Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity.

Recombinant adeno-associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction. Through iterative design-construct-characterization cycles, we have showcased the potential of this synthetic cell production system. Systems characterization of the dynamics of viral transcripts and proteins as well as virus assembly and packaging revealed that the expression level and balance of viral genome and capsid protein are keys to not only the productivity but also the full particle content, an important product quality attribute. Boosting cap gene expression by sequential transfection and integration of multiple copies of the cap gene elevated the rAAV titer to levels on a par with traditional plasmid transfection and virus infection. However, overexpression of the cap gene shifted the balance and kinetics of the genome and capsid. We independently tuned the dynamics of genome amplification and capsid protein synthesis by modulating the induction concentration as well as the time profile, and significantly enhanced full particle content while maintaining a high productivity. This strategy of constructing an inducible stable producer cell line is readily adaptable to rAAV vectors of different serotypes and payloads. It can greatly facilitate scalable production of gene therapy vectors.

Bioconversion of pinoresinol into matairesinol by use of recombinant Escherichia coli.

Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as whole grains and sesame and flax seeds, have anticancer activity and can act as phytoestrogens. The lignans secoisolariciresinol and matairesinol can be converted in the mammalian proximal colon into enterolactone and enterodiol, respectively, which reduce the risk of breast and colon cancer. To establish an efficient bioconversion system to generate matairesinol from pinoresinol, the genes encoding pinoresinol-lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, were expressed in Escherichia coli and purified. The two genes, termed plr-PpH and sdh-PpH, were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 min showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of the mixture of rPLR and rSDH. The percent conversion of (+)-pinoresinol to matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and rSDH. However, conversion of (+)-pinoresinol by fusion protein SDH-PLR stopped at the intermediate product, secoisolariciresinol. In vivo, (+)-pinoresinol was completely converted to matairesinol by living recombinant E. coli expressing PLR-SDH without addition of cofactors.

A comparison of strategies for multiple-gene co-transformation via hairy root induction.

Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.