Shih-Yeh Lin

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

Enterovirus 71 virus-like particle vaccine: Improved production conditions for enhanced yield

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production.

Efficient gene delivery into cell lines and stem cells using baculovirus

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow–derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4–6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor–encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40–75%), but approached ≈90–95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein–Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.

Evaluation of the stability of enterovirus 71 virus-like particle

Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease that caused significant mortality in children, but no vaccine is available yet. EV71 virus-like particle (VLP) is the empty capsid consisting of viral structural proteins but can elicit potent immune responses, rendering VLP a promising EV71 vaccine candidate. To evaluate whether VLP remains stable after long-term storage, which is crucial for advancing the VLP vaccine to the clinical setting, we evaluated the effects of NaCl concentration, buffers and temperatures on the VLP stability. We first validated the use of dynamic light scattering (DLS) for measuring the hydrodynamic diameter (≈30-35 nm) of VLP, which was close to the VLP diameter (≈25-27 nm) as measured by transmission electron microscopy (TEM). Using these techniques, we found that EV71 VLP remained stable for 5 months in sodium phosphate (NaPi) buffers with various NaCl concentrations. EV71 VLP also remained morphologically stable in NaPi, citrate and TE(+) buffers for 5 months, yet the enzyme-linked immunosorbent assay (ELISA) revealed that the VLP stored in citrate and TE(+) buffers partially lost the immunogenicity after 5 months. In contrast, the VLP stored in the NaPi buffer at 4°C remained stable macroscopically and microscopically for 5 months, as judged from the DLS, TEM and ELISA. The VLP stored at 25°C and 37°C also retained stability for 1 month, which would obviate the need of a cold chain during the shipping. These data altogether proved the stability of EV71 VLP and suggested that the VLP is amenable to bioprocessing and storage.

Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens.

After three decades of development, the baculovirus/insect cell expression system is now recognized as a powerful platform for recombinant protein production. With a number of distinct advantages, the baculovirus/insect cell expression system has been extensively used for the production of various vaccine candidates, and several human and veterinary vaccine products have been commercially available. In addition to insect cells, baculovirus is capable of entering a broad range of mammalian cells, lending itself to a promising gene delivery vehicle for antigen expression and display in vivo. The use of baculovirus for antigen expression and delivery has been reviewed in 2008. Rather than a critical evaluation, this paper aims to provide an update of the applications of baculovirus as an in vitro or in vivo antigen expression/delivery vehicle, with special focuses on developments and advances after 2008.

Development of EV71 virus-like particle purification processes

Enterovirus 71 (EV71) causes the outbreaks of hand-foot-and-mouth disease and results in deaths of hundreds of young children. EV71 virus-like particles (VLPs) are empty capsids consisting of viral structural proteins and can elicit potent immune responses, thus holding promise as an EV71 vaccine candidate. However, an efficient, scalable production and purification scheme is missing. For mass production of EV71 VLPs, this study aimed to develop a production and chromatography-based purification process. We first demonstrated the successful EV71 VLPs production in the stirred-tank bioreactor in which High Five™ cells were infected with a recombinant baculovirus co-expressing EV71 structural polyprotein P1 and protease 3CD. The culture supernatant containing the VLPs was subjected to tangential flow filtration (TFF) for concentration/diafiltration, which enabled the removal of >80% of proteins while recovering >80% of VLPs. The concentrated VLPs were next subjected to hydroxyapatite chromatography (HAC) in which the VLPs were mainly found in the flow through. After another TFF concentration/diafiltration, the VLPs were purified by size-exclusion chromatography (SEC) and concentrated/diafiltered by a final TFF. The integrated process yielded an overall VLPs recovery of ≈ 36% and a purity of ≈ 83%, which was better or comparable to the recovery and purity for the purification of live EV71 virus particles. This process thus may move the EV71 VLPs vaccine one step closer to the clinical applications.

Recent patents on the baculovirus systems.

Baculovirus has been widely utilized as a protein production tool in insect cells for nearly 3 decades and has captured growing interests as a vector for gene delivery into mammalian cells and animals over the past decade. This review summarizes important patents pertaining to the use of baculovirus for insect cell infection and mammalian cell transduction, with special emphasis on the vector development, new applications and downstream processing.

Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design

Enterovirus 71 (EV71) is responsible for the outbreaks of hand‐foot‐and‐mouth disease in the Asia‐Pacific region. To produce the virus‐like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co‐express EV71 P1 polypeptide and 3CD protease using the Bac‐to‐Bac® vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v‐cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF‐P1‐C3CD, a recombinant baculovirus constructed using the flashBAC GOLDTM system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High FiveTM cells with BacF‐P1‐C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross‐protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High FiveTM culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. Biotechnol. Bioeng. 2015;112: 2005–2015.

Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference

Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhfr and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells. We next generated stable CHO cell clones coexpressing DHFR, the model protein (EGFP), dCas9 and gRNA targeting dhfr. Combined with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to coamplify more copies of dhfr and egfp genes. Compared with the traditional method relying on MTX selection (up to 250 nM), the CRISPRi approach increased the dhfr copy number ∼3-fold, egfp copy number ∼3.6-fold and enhanced the EGFP expression ∼3.8-fold, without impeding the cell growth. Furthermore, we exploited the CRISPRi approach to enhance the productivity of granulocyte colony stimulating factor (G-CSF) ∼2.3-fold. Our data demonstrate, for the first time, the application of CRISPRi in CHO cells to enhance recombinant protein production and may pave a new avenue to CHO cell engineering.