Modification of T lymphocytes with lentiviral vectors for expression of anti-CD19 chimeric antigen receptor (CAR)

The use of immunotherapy with modified T lymphocytes with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemias and lymphomas resistant to chemotherapy. CAR possess an extracellular domain derived from variable regions of antibodies and costimulation intracellular domains of T lymphocytes. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors as well as normal B cells, but not in other types of cells. Recent clinical studies involving anti-CD19 CAR T-cells have shown excellent responses in a variety of B-cell tumors, even in patients with relapse after high-dose chemotherapy. This study aimed to produce CD4+ lymphocyte lineage Jurkat (ATCC® TIB-152 ™) modified with a second generation anti-CD19 CAR with 4-1BB as intracellular costimulation domain. Lentiviral vectors were produced in HEK293T (ATCC® CRL-3216 ™) transiently transfected with plasmids containing the coding sequence of the CAR, viral envelope VSV-G, and viral capsid. The viral titer was calculated by real time PCR after transduction of HEK293T cells, resulting in 1.65 x 105 IU/mL. The literature indicates an MOI (multiplicity of infection) from 5 to 10 IU/cell for transduction of lymphocytes. A new batch of virus was produced, and the supernatant was ultracentrifuged at 19200 rpm (Beckman Coulter, SW28 rotor) in order to concentrate the viral particles. The viral titer of the concentrated batch was 1.26 x 108 IU/mL. This new titer is compatible with the necessary to infect 107 cells, amount of pre-expansion cells necessary to obtain the number of cells suitable for infusion into patients (2.5 x 109 to 5 x 109 cells). Then, the infection of Jurkat was performed in a 6-well plate with RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 µg/mL Polybrene®, and centrifugation at 1000 rpm for 20 minutes at room temperature. After 16 hours of incubation (37°C, 5% CO2 and 85% humidity), the medium was exchanged for fresh RPMI 1640 10% FBS. After additional 48 hours of incubation under the same conditions, the cells were collected and was their DNA was extracted. We obtained by real-time PCR that the number of integrated viral copies per genome was 35.3 ± 4.5 (mean ± standard deviation) for transduction with MOI of 5 IU/cell. While for MOI of 10 IU/cell, it was obtained 42.6 ± 0.1 copies per genome. It was observed that there was not a significant increase in viral copies when the MOI increased from 5 to 10. This may occur because cell’s surface receptors have been saturated by the large number of viruses. The lentiviral vector used by us has been shown to transduce T lymphocyte satisfactorily. The next steps of the study are the transduction of T lymphocytes from healthy donors and verification of the CAR receptor effectiveness to bind to CD19 of cell B lymphocyte lineages. Grant #2016/08374-5, São Paulo Research Foundation (FAPESP)

New initiatives for Brazilian aquatic plant data management

Initiatives directed at managing information on aquatic plants can support different areas of scientific research, educational practices and political decisions for sustainable management and protection of wetlands and natural resources, as well as reveal knowledge gaps regarding aquatic plants and wetlands. Furthermore, such initiatives can contribute directly to consolidating wetland classification systems in Brazil. The objectives of this work are to present and explore the use of a data repository and online platform created specifically for the management of data on aquatic plants in Brazil. The data repository for aquatic plants was developed to store information on species occurrence records in a standardized form considering: systematics, taxonomy, habit, life form, geographic distribution, hydrographic basin, wetland, bibliographic references and herbarium material. A total of 3,903 records referring to the Southeast Region of Brazil were generated for the data repository. This total encompassed 1,036 species distributed among 132 families and 466 genera. Our results illustrate the diversity of information available regarding aquatic plants, circumscribe this group of species and serve as a source of data about them. Similar databases and electronic infrastructures can be developed for other flora in the future

Establishment of a platform for the production of lentiviral vectors for the modification of anti-CD19 CAR-T cells

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos.Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays

Establishment of a platform for the production of lentiviral vectors for the modification of anti-CD19 CAR-T cells

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos.Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays

Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

Abstract Background A robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells. Results Primary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation. Conclusions Genetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products

Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9

Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations

New initiatives for Brazilian aquatic plant data management

ABSTRACT Initiatives directed at managing information on aquatic plants can support different areas of scientific research, educational practices and political decisions for sustainable management and protection of wetlands and natural resources, as well as reveal knowledge gaps regarding aquatic plants and wetlands. Furthermore, such initiatives can contribute directly to consolidating wetland classification systems in Brazil. The objectives of this work are to present and explore the use of a data repository and online platform created specifically for the management of data on aquatic plants in Brazil. The data repository for aquatic plants was developed to store information on species occurrence records in a standardized form considering: systematics, taxonomy, habit, life form, geographic distribution, hydrographic basin, wetland, bibliographic references and herbarium material. A total of 3,903 records referring to the Southeast Region of Brazil were generated for the data repository. This total encompassed 1,036 species distributed among 132 families and 466 genera. Our results illustrate the diversity of information available regarding aquatic plants, circumscribe this group of species and serve as a source of data about them. Similar databases and electronic infrastructures can be developed for other flora in the future