Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes

Abstract

Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). 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. 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. 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. 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. 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. 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. 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. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…This interesting observation led to a couple of assumptions on the molecular functions of NS2 and NS4; however, the detailed molecular mechanism of how HBoV1 supports AAV replication is not known. The same research group then utilized this bocavirus helper system in order to develop a novel rAAV vector production platform that may have implications in future AAV gene therapy vector production [ 226 ]. They combined a distinct set of AdV and bocavirus helper genes and found that rAAV titers were significantly increased when compared to rAAV vector titers that have been produced with either AdV or bocavirus helper factors alone.…”

Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System

“…Deng et al reported that, with the co-infection of a BEV expressing HBoV1 NP1, the rAAV2/HBoV1 vector was produced in a higher quantity and with a lower percentage of empty particles [ 26 ]. The expression of NP1 led to an increase in rAAV2 replicative-form (RF) DNA intermediates, which may be responsible for the enhanced production [ 11 ]. Another vital reason may be that the ratio of the AAV2 genome and the capsid gene of HBoV1 was not optimal.…”

“…Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia [ 10 ]. The packaging of a rAAV serotype 2 genome into a HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia [ 11 ]. This chimeric vector has shown potential for use in gene therapies for cystic fibrosis (CF) and other lung diseases [ 9 , 10 , 11 ].…”

Transplantation of a Novel Recombinant Adeno-Associated Virus (pAAV-HE1B19K-TE1A) Demonstrates Higher Anti-Tumor Effects in Tumor Cells

“…Moreover, OVs can express toxic proteins, induce inflammatory cytokines such as TNF, and trigger the immune response [ 26 ]. Gene therapy using conditional replicated AAV has become an important focus of tumor gene therapy [ 27 ]. There are 2 main approaches to constructing targeted rAAV.…”