RESUMO
The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.
Assuntos
Genes Transgênicos Suicidas/genética , Terapia Genética , Leucemia/genética , Leucemia/terapia , Oncogenes/genética , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Modelos Animais de Doenças , Ganciclovir/farmacologia , Vetores Genéticos/genética , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Taxa de SobrevidaRESUMO
OBJECTIVE: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. METHODS: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. RESULTS: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. CONCLUSION: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.
Assuntos
Antígenos CD34 , Elementos de DNA Transponíveis , Eletroporação , Expressão Gênica , Células-Tronco Hematopoéticas , Mutagênese Insercional , Transposases/biossíntese , Elementos de DNA Transponíveis/genética , Eletroporação/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Mutagênese Insercional/métodos , Fatores de Tempo , Transposases/genéticaRESUMO
Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos/genética , Lentivirus/genética , Integração Viral , Antígenos CD34/metabolismo , Sequência de Bases , Células Cultivadas , Resistência a Medicamentos , Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , HIV-1/genética , Células HT29 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Jurkat , Canamicina Quinase/genética , Dados de Sequência Molecular , Neomicina/farmacologiaRESUMO
Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34+ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G glycoprotein (VSV-G).
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Antígenos CD34/biossíntese , Linhagem Celular , Produtos do Gene gag/metabolismo , Proteínas de Fluorescência Verde/metabolismo , HIV-1/metabolismo , Humanos , Imunoensaio , Glicoproteínas de Membrana/química , Modelos Genéticos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Células-Tronco/citologia , Fatores de Tempo , Transfecção , Transgenes , Ultracentrifugação , Proteínas do Envelope Viral/químicaRESUMO
Green fluorescent protein (GFP) is employed as a selection marker for gene transduction and to track tumor cells. Transduction of enhanced GFP (eGFP) into human neuroblastoma cell lines via a lentiviral vector significantly sensitized CHLA-20 (wild-type and functional TP53), and to a lesser extent CHLA-90 cells (multidrug-resistant, mutant, and nonfunctional TP53) to carboplatin, doxorubicin, etoposide, or melphalan, relative to cells transduced using the cell surface antigen CD80 as a selection marker. Total glutathione (GSH) was significantly up-regulated (1.8- to 2.8-fold) after eGFP (but not CD80) transduction in cell lines with, but not in those lacking, functional p53. Cytotoxicity of GSH depletion by buthionine sulfoximine in CHLA-20 (but not in CHLA-20-eGFP) was diminished by hypoxia (2% O(2)). Thus, oxidative stress produced by GFP selects for cells with up-regulated GSH in a p53-dependent manner, and also enhanced the cytotoxicity of anticancer drugs in neuroblastoma cell lines. Our data suggest caution when employing GFP-transduced cells to assess drug sensitivity and that using a cell surface antigen as a selection marker for gene transduction may perturb cells less than GFP.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Luminescentes/metabolismo , Neuroblastoma/tratamento farmacológico , Estresse Oxidativo , Transdução Genética , Apoptose/efeitos dos fármacos , Antígeno B7-1/metabolismo , Butionina Sulfoximina/farmacologia , Carboplatina/uso terapêutico , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/uso terapêutico , Glutationa/metabolismo , Proteínas de Fluorescência Verde , Humanos , Lentivirus/genética , Proteínas Luminescentes/genética , Melfalan/uso terapêutico , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.