Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Improving the secretory capacity of CHO producer cells: The effect of controlled Blimp1 expression, a master transcription factor for plasma cells.

With the advent of novel therapeutic proteins with complex structures, cellular bottlenecks in secretory pathways have hampered the high-yield production of difficult-to-express (DTE) proteins in CHO cells. To mitigate their limited secretory capacity, recombinant CHO (rCHO) cells were engineered to express Blimp1, a master regulator orchestrating B cell differentiation into professional secretory plasma cells, using the streamlined CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform. The expression of Blimp1α or Blimp1ß in rCHO cells producing DTE recombinant human bone morphogenetic protein-4 (rhBMP-4) increased specific rhBMP-4 productivity (qrhBMP-4). However, since Blimp1α expression suppressed cell growth more significantly than Blimp1ß expression, only Blimp1ß expression enhanced rhBMP-4 yield. In serum-free suspension culture, Blimp1ß expression significantly increased the rhBMP-4 concentration (>3-fold) and qrhBMP-4 (>4-fold) without significant increase in hBMP-4 transcript levels. In addition, Blimp1ß expression facilitated mature rhBMP-4 secretion by active proteolytic cleavage in the secretory pathway. Transcriptomic profiling (RNA-seq) revealed global changes in gene expression patterns that promote protein processing in secretory organelles. In-depth integrative analysis of the current RNA-seq data, public epigenome/RNA-seq data, and in silico analysis identified 45 potential key regulators of Blimp1 that are consistently up- or down-regulated in Blimp1ß expressing rCHO cells and plasma cells. Blimp1ß expression also enhanced the production of easy-to-express monoclonal antibodies (mAbs) and modulated the expression of key regulators in rCHO cells producing mAb. Taken together, the results show that controlled expression of Blimp1ß improves the production capacity of rCHO cells by regulating secretory machinery and suggest new opportunities for engineering promising targets that are resting in CHO cells.

Feasibility and benefits of a combined programme of exercise and play for paediatric cancer survivors: A pilot study.

OBJECTIVE: This study aimed to determine the feasibility and benefits of a combined programme of exercise and play for childhood cancer survivors on health-related quality of life (HRQOL), post-traumatic growth and physical strength levels. METHODS: Six childhood cancer survivors participated in the 8-week intervention consisting of supervised play and exercise sessions two times per week. The participants performed joint exercises, independently, at home, on the 5 days that they were unable to participate in group exercises. Participants completed measures assessing HRQOL, post-traumatic growth and physical strength levels at baseline and post-intervention. RESULTS: Recruitment, retention and attendance rates in the 8-week combined programme were 87.5%, 85.7% and 89.6%, respectively, with no adverse reactions. Statistically significant improvement was observed in post-traumatic growth (z = -2.20, p = 0.03), subscales of HRQOL school functioning (z = -2.06, p = 0.04) and total score (z = -2.0.3, p = 0.04). Moreover, physical strength measurements using the physical activity promotion system showed that, out of the five total categories, muscle strength (z = -2.02, p = 0.04) and total physical strength (z = -2.03, p = 0.04) scores were statistically significantly improved. CONCLUSION: The 8-week combined programme of exercise and play was feasible and provided preliminary evidence for the benefits of exercise on HRQOL, post-traumatic growth and physical activity levels in childhood cancer survivors.