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Picanco-Castro V.,National Institute of Science and Technology in Stem Cell Therapy | Russo-Carbolante E.M.S.,University of Sao Paulo | Covas D.T.,National Institute of Science and Technology in Stem Cell Therapy | Covas D.T.,University of Sao Paulo
Recent Patents on DNA and Gene Sequences | Year: 2012

Lentiviral vectors are at the forefront of gene delivery systems for research and clinical applications. These vectors have the ability to efficiently transduce nondividing and dividing cells, to insert large genetic segment in the host chromatin, and to sustain stable long-term transgene expression. Most of lentiviral vectors systems in use are derived from HIV-1. Numerous modifications in the basic HIV structure have been made to ensure safety and to promote efficiency to vectors. Lentiviral vectors can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Moreover, these vectors can be used to reprogram cells and generate induced pluripotent stem cells. This review aims to show the patents that resulted in improved safety and efficacy of lentiviral vector with important implications for clinical trials. © 2012 Bentham Science Publishers.

De Castilho Fernandes A.,University of Sao Paulo | De Castilho Fernandes A.,National Institute of Science and Technology in Stem Cell Therapy | Fontes A.,University of Sao Paulo | Fontes A.,National Institute of Science and Technology in Stem Cell Therapy | And 7 more authors.
Biotechnology and Applied Biochemistry | Year: 2011

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. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Picanco-Castro V.,Center for Cell Therapy and Regional Blood Center | Picanco-Castro V.,National Institute of Science and Technology in Stem Cell Therapy | Russo-Carbolante E.,National Institute of Science and Technology in Stem Cell Therapy | Russo-Carbolante E.,University of Sao Paulo | And 15 more authors.
Stem Cells and Development | Year: 2011

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy. © 2011 Mary Ann Liebert, Inc.

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