Andrielle de Castilho Fernandes

Stable and high‐level production of recombinant Factor IX in human hepatic cell line

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.

Simultaneous encapsulation of magnetic nanoparticles and zinc phthalocyanine in poly(methyl methacrylate) nanoparticles by miniemulsion polymerization and in vitro studies.

The aim of this work was the simultaneous encapsulation of magnetic nanoparticles (MNPs) and zinc(II) phthalocyanine (ZnPc) in poly(methyl methacrylate) (PMMA) (MNPsZnPc-PMMA) nanoparticles (NPs) by miniemulsion polymerization and to evaluate the photobiological activity and/or hyperthermia (HPT) against human glioblastoma cells (U87MG). MNPsZnPc-PMMA NPs presented an average diameter of 104 ± 2.5 nm with a polydispersity index (PdI) of 0.14 ± 0.03 and negative surface charge - 47 ± 2.2 mV (pH 7.4 ± 0.1). The encapsulation efficiency (EE%) of ZnPc was 85.7% ± 1.30. The release of ZnPc from PMMA NPs was slow and sustained without the presence of burst effect, indicating a homogeneous distribution of the drug in the polymeric matrix. In the biological assay, MNPsZnPc-PMMA NPs showed considerable cytotoxic effect on U87MG cells only after activation with visible light at 675 nm (photodynamic therapy, PDT) or after application of an alternating magnetic field. The simultaneous encapsulation of MNPs and ZnPc in a drug delivery system with sustained release can be a new alternative for cancer treatment leading to significant tumor regression after minimum doses of heat dissipation and light.

Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells

Abstract293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

Objective Nowadays 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. Methods This 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. Results The 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. Conclusion The 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.

Stem Cells and Cancer Stem Cells

The different organs in human organism are constituted by tissues with mature and specialized cells and its specific stem cells. Stem cells represent only a minuscule fraction of cells that constitute each tissue but they are the only cells with self-renewal capacity. The organ-specific stem cells have specific properties that maintain tissue integrity and are defined mainly by their capacity to undergo self-renewal, as well as differentiation into mature cell types that comprise each organ (Shipitsin & Polyak, 2008). The malignant neoplasias are believed to result from sequential mutations that can occur as a consequence of progressive genetic instability and/or environmental factors. An accordant observation in several investigations has been the association between deregulation of stem cells and carcinogenesis, because there are regulatory mechanisms of self-renewal in normal stem cells that also frequently regulate oncogenesis. In consequence, experimental and clinical evidences that have recently been accumulated support the hypothesis that cancer may arise from mutations in normal stem cell populations, and that these cells would be subject to ongoing genetic and epigenetic changes that could help to establish the disease. The cancer stem cell (CSC) hypothesis states that normal stem cells may be the cells of cancer origin, and that a specific subset of cancer cells with stem cell characteristics can give rise to a hierarchy of proliferative and progressively differentiated bulk of tumoral cells, leading to tumor initiation, progression, and recurrence. In fact, there are several investigations that recently have identified specific CSC markers showing similar expression profiles than the normal stem cells of same organ. Moreover, CSCs can be prospectively isolated based on expression of a specific molecule or combination of molecules, and have the ability to give rise to new tumors when xenografted in immunodeficient mice. Additional confirmations that stem cells can play a role in carcinogenesis are the homologies found between normal adult stem cells and cancer cells. Besides self-renewal capacity, these characteristics include the production of differentiated cells, activation of antiapoptotic pathways, induction of angiogenesis, resistance to apoptosis and drugs (due to active telomerase expression and elevated membrane transporter activity), and the ability to migrate and propagate (Wicha et al., 2006). Notwithstanding, different from normal adult stem cells that remain constant in number, CSCs can increase as the tumors grow, and originate the progeny that can be both locally invasive and/or colonize distant sites. Therefore, the consolidation of CSCs knowledge into our current view of multistep cancer development has important implications for defining the target population for therapeutical

Human Papillomavirus Infection and Penile Cancer: Past, Present and Future

Penile squamous cell carcinoma (PSSC) is an uncommon malignant tumor, which accounts for less than 1% of adult male cancers in North America and Europe, but is markedly higher in developing locations, such as Asia, Africa and South America, representing up to 10% of tumors in men. Human papillomavirus (HPV) infection has shown an important role in penile cancer pathogenesis. In 2009, a systematic review of published literature found that 40% of penile tumors were HPV-related, and that type 16 HPV was the most common subtype in this group (Backes et al., 2009). Another interesting relation between HPV infection and penile cancer is the finding that specific histological subtypes are associated with HPV infection. Penile carcinomas with basaloid differentiation and warty features have shown a strong association with HPV infection, with recent studies showing that HPV infection is present in 76% of basaloid tumors, while the presence in verrucous cancer was 24.5% (Backes et al., 2009).

O uso de células-tronco mesenquimais como nova perspectiva de tratamento para hemofilia b.

A Hemofilia B e uma doenca genetica ligada ao cromossomo X e consiste na deficiencia do fator IX (FIX) da coagulacao sanguinea. Clinicamente, essa doenca e caracterizada por episodios de sangramento principalmente nos musculos, articulacoes e tecidos moles. O tratamento se da pela terapia de reposicao do FIX derivado do plasma (pdFIX) ou FIX humano recombinante (rhFIX). As inconvenientes infusoes intravenosas, juntamente com o alto custo dos pdFIX e/ou rhFIX, incentivaram o desenvolvimento de novas abordagens terapeuticas que permitissem resultados mais estaveis e duradouros. Postulava-se que a terapia genica, insercao do genes funcional em celulas e/ou tecidos do paciente, pudesse suprir a necessidade fisiologica do FIX ativo em portador de hemofilia B, contudo as barreiras imunologicas do paciente representaram um grande obstaculo para esta terapia. Logo, os resultados insatisfatorios obtidos com a terapia genica acrescido da possibilidade de uso terapeutico das celulas-tronco mesenquimais (CTMs), celulas consideradas imunoprivilegiadas, direcionou-se as abordagens terapeuticas para a combinacao das terapias genica e celular, ou seja, a manipulacao genetica de CTMs ex vivo com a finalidade de transplanta-las em hospedeiros, e dessa forma corrigir de forma eficiente e eficaz o fenotipo da hemofilia B. Dessa forma, este artigo revisa as caracteristicas da hemofilia, os tratamentos disponiveis e as perspectivas para uso de CTMs no tratamento da hemofilia B.