It can provide a unique chimeric virus vector with more pertinency for gene therapy of lung diseases.In this study,we first verified two baculovirus transfer vectors p Fast Bac-AAV2ITR-e GFP and p Fast Bac-AAV2Rep-HBo V1Cap,and according to the Bac-to-Bac system,we obtained two recombinant Bm NPVs:Bm NPV/AAV2ITR-e GFP and Bm NPV/AAV2Rep-HBo V1Cap.Continuous co-infection experiments of these two r Bm NPVs at the cellular level showed that e GFP,Rep,and Cap proteins could be expressed stably until the P4-P5 passages.The expression stability decreased after P6 On the first day of 5^thinstar,silkworm larvae(Jingsong-Zhenjiang)have been co-infected by the P3 r Bm NPVs.After 72h of co-infection,the e GFP was observed highly expressed in the larvae.Meanwhile,dissection of the infected larvae showed the highest protein expression in fat body and hemolymph.The silkworm pupae were also co-infected by direct injection of recombinant baculoviruses.The protein(e GFP)expression appeared after 48h of infection,indicating that the protein expression efficiency in silkworm pupae was higher than that in larvae.The purification method of r AAV has a great influence on the yield and quality of the vector.Density gradient ultracentrifugation is one of the most extensive methods for purifying r AAV in the laboratory.It is suitable for all r AAV serotypes and is beneficial for the removal of empty virions.We compared the purification effects of cesium chloride(Cs Cl)density gradient ultracentrifugation and iodixanol density gradient ultracentrifugation to purify r AAV2/HBo V1 and found that the purification quality of the iodixanol method was better than that of the Cs Cl method in this experiment.Not only the separation time is shorter,but the purity is much higher than the Cs Cl method.We have also quantified and analyzed the transduction activity of the purified r AAV2/HBo V1.According to q PCR quantitative analysis data,the average yield of r AAV2/HBo V1 in Bm N cell samples was about 2×10⁴VG/cell,the average yield per larva was 2.5×10¹²VG,and the average yield per silkworm pupa can reach to4.6×10¹²VG.The results showed that the production efficiency of r AAV2/HBo V1 in the silkworm bioreactor was very high,which can meet the requirements of large-scale and low-cost production of r AAV vectors.Transmission electron microscopy(TEM)showed that the empty virions ratio of purified r AAV2/HBo V1virus was still high,which means the optimization of this production system is required in the future.For example,introduce the expression of the NP1 protein which is important for the packaging of the HBo V1 virus capsid,or adjust the composition ratio of the AAV2 genome and the HBo V1 capsid gene.In human bronchial epithelial cells(16HBE),the purified r AAV2/HBo V1 showed certain transduction activity,but the fluorescence expression was low.It is speculated that r AAV2/HBo V1 did not specifically infect 16HBE cells.Polarized human airway epithelial cells(HAE-ALI)cultured on the air-liquid interface are the best cell line for wild-type HBo V1 infection and in vitro transduction of r AAV2/HBo V1 vectors.Next,we will try to use FACS to accurately detect the transduction activity of r AAV2/HBo V1 in HAE-ALI cells.In conclusion,we have established a BEV/insect production system for the production of a novel chimeric r AAV2/HBo V1 vector in the Bombyx mori baculovirus expression system and summarized an efficient vector purification method.The new system can successfully pseudo-package the r AAV2 genome in the HBo V1 capsid to form a r AAV2/HBo V1 vector for the treatment of all kinds of human lung diseases.This work demonstrates that the silkworm insect bioreactor has great application potential in the large-scale production of r AAV vectors.The established system also provides a new idea for the r AAV vectors production.