RESUMEN
A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.
Asunto(s)
Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales/métodos , Linfoma/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Animales , Biomarcadores , Línea Celular Tumoral , Ciclofosfamida/análogos & derivados , Ciclofosfamida/química , Perros , Doxorrubicina/química , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Prednisolona/química , Serina-Treonina Quinasas TOR/metabolismo , Vincristina/químicaRESUMEN
Minimal conditioning or even no conditioning would be the preferred preparation for most gene therapy applications for nonmalignant diseases. However, reduced intensity conditioning (RIC) regimens in patients with nonhematologic malignancies have not led to long-term engraftment unless a selective advantage was present for the transplanted donor cells. Similar findings have also been observed in a number of large animal studies. Inadequate myelosuppression levels were thought to be responsible for the outcomes. To address this issue several innovative protocols in small animals have been presented with selective hematopoietic myelosuppression and less systemic toxicity. Such protocols promised to curb the transplant-related morbidity and mortality in myeloablative conditioning and provide effective long-term engraftment, especially in patients with gene-corrected autografts. In the present study we have tested some of these promising RIC regimens in nonhuman primates, a clinically relevant large animal model. Our data suggest that transient myelosuppression induced by anti-c-Kit antibody in conjunction with low-dose irradiation may lead to long-term engraftment, albeit at low levels. The animals with busulfan conditioning with or without anti-c-Kit that received gene-modified autologous transplants with green fluorescent protein expression had similar myelosuppression, but failed long-term engraftment and despite immunosuppressive treatment had all the hallmarks seen previously in similar models without immunosuppression. Our preliminary data expand current knowledge of RIC and emphasize the need to explore whether specific and directed myelosuppression alone is adequate in the absence of microenvironmental modulation, or whether innovative combinations are necessary for safe and effective engraftment.
Asunto(s)
Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Inmunosupresores/administración & dosificación , Acondicionamiento Pretrasplante , Animales , Antígenos CD34/inmunología , Modelos Animales de Enfermedad , Humanos , Terapia de Inmunosupresión/métodos , Lentivirus/genética , Lentivirus/inmunología , Macaca/inmunología , Trasplante AutólogoRESUMEN
Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.
Asunto(s)
Proteínas de Homeodominio/genética , Modelos Animales , Trasplante de Células Madre , Factores de Transcripción/genética , Animales , Marcadores Genéticos , Humanos , Macaca nemestrina , PrimatesRESUMEN
Umbilical cord blood (CB) transplantation is a promising therapeutic approach but continues to be associated with delayed engraftment and infections. Here, we explored in our macaque CB transplant model expansion and engraftment kinetics of cells expanded with the combination of HOXB4 and Delta-1. CB cells were divided into two equal fractions; one fraction was transduced with HOXB4 yellow fluorescent protein (YFP) and expanded on control OP9 cells, and the other was transduced with HOXB4 green fluorescent protein (GFP) and expanded on Delta-expressing OP9 cells (OP9-DL1). Both fractions were transplanted into myeloablated subjects. Neutrophil and platelet recovery occurred within 7 and 19 days respectively, which was significantly earlier than in our previous study using cells expanded with HOXB4 alone, which resulted in neutrophil recovery within 12 days (P = 0.05) and platelet recovery within 37 days (P = 0.02). Furthermore, two of three animals in the current study remained fully transfusion-independent after transplantation. By day 30, reconstitution of lymphocytes was significantly greater with the HOXB4/OP9-DL1 expanded cells in all animals (P = 0.05). In conclusion, our data show that the combination of OP9-DL1 and HOXB4 can result in increased numbers of repopulating cells, thus leading to rapid engraftment and transfusion independence in macaques transplanted with autologous, expanded CB cells.
Asunto(s)
Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Macaca nemestrina/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Animales , Antígenos CD34/metabolismo , Plaquetas/metabolismo , Transfusión Sanguínea/métodos , Diferenciación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Sangre Fetal/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macaca nemestrina/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages.
Asunto(s)
Linaje de la Célula , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Animales , Proteínas Bacterianas/genética , Ensayo de Unidades Formadoras de Colonias , Femenino , Genes Reporteros , Vectores Genéticos/genética , Supervivencia de Injerto , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Proteínas Luminiscentes/genética , Macaca nemestrina , Masculino , Modelos Animales , Mielopoyesis , Complicaciones Posoperatorias , Embarazo , Proteínas Recombinantes de Fusión/fisiología , Retroviridae/genética , Trombopoyesis , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transducción Genética , Acondicionamiento Pretrasplante , Integración ViralRESUMEN
Expansion of hematopoietic stem cells (HSCs) is beneficial in settings where HSC numbers are limited, such as cord blood transplantation. The human homeobox transcription factor HOXB4 has been shown to enhance stem cell expansion in several experimental models. We have shown previously that HOXB4 overexpression in monkey CD34(+) cells has a dramatic effect on expansion and engraftment of short-term repopulating cells. Here, we wished to compare the effects of HOXB4 and another candidate gene, NUP98-HOXA10hd (NA10hd). We used a competitive repopulation assay in pigtailed macaques to study engraftment of CD34(+) cells modified with gammaretroviral HOXB4YFP or NA10hdGFP. We found that HOXB4YFP contributed more to early hematopoiesis (<30 days), whereas NA10hdGFP contributed more to later hematopoiesis. In each case, we observed two distinct peaks in engraftment of NA10hd-transduced cells, one within 20 days post transplant and another after 5-6 months. Analysis of CD14(+), CD3(+), and CD20(+) subsets confirmed that higher percentages of cells of each lineage were derived from NA10hdGFP(+) progenitors than from HOXB4YFP(+) progenitors. In conclusion, we show that HOXB4 and NA10hd both have a significant impact on hematopoietic reconstitution; however, these effects are differential and therefore may offer complementary strategies for HSC expansion.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Factores de Transcripción/metabolismo , Animales , Antígenos CD34/metabolismo , Proteínas Bacterianas/metabolismo , Médula Ósea/metabolismo , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Eritrocitos/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Trasplante de Células Madre Hematopoyéticas , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Humanos , Leucocitos/metabolismo , Proteínas Luminiscentes/metabolismo , Macaca nemestrina , Modelos Animales , Proteínas de Complejo Poro Nuclear/genética , Retroviridae/genética , Factores de Transcripción/genéticaRESUMEN
The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells.
Asunto(s)
Genes Transgénicos Suicidas , Vectores Genéticos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Teratoma/metabolismo , Animales , Southern Blotting , Diferenciación Celular , Línea Celular , Proliferación Celular , Clonación Molecular , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lentivirus/genética , Macaca/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Mutagénesis Insercional , Medicina Regenerativa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Induced pluripotent stem (iPS) cells have great potential for regenerative medicine and gene therapy. Thus far, iPS cells have typically been generated using integrating viral vectors expressing various reprogramming transcription factors; nonintegrating methods have been less effective and efficient. Because there is a significant risk of malignant transformation and cancer involved with the use of iPS cells, careful evaluation of transplanted iPS cells will be necessary in small and large animal studies before clinical application. Here, we have generated and characterized nonhuman primate iPS cells with the goal of evaluating iPS cell transplantation in a clinically relevant large animal model. We developed stable Phoenix-RD114-based packaging cell lines that produce OCT4, SOX2, c-MYC, and KLF4 (OSCK) expressing gammaretroviral vectors. Using these vectors in combination with small molecules, we were able to efficiently and reproducibly generate nonhuman primate iPS cells from pigtailed macaques (Macaca nemestrina). The established nonhuman primate iPS cells exhibited pluripotency and extensive self-renewal capacity. The facile and reproducible generation of nonhuman primate iPS cells using defined producer cells as a source of individual reprogramming factors should provide an important resource to optimize and evaluate iPS cell technology for studies involving stem cell biology and regenerative medicine.
Asunto(s)
Reprogramación Celular , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas , Macaca nemestrina/genética , Animales , Diferenciación Celular/genética , Línea Celular , Reprogramación Celular/genética , Femenino , Fibroblastos/citología , Gammaretrovirus/genética , Gammaretrovirus/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Macaca nemestrina/metabolismo , Masculino , Ratones , Análisis por Micromatrices , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Medicina Regenerativa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Riesgo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , TransfecciónRESUMEN
Umbilical cord blood (UCB) is an attractive cell source for hematopoietic cell transplantation (HCT). Here we examine whether the combination of homeobox B4 (HOXB4) and Delta-1 ligand (DL) synergize when used together. Monkey and human UCB CD34(+) cells were transduced with a HOXB4-expressing gammaretroviral vector and cultured with DL. Individual and combined effects of HOXB4 and DL were assessed by colony-forming unit assays, flow cytometry, and nonobese diabetic/severe combined immune deficienct mouse transplantation. The presence of DL yielded higher percentage of CD34(+) and CD7(+) cells and lower percentages of CD14(+) cells than non-DL cultures. Furthermore, HOXB4 yielded higher percentages of CD34(+) and CD14(+) cells than non-HOXB4 cultures. Interestingly, coculture with DL-expressing OP9 cells resulted in better maintenance of HOXB4 than culture in DL-conditioned medium. Culture of HOXB4-transduced human cells in the presence of DL yielded enhanced generation of repopulating cells with higher levels of engraftment of human CD45(+), CD34(+), CD3(+), CD20(+), and CD41(+) cells compared with either factor individually. Our results demonstrate enhanced generation of hematopoietic progenitors by combining HOXB4 and DL; addition of DL further enhances expansion of multipotent cells capable of repopulating lymphoid and megakaryocyte lineages, which is not observed with HOXB4 alone.
Asunto(s)
Sangre Fetal/citología , Sangre Fetal/trasplante , Proteínas de Homeodominio/fisiología , Proteínas de la Membrana/fisiología , Factores de Transcripción/fisiología , Animales , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados/farmacología , Sangre Fetal/metabolismo , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Linfocitos/metabolismo , Macaca nemestrina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Mieloides/metabolismo , FenotipoRESUMEN
Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR). Cocal-pseudotyped lentiviral vectors can be produced at titers as high as with VSV-G, have a broad tropism, and are stable, allowing for efficient concentration by centrifugation. Additionally, cocal vectors are more resistant to inactivation by human serum than VSV-G-pseudotyped vectors, and efficiently transduce human CD34(+) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating cells (SRCs), and long-term primate hematopoietic repopulating cells. These studies establish the potential of cocal-pseudotyped lentiviral vectors for a variety of scientific and therapeutic gene transfer applications, including in vivo gene delivery and hematopoietic stem cell (HSC) gene therapy.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/inmunología , Células Madre Hematopoyéticas , Lentivirus/inmunología , Glicoproteínas de Membrana/inmunología , Células Receptoras Sensoriales , Animales , Antígenos CD34/genética , Gatos , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Macaca , Glicoproteínas de Membrana/genética , Ratones , Ratones SCID , Transducción Genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The important contributions of the alpha4 integrin VLA-4 and the CXCR4/SDF-1 axis in mobilization have been demonstrated and thereby, these pathways can be suggested as rational targets for clinical stem cell mobilization in the absence of cytokine use. alpha4-blockade alone (in humans, macaques and mice), or genetic ablation of alpha4-integrin in mice, provides reproducible, but modest mobilization. Similarly, CXCR4 blockade with small-molecule antagonists mobilizes hematopoietic stem cells in all three species, but at least with the established single-injection schedule, the mobilization efficiency is marginally sufficient for clinical purposes. Hypothesizing that the different molecular targets (alpha4-integrin vs. CXCR4) might allow for additive mobilization effects, we therefore tested the efficacy of the combination of alpha4-integrin blockade with anti-functional antibodies and CXCR4 blockade with the small-molecule inhibitor AMD3100 in macaques, or the combination of conditional alpha4-integrin ablation and AMD3100 in mice. Mobilization was at least additive. While the prolonged effects of alpha4-blocking antibodies may not be suitable for clinical mobilization, future availability of small-molecule alpha4-antagonists in combination with AMD3100 could provide an alternative to granulocyte colony-stimulating factor.