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1.
Life Sci ; 270: 119142, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33524419

RESUMO

Adeno-associated virus (AAV) vector, an excellent gene therapy vector, has been widely used in the treatment of various central nervous system (CNS) diseases. Due to the presence of the blood-brain barrier (BBB), early attempts at AAV-based CNS diseases treatment were mainly performed through intracranial injections. Subsequently, systemic injections of AAV9, the first AAV that was shown to have BBB-crossing ability in newborn and adult mice, were assessed in clinical trials for multiple CNS diseases. However, the development of systemic AAV injections to treat CNS diseases is still associated with many challenges, such as the efficiency of AAV in crossing the BBB, the peripheral toxicity caused by the expression of AAV-delivered genes, and the immune barrier against AAV in the blood. In this review, we will introduce the biology of the AAV vector and the advantages of systemic AAV injections to treat CNS diseases. Most importantly, we will introduce the challenges associated with systemic injection of therapeutic AAV in treating CNS diseases and suggest feasible solutions.


Assuntos
Doenças do Sistema Nervoso Central/terapia , Dependovirus/genética , Terapia Genética/métodos , Absorção Fisiológica , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Doenças do Sistema Nervoso Central/genética , Dependovirus/metabolismo , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/tendências , Vetores Genéticos/genética , Humanos , Camundongos , Transdução Genética/métodos , Transgenes
2.
Nucleic Acids Res ; 48(14): 8178-8187, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32619241

RESUMO

The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Elementos de Resposta , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Nanopartículas/química , Células THP-1 , Transdução Genética/métodos
3.
Sci Rep ; 10(1): 1812, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32020016

RESUMO

Haematopoietic stem cells (HSCs) have the potential for lifetime production of blood and immune cells. The introduction of transgenes into HSCs is important for basic research, as well as for multiple clinical applications, because HSC transplantation is an already established procedure. Recently, a major advancement has been reported in the use of cyclosporine H (CsH), which can significantly enhance the lentivirus (LV) transduction of human haematopoietic stem and progenitor cells (HSPCs). In this study, we employed CsH for LV transduction of murine HSCs and defined haematopoietic progenitors, confirming previous findings in more specific subsets of primitive haematopoietic cells. Our data confirm increased efficiencies, in agreement with the published data. We further experimented with the transduction with the simultaneous use of several vectors. The use of CsH yielded an even more robust increase in rates of multi-vector infection than the increase for a single-vector. CsH was reported to reduce the innate resistance mechanism against LV infection. We indeed found that additional pretreatment could increase the efficiency of transduction, in agreement with the originally reported results. Our data also suggest that CsH does not reduce the efficiency of transplantation into immune-competent hosts or the differentiation of HSCs while enhancing stable long-term expression in vivo. This new additive will surely help many studies in animal models and might be very useful for the development of novel HSC gene therapy approaches.


Assuntos
Ciclosporina , Células-Tronco Hematopoéticas/metabolismo , Transdução Genética/métodos , Animais , Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus , Camundongos
4.
Gene ; 724: 144157, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629820

RESUMO

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.


Assuntos
Dependovirus/genética , MicroRNAs/genética , Infecções por Parvoviridae/genética , Epitélio Pigmentado da Retina/virologia , Transdução Genética/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Parvovirinae/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/citologia , Transgenes
5.
J Allergy Clin Immunol ; 145(2): 679-697.e5, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31513879

RESUMO

BACKGROUND: Patients with T-cell immunodeficiencies are generally treated with allogeneic hematopoietic stem cell transplantation, but alternatives are needed for patients without matched donors. An innovative intrathymic gene therapy approach that directly targets the thymus might improve outcomes. OBJECTIVE: We sought to determine the efficacy of intrathymic adeno-associated virus (AAV) serotypes to transduce thymocyte subsets and correct the T-cell immunodeficiency in a zeta-associated protein of 70 kDa (ZAP-70)-deficient murine model. METHODS: AAV serotypes were injected intrathymically into wild-type mice, and gene transfer efficiency was monitored. ZAP-70-/- mice were intrathymically injected with an AAV8 vector harboring the ZAP70 gene. Thymus structure, immunophenotyping, T-cell receptor clonotypes, T-cell function, immune responses to transgenes and autoantibodies, vector copy number, and integration were evaluated. RESULTS: AAV8, AAV9, and AAV10 serotypes all transduced thymocyte subsets after in situ gene transfer, with transduction of up to 5% of cells. Intrathymic injection of an AAV8-ZAP-70 vector into ZAP-70-/- mice resulted in a rapid thymocyte differentiation associated with the development of a thymic medulla. Strikingly, medullary thymic epithelial cells expressing the autoimmune regulator were detected within 10 days of gene transfer, correlating with the presence of functional effector and regulatory T-cell subsets with diverse T-cell receptor clonotypes in the periphery. Although thymocyte reconstitution was transient, gene-corrected peripheral T cells harboring approximately 1 AAV genome per cell persisted for more than 40 weeks, and AAV vector integration was detected. CONCLUSIONS: Intrathymic AAV-transduced progenitors promote a rapid restoration of the thymic architecture, with a single wave of thymopoiesis generating long-term peripheral T-cell function.


Assuntos
Terapia Genética/métodos , Timócitos , Transdução Genética/métodos , Proteína-Tirosina Quinase ZAP-70 , Animais , Dependovirus , Vetores Genéticos , Síndromes de Imunodeficiência/terapia , Camundongos , Camundongos Knockout , Proteína-Tirosina Quinase ZAP-70/administração & dosagem , Proteína-Tirosina Quinase ZAP-70/genética
6.
Methods Mol Biol ; 2043: 137-155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463909

RESUMO

Lentiviral systems have proven advantageous in the delivery and long-term integration of gene sequences into the genome of several cell types in vitro, in vivo, as well as in clinical trials. Here we detail the protocols involved in the molecular cloning of ADAMTSL2 and ADAMTSL4 into the human immunodeficiency virus (HIV)-derived pCDH lentiviral system. We also describe the lentiviral transduction of ADAMTSL2 and ADAMTSL4 into mammalian HEK293-EBNA cells to create stable cell lines, as well as their recombinant expression.


Assuntos
Proteínas ADAMTS/genética , Clonagem Molecular/métodos , Transdução Genética/métodos , Proteínas ADAMTS/metabolismo , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Proteínas Recombinantes/metabolismo
7.
Cytotherapy ; 21(12): 1246-1257, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31837737

RESUMO

BACKGROUND: Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS: The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION: This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Organismos Geneticamente Modificados , Linfócitos T/fisiologia , Engenharia Tecidual/métodos , Transdução Genética/métodos , Reatores Biológicos/normas , Técnicas de Cultura de Células/normas , Diferenciação Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/normas , Células Cultivadas , Desenho de Equipamento , Gases/farmacocinética , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/fisiologia , Ativação Linfocitária/imunologia , Organismos Geneticamente Modificados/citologia , Permeabilidade , Receptores de Antígenos Quiméricos/genética , Linfócitos T/citologia , Transdução Genética/normas
8.
Sci Rep ; 9(1): 17637, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776415

RESUMO

Retroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Here, we show that human cells transduced to stably express GFP transfer GFP gene to non-transduced cells. This horizontal gene transfer was mediated by a fraction of extracellular membrane vesicles that were released by the transduced cells. These vesicles carried endogenous retroviral envelope protein syncytin 1 and essentially acted as replication-competent retroviruses. The ability to transfer the GFP gene correlated with the levels of syncytin 1 expression in the transduced cells and depended on the fusogenic activity of this protein, substantiating the hypothesis that endogenous syncytin 1 mediates fusion stage in the delivery of extracellular vesicle cargo into target cells. Our findings suggest that testing for replication-competent retroviruses, a routine safety test for transduced cell products in clinical studies, should be also carried out for cell lines generated by retroviral vectors in in vitro studies.


Assuntos
Produtos do Gene env/metabolismo , Proteínas da Gravidez/metabolismo , Retroviridae/genética , Transdução Genética/métodos , Animais , Western Blotting , Linhagem Celular , Marcadores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Sci Rep ; 9(1): 15101, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641163

RESUMO

The development and approval of engineered cellular therapies are revolutionizing approaches to treatment of diseases. However, these life-saving therapies require extensive use of inefficient bioprocessing equipment and specialized reagents that can drive up the price of treatment. Integration of new genetic material into the target cells, such as viral transduction, is one of the most costly and labor-intensive steps in the production of cellular therapies. Approaches to reducing the costs associated with gene delivery have been developed using microfluidic devices to increase overall efficiency. However, these microfluidic approaches either require large quantities of virus or pre-concentration of cells with high-titer viral particles. Here, we describe the development of a microfluidic transduction device (MTD) that combines microfluidic spatial confinement with advective flow through a membrane to efficiently colocalize target cells and virus particles. We demonstrate that the MTD can improve the efficiency of lentiviral transduction for both T-cell and hematopoietic stem-cell (HSC) targets by greater than two fold relative to static controls. Furthermore, transduction saturation in the MTD is reached with only half the virus required to reach saturation under static conditions. Moreover, we show that MTD transduction does not adversely affect cell viability or expansion potential.


Assuntos
Lentivirus/genética , Microfluídica/métodos , Células-Tronco de Sangue Periférico/metabolismo , Transdução Genética/métodos , Células Cultivadas , Vetores Genéticos/genética , Humanos , Microfluídica/instrumentação , Transplante de Células-Tronco de Sangue Periférico/métodos , Transdução Genética/instrumentação
10.
Mol Pharm ; 16(11): 4738-4750, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31596095

RESUMO

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (∼74%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3-2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (∼2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Hemofilia A/genética , Degeneração Retiniana/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Hemofilia A/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Retina/metabolismo , Degeneração Retiniana/metabolismo , Transdução Genética/métodos , Transgenes/genética
11.
Methods Mol Biol ; 2048: 27-39, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396926

RESUMO

Cancer immunotherapy has been developed and established as a new treatment modality. Recently, adoptive transfer therapy using T cells redirected with antigen-specific antitumor receptors, such as T-cell receptor (TCR) and chimeric antigen receptor (CAR), has demonstrated clinical benefits even in patients with refractory malignancies. To advance this treatment modality, both generation of gene-modified T cells and evaluation of their reactivity with high quality in vitro are required. To achieve this, it is important to establish the ways (1) to generate optimal viral particle for T-cell transduction, (2) to transduce antitumor receptors into T cells and expand redirected T cells efficiently, and (3) to assess the functionality of antigen-specific gene-modified T cells precisely. Here, we summarize established protocols to generate and analyze antitumor receptor-transduced T cells. These procedures help to further assess characteristics of gene-modified T cells, resulting in promotion of translational research for cancer immunotherapy.


Assuntos
Citometria de Fluxo/métodos , Proteínas do Tecido Nervoso/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Fator de Crescimento Neural/genética , Linfócitos T/transplante , Transdução Genética/métodos , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/instrumentação , Técnica Direta de Fluorescência para Anticorpo/instrumentação , Técnica Direta de Fluorescência para Anticorpo/métodos , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária , Neoplasias/imunologia , Neoplasias/terapia , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Fator de Crescimento Neural/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo
12.
Methods Mol Biol ; 2048: 41-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396927

RESUMO

Adoptive T cell therapy is an attractive strategy in tumor immunotherapy. The transfer of in vitro expanded tumor-associated antigen (TAA)-specific T cells from patients may effectively destroy the original tumor cells. One of the limitations is a rapid acquisition of tolerant (anergy, deletion, dysfunctional, and/or exhausted) phenotypes. We and others found that stem cell memory T (TSCM) cells are strongly resistant to tolerance, showing strong expansion and persistence in vivo and providing long-lasting antitumor effects. We previously established that phenotypically TSCM cells (iTSCM) can be induced using a simple coculture of activated T cells with OP9 stroma cells expressing a Notch ligand. Here, we describe a defined protocol for generating human iTSCM cells, including reagents, culture setting, and procedure.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Separação Celular/métodos , Citometria de Fluxo/métodos , Cultura Primária de Células/métodos , Animais , Antígenos de Neoplasias/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular/métodos , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Citometria de Fluxo/instrumentação , Técnica Direta de Fluorescência para Anticorpo , Voluntários Saudáveis , Humanos , Memória Imunológica , Imunoterapia Adotiva/métodos , Ativação Linfocitária , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais , Camundongos , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução Genética/métodos
13.
Methods Mol Biol ; 2048: 53-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396928

RESUMO

The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that encounter embryonic stem cells. Adoptive T cell immunotherapy is a form of cellular immunotherapy that involves transfusion of functional T cells. However, this approach requires T cell expansion and the process causes T cell exhaustion. As a result, highly expanded T cells have not proven to be particularly effective for treatments. This exhaustion issue could be overcome due to rejuvenation of T cells by reprogramming to pluripotency and redifferentiation to T cells. This is a potential therapeutic strategy for combating various types of cancer.


Assuntos
Reprogramação Celular/imunologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Cultura Primária de Células/métodos , Linfócitos T Citotóxicos/fisiologia , Reprogramação Celular/genética , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes/genética , Vírus Sendai/genética , Fatores de Transcrição/genética , Transdução Genética/métodos
14.
Methods Mol Biol ; 2048: 81-84, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396932

RESUMO

The use of induced pluripotent stem cells (iPSCs) as a cell source for producing cytotoxic T lymphocytes (CTLs) is expected to have advantages in the antigen specificity, rejuvenation profile, and reproducible number of CTLs. We have developed the way to differentiate CD8αß T cells from TCR-transduced iPSCs (TCR-iPSCs). These T cells express monoclonal expression of the transduced TCR. Generating CD8αß CTLs from TCR-iPSC could contribute to safe and effective allogeneic regenerative T cell immunotherapies.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Linfócitos T Citotóxicos/transplante , Transdução Genética/métodos , Animais , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem Celular , Separação Celular/instrumentação , Separação Celular/métodos , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Meios de Cultura/metabolismo , Citocinas/metabolismo , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Lentivirus/genética , Células-Tronco Mesenquimais , Camundongos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Transdução Genética/instrumentação , Transplante Homólogo/métodos
15.
Methods Mol Biol ; 2048: 131-141, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396937

RESUMO

In recent years cancer immunotherapy, especially the cell-based immunotherapy, has reached several milestones and achieved a lot of cancer remission in the clinics. Obtaining a more potent and effective cytotoxic T lymphocytes (CTLs) for cancer immunotherapy is always the ultimate goal for the researchers. However, the difficulty in harvesting a large number of tumor antigen-specific CTLs from the tumor patient is still a major obstacle we need to overcome. In our previous studies, it is shown that pluripotent stem cell-derived CTL-especially the genetically engineered antigen-specific CTLs-may serve as a good source of unlimited number of highly reactive and antigen-specific CTLs. Here we present a two-step method for the generation of antigen-specific T lymphocytes from iPS cells by in vitro priming and in vivo maturation.


Assuntos
Técnicas de Cultura de Células/métodos , Imunoterapia Adotiva/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Melanoma Experimental/terapia , Linfócitos T Citotóxicos/transplante , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Meios de Cultura/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Vetores Genéticos/genética , Proteínas de Homeodomínio/genética , Humanos , Melanoma Experimental/imunologia , Camundongos , Camundongos Knockout , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Linfócitos T Citotóxicos/fisiologia , Transdução Genética/instrumentação , Transdução Genética/métodos
16.
Bone ; 128: 115032, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31398502

RESUMO

Traditionally, ex vivo gene therapy involves a two-step approach, with culture expansion of cells prior to transduction and implantation. We have tried to simplify this strategy and eliminate the time and cost associated with culture expansion, by introducing "next-day" regional gene therapy using human bone marrow cells. The purpose of this study was to determine whether a lentiviral vector (LV) carrying the cDNA for BMP-2 can transduce freshly isolated human BM cells, leading to abundant BMP production and bone formation in vivo, and evaluate the in vivo osteoinductive potential of "next-day" gene therapy and the standard "two-step" tissue culture expansion approach. To this end, human bone marrow cells (HBMC) from patients undergoing total hip arthroplasty were harvested, transduced with a BMP-2-expressing LV either overnight ("next day" gene therapy; ND) or after culture expansion (cultured "two-step" approach; C) and then implanted into a rat critical-sized femoral defect. The animals were randomly assigned to one of the following groups: I; ND-HBMC transduced with LV-TSTA BMP-2, II; ND-HBMC transduced with LV-TSTA GFP, III; non-transduced ND-HBMC; IV; C-HBMC transduced with LV-TSTA BMP-2, V; C-HBMC transduced with LV-TSTA-GFP, VI; non-transduced C-HBMC. Treatment with either "next-day" or cultured HBMC demonstrated a significant increase in new bone formation compared with all negative control groups as seen in plain radiographs, microCT and histologic/histomorphometric analysis. At 12 weeks post-op, complete defect union on plain X-rays occurred in 7/14 animals in the ND-HBMC/BMP-2 group and 12/14 in the C-HBMC/BMP-2 treated rats. The two-step approach was associated with more consistent results, a higher union rate, and superiority with regards to all of the studied bone healing parameters. In this study we demonstrate proof of concept that BMP-2-transduced human bone marrow cells can be used to enhance bone healing in segmental bone defects, and that regional gene therapy using lentiviral transduction has the osteoinductive potential to heal large bone defects in clinical settings.


Assuntos
Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Terapia Genética/métodos , Adulto , Idoso , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/terapia , Proteína Morfogenética Óssea 2/genética , Células Cultivadas , Feminino , Humanos , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , Osteogênese/genética , Osteogênese/fisiologia , Ratos , Ratos Nus , Estresse Mecânico , Transdução Genética/métodos , Microtomografia por Raio-X
17.
Hum Gene Ther ; 30(10): 1284-1296, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31407607

RESUMO

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.


Assuntos
Capsídeo/química , Dependovirus/genética , Engenharia Genética/métodos , Vetores Genéticos/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transdução Genética/métodos , Sequência de Aminoácidos , Capsídeo/metabolismo , Diferenciação Celular , Dependovirus/metabolismo , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Biblioteca de Peptídeos
18.
Proc Natl Acad Sci U S A ; 116(37): 18571-18577, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31375630

RESUMO

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.


Assuntos
Bacteriófago M13/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias/terapia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bacteriófago M13/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral , Dependovirus/imunologia , Endossomos/imunologia , Endossomos/virologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Lisossomos/imunologia , Lisossomos/virologia , Camundongos , Neoplasias/genética , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Estudo de Prova de Conceito , Ratos , Transdução Genética/métodos , Internalização do Vírus , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Physiol Genomics ; 51(9): 449-461, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31398086

RESUMO

A resurgence in the development of newer gene therapy systems has led to recent successes in the treatment of B cell cancers, retinal degeneration and neuromuscular atrophy. Gene therapy offers the ability to treat the patient at the root cause of their malady by restoring normal gene function and arresting the pathological progression of their genetic disease. The current standard of care for most genetic diseases is based upon the symptomatic treatment with polypharmacy while minimizing any potential adverse effects attributed to the off-target and drug-drug interactions on the target or other organs. In the kidney, however, the development of gene therapy modifications to specific renal cells has lagged far behind those in other organ systems. Some positive strides in the past few years provide continued enthusiasm to invest the time and effort in the development of new gene therapy vectors for medical intervention to treat kidney diseases. This mini-review will systematically describe the pros and cons of the most commonly tested gene therapy vector systems derived from adenovirus, retrovirus, and adeno-associated virus and provide insight about their potential utility as a therapy for various types of genetic diseases in the kidney.


Assuntos
Terapia Genética/métodos , Nefropatias/terapia , Adenoviridae/genética , Animais , Proteínas do Capsídeo/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Humanos , Lentivirus/genética , Camundongos , Transdução Genética/métodos
20.
Blood ; 134(16): 1298-1311, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31416800

RESUMO

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Proteínas de Membrana/efeitos dos fármacos , Resveratrol/farmacologia , Transdução Genética/métodos , Animais , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Vetores Genéticos , Xenoenxertos , Humanos , Lentivirus , Proteínas de Membrana/metabolismo , Camundongos , Transporte Proteico/efeitos dos fármacos
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