RESUMEN
Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.
Asunto(s)
Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Glicoproteínas de Membrana/genética , Linfocitos T/metabolismo , Transducción Genética , Técnicas de Cultivo de Célula , Expresión Génica , Genes Reporteros , Ingeniería Genética , Células HEK293 , Humanos , Lentivirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de LDL/metabolismo , Receptores Virales/metabolismo , Transgenes , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Lentivirus/efectos de los fármacos , Lentivirus/genética , Sirolimus/farmacología , Transducción Genética/métodos , Animales , Vectores Genéticos/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.
Asunto(s)
Microambiente Celular , Microvasos/metabolismo , Trombopoyesis/fisiología , Anticuerpos/inmunología , Antígenos CD34/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Movimiento Celular , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Megacariocitos/citología , Megacariocitos/metabolismo , Microscopía Confocal , Microscopía Electrónica , Receptores CXCR4/inmunología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
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.