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1.
Blood ; 123(10): 1483-6, 2014 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-24429337

RESUMO

We conducted a pilot trial to investigate the safety and effectiveness of mobilizing CD34(+) hematopoietic progenitor cells (HPCs) in adults with ß-thalassemia major. We further assessed whether thalassemia patient CD34(+) HPCs could be transduced with a globin lentiviral vector under clinical conditions at levels sufficient for therapeutic implementation. All patients tolerated granulocyte colony-stimulating factor well with minimal side effects. All cell collections exceeded 8 × 10(6) CD34(+) cells/kg. Using clinical grade TNS9.3.55 vector, we demonstrated globin gene transfer averaging 0.53 in 3 validation runs performed under current good manufacturing practice conditions. Normalized to vector copy, the vector-encoded ß-chain was expressed at a level approximating normal hemizygous protein output. Importantly, stable vector copy number (0.2-0.6) and undiminished vector expression were obtained in NSG mice 6 months posttransplant. Thus, we validated a safe and effective procedure for ß-globin gene transfer in thalassemia patient CD34(+) HPCs, which we will implement in the first US trial in patients with severe inherited globin disorders. This trial is registered at www.clinicaltrials.gov as #NCT01639690.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/terapia , Animais , Antígenos CD34/metabolismo , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Células Precursoras Eritroides/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Xenoenxertos , Humanos , Camundongos , Transdução Genética , Globinas beta/biossíntese , Talassemia beta/metabolismo
2.
Future Oncol ; 9(5): 713-26, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23647299

RESUMO

Metastatic melanoma accounts for approximately 80% of skin cancer-related deaths. Up to now there has been no effective treatment for stage IV melanoma patients due to the complexity and dissemination potential of this disease. Melanomas are heterogeneous tumors in which conventional therapies fail to improve overall survival. Targeted therapies are being developed, but the final outcome can be hampered by the incomplete knowledge of the process of melanoma progression. Even if the intracellular pathways are similar, the interaction of the cells with the surrounding environment should be taken into consideration. This article seeks to highlight some of the advances in the understanding of the molecular mechanisms underlying melanoma dissemination.


Assuntos
Melanoma/metabolismo , Redes e Vias Metabólicas/genética , Metástase Neoplásica/patologia , Neoplasias Cutâneas/metabolismo , Adesão Celular/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular/genética , Humanos , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Neoplasias Cutâneas/patologia
3.
Mol Ther ; 17(8): 1434-41, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19352322

RESUMO

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.


Assuntos
Cromatina/metabolismo , Vetores Genéticos/genética , Elementos Isolantes/fisiologia , Retroviridae/genética , Ouriços-do-Mar/genética , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Efeitos da Posição Cromossômica , Fator de Transcrição GATA1/metabolismo , Elementos Isolantes/genética , Camundongos , Células NIH 3T3 , Fator 1 de Transcrição de Octâmero/metabolismo , Ligação Proteica
4.
Stem Cells Int ; 2011: 673752, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21912558

RESUMO

Ex vivo expanded erythroblasts (EBs) may serve as advanced transfusion products provided that lodgment occurs in the macrophage-niche of the marrow permitting maturation. EBs expanded from adult and cord blood expressed the receptors (CXCR4, VLA-4, and P-selectin ligand 1) necessary for interaction with macrophages. However, 4-days following transfusion to intact NOD/SCID/IL2Rγ(null) mice, CD235a(pos) EBs were observed inside CD235a(neg) splenic cells suggesting that they underwent phagocytosis. When splenectomized and intact NOD/SCID/IL2Rγ(null) mice were transfused using retrovirally labeled human EBs, human cells were visualized by bioluminescence imaging only in splenectomized animals. Four days after injection, human CD235a(pos) cells were detected in marrow and liver of splenectomized mice but only in spleen of controls. Human CD235a(pos) erythrocytes in blood remained low in all cases. These studies establish splenectomized NOD/SCID/IL2Rγ(null) mice as a suitable model for tracking and quantification of human EBs in vivo.

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