RESUMO
Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.
Assuntos
Edição de Genes , Imunodeficiência Combinada Severa/genética , Animais , Proteína Quinase Ativada por DNA/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCID , Proteínas Nucleares/genéticaRESUMO
Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.
Assuntos
Proteína BRCA2/genética , Anemia de Fanconi/genética , Terapia Genética , Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Proteína BRCA2/biossíntese , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular , Dano ao DNA/genética , Anemia de Fanconi/patologia , Anemia de Fanconi/terapia , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , CamundongosRESUMO
Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.
Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Cadeias kappa de Imunoglobulina/isolamento & purificação , Imunotoxinas/isolamento & purificação , Fatores de Virulência/imunologia , ADP Ribose Transferases/uso terapêutico , Animais , Toxinas Bacterianas/uso terapêutico , Linhagem Celular , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Exotoxinas/uso terapêutico , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/uso terapêutico , Imunotoxinas/química , Imunotoxinas/uso terapêutico , Camundongos , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Fatores de Virulência/uso terapêutico , Exotoxina A de Pseudomonas aeruginosaRESUMO
The dissection of the molecular pathways participating in genetic instability disorders has rendered invaluable information about the mechanisms of cancer pathogenesis and progression, and is offering a unique opportunity to establish targeted anticancer therapies. Fanconi anaemia (FA) is a paradigm of cancer-prone inherited monogenic disorders. Moreover, accumulated evidence indicates that genetic and epigenetic alterations in FA genes can also play an important role in sporadic cancer in the general population. Here, we summarise current progress in the understanding of the molecular biology of FA and review the principal mechanisms accounting for a disrupted FA pathway in sporadic cancer. Additionally, we discuss the impact of these findings in the development of new anticancer therapies, particularly with DNA interstrand crosslinkers and with new inhibitors of the FA and/or alternative DNA repair pathway (AU)
Assuntos
Humanos , Masculino , Feminino , Anemia de Fanconi/complicações , Anemia de Fanconi/genética , Neoplasias/genética , Lesões Pré-Cancerosas/complicações , Lesões Pré-Cancerosas/genética , Transformação Celular Neoplásica/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The efficacy of gene therapy for the treatment of inherited immunodeficiency has been highlighted in recent clinical trials, although in some cases complicated by insertional mutagenesis and silencing of vector genomes through methylation. To minimize these effects, we have evaluated the use of regulatory elements that confer reliability of gene expression, but also lack potent indiscriminate enhancer activity. The Vav1 proximal promoter is particularly attractive in this regard and may be useful in situations where high-level or complex regulation of gene expression is not necessary. X-linked severe combined immunodeficiency (SCID-X1) is a good candidate for such an approach, particularly as there may be additional disease-related intrinsic risks of leukemogenesis, and where safety is therefore a paramount concern. We have tested whether lentiviral vectors expressing the common cytokine receptor gamma chain under the control of the proximal Vav1 gene promoter are effective for correction of signaling defects and the disease phenotype. Despite low-level gene expression, we observed near-complete restoration of cytokine-mediated STAT5 phosphorylation in a model cell line. Furthermore, at low vector copy number, highly effective T- and B-lymphocyte reconstitution was achieved in vivo in a murine model of SCID-X1, in both primary and secondary graft recipients. This vector configuration deserves further evaluation and consideration for future clinical trials.
Assuntos
Terapia Genética , Subunidade gama Comum de Receptores de Interleucina/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-vav/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Ordem dos Genes , Vetores Genéticos/genética , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Transdução de Sinais , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapiaRESUMO
In utero cell and gene therapies constitute alternative strategies to the postnatal treatment of inherited diseases. Fetal hematopoietic progenitors could be a potential source of donor cells for these strategies. In this study, hematopoietic lineage-negative fetal liver cells from 14.5-day-old fetuses were transduced under different cytokine and culture combinations using a lentiviral vector expressing the enhanced green fluorescent protein (EGFP). When cells were transduced for 6 h in the presence of mSCF, hTPO and FLT3-L in retronectin-coated dishes at a multiplicity of infection of 10 transduction units/cell, up to 70% of granulo-macrophage colony-forming cells expressed the EGFP reporter gene. In utero transplantation experiments revealed that conditions leading to high transduction efficiencies were associated with poor engraftments of syngeneic recipients. Significantly, this effect was associated with the detection of a humoral and cellular immunoresponse against the transgenic protein. Moreover, the humoral response against EGFP was detected not only in in utero transplanted recipients but also in the operated mothers, suggesting the maternal origin of the anti-EGFP immunoresponse. These observations reinforce the necessity of carefully studying the potential immunoresponses in future prenatal gene therapy protocols.
Assuntos
Formação de Anticorpos , Feto , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Celular , Fígado/embriologia , Prenhez , Transgenes/imunologia , Animais , Feminino , Terapia Genética/métodos , Sobrevivência de Enxerto , Lentivirus/genética , Camundongos , Camundongos SCID , Gravidez , Transdução GenéticaRESUMO
AIMS/HYPOTHESIS: Recovery from diabetes requires restoration of beta cell mass. Igf1 expression in beta cells of transgenic mice regenerates the endocrine pancreas during type 1 diabetes. However, the IGF-I-mediated mechanism(s) restoring beta cell mass are not fully understood. Here, we examined the contribution of pre-existing beta cell proliferation and transdifferentiation of progenitor cells from bone marrow in IGF-I-induced islet regeneration. METHODS: Streptozotocin (STZ)-treated Igf1-expressing transgenic mice transplanted with green fluorescent protein (GFP)-expressing bone marrow cells were used. Bone marrow cell transdifferentiation and beta cell replication were measured by GFP/insulin and by the antigen identified by monoclonal antibody Ki67/insulin immunostaining of pancreatic sections respectively. Key cell cycle proteins were measured by western blot, quantitative RT-PCR and immunohistochemistry. RESULTS: Despite elevated IGF-I production, recruitment and differentiation of bone marrow cells to beta cells was not increased either in healthy or STZ-treated transgenic mice. In contrast, after STZ treatment, IGF-I overproduction decreased beta cell apoptosis and increased beta cell replication by modulating key cell cycle proteins. Decreased nuclear levels of cyclin-dependent kinase inhibitor 1B (p27) and increased nuclear localisation of cyclin-dependent kinase (CDK)-4 were consistent with increased beta cell proliferation. However, islet expression of cyclin D1 increased only after STZ treatment. In contrast, higher levels of cyclin-dependent kinase inhibitor 1A (p21) were detected in islets from non-STZ-treated transgenic mice. CONCLUSIONS/INTERPRETATION: These findings indicate that IGF-I modulates cell cycle proteins and increases replication of pre-existing beta cells after damage. Therefore, our study suggests that local production of IGF-I may be a safe approach to regenerate endocrine pancreas to reverse diabetes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estreptozocina/farmacologiaRESUMO
Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.
Assuntos
Eritrócitos/enzimologia , Células Precursoras Eritroides/metabolismo , Expressão Gênica , Terapia Genética , Piruvato Quinase/genética , Transgenes/genética , Animais , Antígenos CD34/análise , Western Blotting , Diferenciação Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Piruvato Quinase/farmacologia , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. METHODS: We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. RESULTS: Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. CONCLUSIONS: The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.
Assuntos
Vetores Genéticos , Vírus da Leucemia Murina/genética , Transdução Genética , Células 3T3 , Animais , Sequência de Bases , Transplante de Medula Óssea , Linhagem Celular , Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Provírus/genética , RNA Mensageiro/genéticaRESUMO
Fanconi anemia (FA) is an inherited DNA repair disorder characterized by genetic instability of cells lacking a functional FA/BRCA pathway. Previous studies have shown that in vitro stimulation of bone marrow cells (BMCs) from FA mice promotes apoptosis, reduces the reconstitution ability of the stem cells, and induces myelodysplasia and myeloid leukemia upon reinfusion of the cells. This suggests the convenience of adapting standard protocols of gene therapy to FA. Here we show that the reserve of BM progenitors in FA patients is generally below 20% of normal values. Because this reduced reserve could activate the cycling of BM progenitors, we developed a simplified protocol to transduce BMCs from FA patients with gammaretroviral vectors. We demonstrate that a short in vitro manipulation (12-24 hr) of fresh mononuclear BMCs is sufficient to transduce 42% of hematopoietic progenitors from FA-A patients, in the absence of in vitro prestimulation. When FANCA-expressing vectors were used, this simple procedure reversed the phenotype of the BM progenitors from these patients. We propose that our approach will be more efficient and safer compared with standard gene therapy protocols for FA.
Assuntos
Anemia de Fanconi/terapia , Terapia Genética/métodos , Células-Tronco Hematopoéticas/fisiologia , Transdução Genética/métodos , Células da Medula Óssea/fisiologia , Células Cultivadas , Anemia de Fanconi/sangue , Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Humanos , Valores de ReferênciaRESUMO
Using an experimental mouse model, we have investigated the kinetics of hematopoietic reconstitution of recipients transplanted during fetal development with fresh and transduced hematopoietic stem cells (HSCs). Total bone marrow (BM) and purified Lin(-)Sca-1(+) cells, either fresh or transduced ex vivo with enhanced green fluorescent protein (EGFP)-encoding retroviral vectors, were in utero transplanted (IUT) into fetal mice. Data obtained 2 months after transplantation showed a similar proportion of engrafted animals, regardless of the fact that samples were purified or not on HSCs, and subjected or not to ex vivo transduction with retroviral vectors. The transplantation of grafts enriched in HSCs, either fresh or transduced, always improved the levels of donor chimerism of IUT mice in comparison with results obtained in mice transplanted with unpurified BM grafts (6.8 and 7.3% versus 1.15% median values, respectively). Significantly, engrafted recipients that were transplanted with the transduced graft always contained transduced EGFP(+) cells in peripheral blood (around 5% of donor cells were EGFP(+) at 2 months post-transplantation). This proportion was essentially maintained at longer times post-transplantation, as well as in secondary recipients transplanted with the BM of IUT mice. Our study describes for the first time a significant and stable engraftment of unconditioned mice subjected to IUT with HSCs transduced with retroviral vectors.
Assuntos
Doenças Fetais/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Retroviridae/genética , Transdução Genética/métodos , Animais , Quimera , Feminino , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Modelos Animais , GravidezRESUMO
We have reported short periods of post transplant neutropenia in human patients co-transplanted with cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells. To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have co-transplanted fixed numbers of human CB CD34+ cells mixed with different numbers of MPB CD34+ cells into NOD/SCID mice. We periodically quantified the proportion of human cells and the relative contribution of MPB and CB cells to the human engraftment on marrow aspirates. At the lowest MPB/CB ratios (5 : 1, 10 : 1), the contribution of CB cells predominated at all time points analyzed, and in three out of four experiments MPB cell contributions progressively decreased from day +15. At higher MPB/CB ratios, MPB cells had a more important contribution to both early and late engraftment, with the highest cell ratio resulting in only marginal CB cell engraftment. Therefore, our results showed greater potential, on a per cell basis, of human CB vs MPB cells for competitive sustained engraftment in the xenogeneic model used, which was only abrogated by the co-infusion of very high numbers of MPB cells.
Assuntos
Antígenos CD34 , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sobrevivência de Enxerto , Transplante de Células-Tronco de Sangue Periférico , Animais , Humanos , Cinética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Animais , Neutropenia/etiologia , Transplante HeterólogoRESUMO
Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate.
Assuntos
Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Deleção de Genes , Reação em Cadeia da Polimerase/métodos , Elementos Alu , Análise Mutacional de DNA , Éxons/genética , Anemia de Fanconi/epidemiologia , Proteína do Grupo de Complementação A da Anemia de Fanconi , Heterogeneidade Genética , Heterozigoto , Humanos , Espanha/epidemiologiaRESUMO
The insertion of suicide genes in donor T lymphocytes constitutes the basis of new approaches aiming at the treatment of the graft-versus-host disease (GVHD), a frequent complication in recipients of allogeneic haematopoietic grafts. In this study we investigated the impact that the ex vivo manipulation required for the retroviral transduction of T cells had on the functionality and differentiation of these cells. Compared to fresh T cells, samples that had been subjected to standard activation (1 microg/ml of both anti-CD3i and anti-CD28i MoAbs) followed by transduction with vectors encoding for the HSV-tk and tNGFR genes maintained the proliferative response to an allogeneic stimulus. These cells, however, had a significantly lower cytotoxic response to allogeneic cells compared to fresh samples. When the concentration of anti-CD3i was reduced to up to 1000-fold (1 ng/ml), similar T-cell transductions were obtained, while the cytotoxicity of the ex vivo manipulated samples was significantly recovered, when assessed either at 7 or 14 days of culture. In all instances, a similar functionality was observed in transduced samples not subjected to immunomagnetic cell sorting, compared to purified fractions enriched in NGFR(+) and NFGR(-) cells. The analysis of CD45RA and CCR7 markers in samples transduced under standard stimulatory conditions showed a differentiation of fresh CD8(+) CD45RA(+)/CCR7(+) naive cells to cells having a predominant central CD45RA(-)/CCR7(+) and effector CD45RA(-)/CCR7(-) memory phenotype. However, when samples were activated with low doses of anti-CD3i, a significant population of naive cells became apparent. Although activation with high doses of anti-CD3i/anti-CD28i resulted in a similar phenotype in both NGFR(+) and NFGR(-) populations, the naive population observed in samples activated with low concentrations of anti-CD3i was almost restricted to the NGFR(-) population. These results show that reducing the stimulation mediated by anti-CD3i in protocols of T-cell retroviral gene transfer significantly helps to preserve the cytotoxic capacity of these cells to allogeneic cells, without affecting the susceptibility of these cells to the retroviral vector. In addition, we observed that modulating the activation of transduced T cells implies the generation of changes in the differentiation of CD8(+) cells, although we could not establish a direct relationship between the CD45RA/CCR7 phenotype of these cells and their cytotoxic reactivity to an allogeneic stimulus.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/imunologia , Retroviridae/genética , Subpopulações de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Citotoxicidade Imunológica , Humanos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/virologia , Transdução GenéticaRESUMO
Autologous bone marrow transplantation is an alternative therapeutic option for acute myeloid leukemia patients lacking a compatible donor. However, bone marrow from these patients may contain residual leukemic cells that should be ideally eliminated prior to the infusion of the graft. With the aim of developing more efficient protocols of graft purging, adenoviral-mediated gene transfer protocols have been conducted. We studied whether suicide adenoviral vectors expressing the cytosine deaminase gene (AdCD) could be used for selectively killing leukemic WEHI-3B cells. The AdCD transduction followed by the 5-fluorocytosine exposure abrogated the growth of WEHI-3B cells in vitro, with a minimal effect on normal hematopietic progenitors. To test the efficacy of the purging protocol in vivo, bone marrow cells were mixed with syngenic WEHI-3B cells and this chimeric cell population was transduced with AdCD vectors. Infected cells were injected into myeloablated Balb-c mice, which then received a 5-fluorocytosine treatment for 4 days. All mice transplanted with unpurged bone marrow developed leukemia and died. However, 90% of recipients receiving the purging treatment were healthy up to 9 months post-transplantation and had a perfectly re-established hematopoietic system, without any signal of leukemic cell presence. In conclusion, suicide adenoviral vectors are proposed as a tool for the purging of Adenoviral-susceptible myeloid leukemia cells contaminating autologous bone marrow grafts.
Assuntos
Adenoviridae/genética , Purging da Medula Óssea/métodos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Leucemia/terapia , Transdução Genética/métodos , Animais , Transplante de Medula Óssea , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Transplante AutólogoRESUMO
In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Dose Máxima Tolerável , Neutropenia/induzido quimicamente , Valor Preditivo dos Testes , Xenobióticos/toxicidade , Doença Aguda , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Europa (Continente) , Sangue Fetal/citologia , Humanos , Cooperação Internacional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutropenia/patologia , Reprodutibilidade dos Testes , Método Simples-Cego , Estados Unidos , Xenobióticos/classificaçãoAssuntos
Genes Virais , Marcadores Genéticos , Vetores Genéticos/efeitos adversos , Receptor de Fator de Crescimento Neural/análise , Retroviridae/genética , Animais , Transplante de Medula Óssea , Cães , Humanos , Leucemia/etiologia , Leucemia/prevenção & controle , Camundongos , Ratos , Transdução GenéticaRESUMO
Ecteinascidine-743 (ET-743) and aplidine are two marine-derived antineoplastics currently in phase II development. With the aim of evaluating whether in vitro haematopoietic studies can predict the toxicity of these two drugs in patients, human bone marrow (BM) samples were incubated with these drugs under conditions which mimicked the administration exposures used in the clinics. As it was observed in different cancer cell lines, ET-743 was more toxic on an equimolar basis in human hematopoietic progenitors (inhibitory concentration reducing the viability to 50% after 24 h exposures; IC50(24h): 10-50 nM) compared with doxorubicin (IC50(24h) values: 280-460 nM), used as a control anticancer drug. In contrast to the high haematotoxic effects observed for ET-743, similar IC values were obtained for aplidine (IC50(24h): 150-530 nM) compared with doxorubicin. For both ET-743 and aplidine, the megakaryocytic progenitor was the most sensitive, compared with the other haematopoietic progenitors (IC50 values were 3- to 5-fold lower in the CFU-Megs compared with the CFU-GMs). The observation that the Cmax observed in patients treated with the aplidine maximum tolerated dose (MTD) (7.1 nM) was 21-75 fold lower than the IC50(24h) value observed for the different haematopoietic progenitors is highly consistent with the lack of haematotoxicity observed in patients treated with this drug. In the case of ET-743, differences between the Cmax value corresponding to the MTD (2.6 nM) and the in vitro IC50 values corresponding to the different progenitors were much lower (4-19-fold), also consistent with the haematotoxicity that was observed in patients treated at recommended doses (RDs) and MTDs. Although CFU-Megs were more sensitive than CFU-GM progenitors to ET-743 in vitro, clinical data showed that neutropenic events were more frequent than thrombocytopenic episodes. Aiming to further improve the predictive value of in vitro IC values corresponding to the different haematopoietic progenitors, additional refinement parameters derived from pharmacokinetic and animal studies are proposed.
Assuntos
Antineoplásicos/efeitos adversos , Doenças da Medula Óssea/induzido quimicamente , Depsipeptídeos , Dioxóis/efeitos adversos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Isoquinolinas/efeitos adversos , Neoplasias/tratamento farmacológico , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Doxorrubicina/efeitos adversos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Peptídeos Cíclicos/efeitos adversos , Tetra-Hidroisoquinolinas , Trabectedina , Células Tumorais CultivadasRESUMO
We have investigated the influence of ex vivo expansion of human CD34(+) cord blood cells on the expression and function of adhesion molecules involved in the homing and engraftment of haematopoietic progenitors. Ex vivo expansion of umbilical cord blood CD34(+) cells for 6 d in the presence of interleukin 3 (IL-3), IL-6 and stem cell factor (SCF) or IL-11, SCF and Flt-3L resulted in increased expression of alpha 4, alpha 5, beta 1, alpha M and beta 2 integrins. However, a significant decrease in the adhesion of progenitor cells to fibronectin was observed after the ex vivo culture (adhesion of granulocyte-macrophage colony-forming units (CFU-GM) was 22 +/- 4% in fresh cells versus 5 +/- 2% and 2 +/- 2% in each combination of cytokines). Incubation with the beta 1 integrin-activating antibody TS2/16 restored adhesion to fibronectin. Transplantation of ex vivo expanded umbilical cord blood CD34(+) cells was associated with an early delayed engraftment in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Incubation of cells with the monoclonal antibody TS2/16 before transplantation almost completely abrogated NOD/SCID repopulating ability of both fresh and expanded CD34(+) cells. The seeding efficiency of fresh and expanded CD34(+) cells was similar, but markedly reduced after incubation with the TS2/16 monoclonal antibody. Our results show that functional activation of beta 1 integrins could overcome the decreased very late antigen (VLA)-4- and VLA-5-mediated adhesion observed after ex vivo expansion of haematopoietic progenitors. However, in vivo, these effects induced an almost complete abrogation of the homing and repopulating ability of CD34(+) UCB cells.