The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.
Schwannomas are benign Schwann cell tumors on the peripheral nerves that occur in the autosomal dominant conditions of NF2 (neurofibromatosis type 2) or schwannomatosis. NF2 is caused by germline mutations in the NF2 gene, and patients can form tumors when the remaining normal gene copy is mutated. In schwannomatosis, two genes are known to have germline mutations: SMARCB1 and LZTR1, both near the NF2 gene on chromosome 22. Schwannomas in schwannomatosis have variable somatic deletions of multiple genes, and the tumors also have a risk of becoming malignant. These tumors can cause deformities, functional problems, substantial pain, and even lead to death. Schwannomas are difficult to treat because surgery involves cutting the affected nerves. There are currently no drug therapies, and due to the paucity of cell cultures and cell lines, there is a lack of molecular and cell biology data about schwannomas. The goal of this work was to better characterize a set of 20 tumors, to contribute information helpful in developing more targeted therapies. This involved loss of heterozygosity study using polymorphisms in or near the genes NF2, LZTR1 and SMARCB1 to identify somatic deletions. DNA sequencing of SMARCB1 exons was also carried out to search for germline and somatic mutations. This work revealed several mutations that contribute new knowledge to the field.
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