The crude lysates from the experiments in (B) and (C) were mixed with 2× SDS loading buffer, respectively, boiled and resolved in 10% SDS-PAGE. (D and E) BocaHelper stimulates less AAV viral protein expression than pAdHelper does. DRP of vector produced per cell is shown. Cells were lysed at 72 hr post-transfection and DNase-resistant particles (DRP) were extracted and quantitated by real-time PCR. HEK293 cells were co-transfected with rAAV proviral vector (pAV2Ftg83lucCMVmCherry), with the AAV helper pAAVRep2Cap2 (B) or pAAVRep2Cap5 (C) plasmids and with pAdHelper or pBocaHelper. (B and C) Comparison of vector production helped with pAdHelper versus pBocaHelper. rAAV proviral genome, AAV helper (pAAVRep2Cap2 or pAAVRep2Cap5), adenovirus pAdHelper and human bocavirus pBocaHelper plasmids used in this study are diagramed. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.ĭNA replication HBoV1 gene expression helper gene rAAV.īocaHelper Facilitates the Production of rAAV2 and rAAV5 Vector (A) Schematic diagram of plasmids. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3-7 times higher than that using pAdHelper. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%-70% of that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. Despite a production yield of 1-2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper.
We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells.
In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2).
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