Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.

ObjectiveNowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.MethodsThis study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.ResultsThe retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.ConclusionThe results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

No abstract

RESUMO:Os gliomas são considerados tumores primários do sistema nervoso central, representando apenas 2% dos casos de câncer e o glioblastoma multiforme (GBM) é a forma mais comum e com pior prognóstico dos tumores da glia do sistema nervoso central. O GBM é caracterizado pela alta taxa proliferativa e migratória juntamente com a ausência de apoptose, o que o torna altamente agressivo. Atualmente o tratamento para o glioblastoma depende da localização, do tipo de célula e do grau de malignidade da mesma. Entretanto, não existe um tratamento eficiente disponível para o GBM, apesar dos inúmeros avanços nas técnicas de cirurgia, radioterapia e quimioterapia, este tipo de neoplasia, ainda continua sendo, um dos maiores desafios para a oncologia. A Terapia Fotodinâmica (PDT) vem sendo amplamente utilizada nos tratamentos de tumores sólidos e sua ação terapêutica se dá pela absorção de luz por um fármaco fotossensibilizante, o que resulta numa cascata de eventos fotofísicos, fotoquímicos e fotobiológicos, que induzem em última instancia a apoptose e/ou necrose das células que foram marcadas de forma eficiente pelo fármaco fotosensibilizante. Dessa forma, este trabalho de revisão bibliográfica aborda as principais características do glioblastoma e as perspectivas de novos tratamentos para este tipo de tumor, os quais utilizam a PDT. Palavras-chave: Glioblastoma, sistema nervoso central, Terapia Fotodinâmica. ABSTRACT:Gliomas are considered primary tumors of the central nervous system, representing only 2% of cancers. Glioblastoma multiforme (GBM) is the most common form and with poor prognosis of tumors of the central nervous system. The GBM is characterized by high proliferative rate and migration, besides the absence of apoptosis, which makes it highly aggressive. Currently treatment to glioblastoma depends on the location cell type and degree of malignancy. However, there is no effective treatment available for GBM, despite numerous advances in surgical techniques, radiotherapy and chemotherapy, this type of cancer, yet remains one of the biggest challenges for oncology. Photodynamic Therapy (PDT) has been widely used in the treatment of solid tumors and its therapeutic action is due to the absorption of light by a photosensitizer drug, resulting in a cascade of events photophysical, photochemical and photobiological, ultimately inducing apoptosis and / or necrosis of cells that were efficiently labeled by the photosensitizing drug. Thus, this review discusses the main features of glioblastoma and prospects for new treatments for this type of tumor, which use the PDT.