Dataset Information

OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy.

ABSTRACT: Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1-12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1-12 rep and cap genes induced upon infection with a single baculovirus that also carries the rAAV genome. rAAV burst sizes reach up to 5 × 10(5) benzonase-resistant, highly infectious genomic particles per cell, exceeding typical yields of current rAAV production systems. In contrast to recombinant rep/cap baculovirus strains currently employed for large-scale rAAV production, the Sf9rep/cap cell lines are genetically stable, leading to undiminished rAAV burst sizes over serial passages. Thus, OneBac combines full AAV serotype options with the capacity for stable scale-up production, the current bottleneck for the transition of AAV from gene therapy trials to routine clinical treatment.

SUBMITTER: Mietzsch M

PROVIDER: S-EPMC3955967 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy.

Mietzsch Mario M Grasse Sabrina S Zurawski Catherine C Weger Stefan S Bennett Antonette A Agbandje-McKenna Mavis M Muzyczka Nicholas N Zolotukhin Sergei S Heilbronn Regine R

Human gene therapy 20140123 3

Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1-12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1-12 rep and cap genes induced upon infection with a single baculovirus that also carri ...[more]

PMID: 24299301

Similar Datasets

Project description:Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.

Dataset Information

OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy.

Publications

OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1-12 vectors for gene therapy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets