RESUMO
Retroviral overexpression of NF-Ya, the regulatory subunit of the transcription factor NF-Y, activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer, more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4, a target gene of NF-Ya, using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction, the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover, TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated, transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy.
Assuntos
Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Proliferação de Células , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Humanos , Técnicas In Vitro , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Transplante HeterólogoRESUMO
The clinical potential of therapeutic quantities of primary hematopoietic cells, either unmodified or altered via genetic modification, has stimulated the search for techniques that allow the production of large numbers of hematopoietic precursors, more primitive progenitors, and perhaps hematopoietic stem cells (HSC) themselves. Modifications of in vitro culture conditions to promote progenitor cell expansion have included combinations of polypeptide cytokines, small molecules, and transcription factors. Here we describe the methods for use of the transcription factor linked to a TAT-based protein transcription domain, in combination with cytokines and serum-free culture condition to stimulate the proliferation of primary cells. Human peripheral blood (PB) CD34(+) cells treated with TAT-NF-Ya fusion protein and grown in vitro for 1 month proliferate four times more than did cells in cultures that contained only cytokines, including increased production of hematopoietic cells of all maturities. These results and techniques should be suitable for multiple applications of ex vivo generation of hematopoietic cells using protein transduction.