RESUMO
One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.
Assuntos
Engenharia Genética , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Lentivirus/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Citometria de Fluxo , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética , Vetores Genéticos/isolamento & purificação , Humanos , Imunoterapia Adotiva , Transdução Genética , Transfecção , Transgenes , Ultracentrifugação/métodosRESUMO
Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.
Assuntos
Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Imunoterapia Adotiva , Lentivirus/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos/isolamento & purificação , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , TransgenesRESUMO
Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Fator VIII/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Recombinantes/biossíntese , Transfecção/métodos , Animais , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Vetores Genéticos/biossíntese , Humanos , Plasmídeos/biossíntese , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , Proteína 1 de Ligação a X-BoxRESUMO
Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil's scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.
Assuntos
Biofarmácia , Comunicação Interdisciplinar , Proteínas Recombinantes/biossíntese , Transferência de Tecnologia , Amiloide/biossíntese , Animais , Baculoviridae/metabolismo , Biotecnologia , Proteínas Morfogenéticas Ósseas/biossíntese , Brasil , Linhagem Celular , Fator IX/biossíntese , Fator VIII/biossíntese , Hormônio Foliculoestimulante/biossíntese , Engenharia Genética , Vetores Genéticos/biossíntese , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Pesquisa/economia , Pesquisa/organização & administração , Spodoptera/virologiaRESUMO
Growth manipulation of fish is an important task in aquatic biotechnology. The growth promoting effect of recombinant Pichia pastoris expressing tilapia growth hormone was demonstrated in red tilapia fry (Oreochromis sp.), which were immersed into water containing intact cells of the recombinant yeast. The weight increase of the treated group was 171% relative to the control group after 6 weeks.
Assuntos
Aquicultura/métodos , Hormônio do Crescimento/biossíntese , Proteínas Recombinantes/biossíntese , Tilápia/crescimento & desenvolvimento , Animais , Vetores Genéticos/biossíntese , Hormônio do Crescimento/isolamento & purificação , Hormônio do Crescimento/farmacologia , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Survivin, a 16.5 kDa tumor associated antigen, is the smallest member of the inhibitor of apoptosis family that is abundantly expressed during development but essentially absent in normal adult tissues. Interestingly, survivin expression is up-regulated in virtually all types of cancers studied, as well as in vascular endothelial cells during tumor associated angiogenesis. Survivin links apoptosis to cell cycle progression and plays a pivotal role in regulation of cell proliferation. These characteristics make survivin a potentially promising generic target for cancer immunotherapy. Hence, a genetic immunization strategy to induce tumor-specific immune responses against human survivin in a pre-clinical animal model was developed. In initial studies, BALB/c mice were immunized by intramuscular injection with DNA coding for human survivin (pcDNA3.1/hSurv). In addition, a construct encoding a secreted version of survivin (pSecTag2B/hSurv) was designed. A plasmid coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-injected in both cases as a molecular adjuvant. Expression of survivin following transfection in mouse cells was corroborated. Humoral responses against human survivin were detected in mice sera using two immunization protocols (injections at 2- or 3-week intervals). The humoral response was markedly improved by secretion of survivin and co-expression of GM-CSF. The predominant antibody subclass detected in responsive mice was IgG2a, suggesting that a Th1-CD4+ cellular response had been induced. Furthermore, DNA immunization with survivin encoding vectors generated an effective CD8+ T cell response measured as an increase of cytotoxic Interferon-gamma (IFN-gamma) secreting CD8+ T cells. In conclusion, intramuscular genetic immunization of mice with human survivin encoding plasmids induced a survivin-specific humoral as well as cellular immune response in recipient mice. Secretion of survivin and co-injection of GM-CSF as a genetic adjuvant appear to be more important in generating an humoral than a cellular immune response.
Assuntos
Anticorpos Antineoplásicos/biossíntese , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Técnicas de Transferência de Genes , Imunidade Celular , Proteínas Associadas aos Microtúbulos/imunologia , Vacinas de DNA/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Proteínas Inibidoras de Apoptose , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Repressoras , Survivina , Vacinas de DNA/administração & dosagemRESUMO
A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA imcompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submitidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüênciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 710 resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado...