RESUMEN
The inability of adenovirus to infect primitive hematopoietic cells presents an obstacle to the use of adenovirus vectors for gene transfer to these cell types. Therefore, expanding the tropism of adenovirus vectors to unique cell surface antigens would be an important development for gene therapy protocols. In this study, we sought to redirect infection of adenovirus vectors to primitive human hematopoietic cells that universally express the c-Kit receptor on their cell surface. To accomplish this, a vector was constructed by covalently linking biotin molecules to recombinant adenovirus, followed by addition of the biotinylated ligand for the c-Kit receptor, stem cell factor (SCF), through an avidin bridge. Gene transfer was directed specifically to c-Kit-positive hematopoietic cell lines, resulting in up to a 2,440-fold increase in luciferase expression with frequencies equivalent to recombinant virus infection of permissive cells. Substitution of biotinylated antibodies directed against c-Kit, CD34 (binds L-selectin), and CD44 (hyaluronate receptor) receptors for biotinylated SCF resulted in 50-, 8-, and 260-fold increases in reporter gene expression, respectively, demonstrating that infection also could be redirected through antibody-antigen interactions and through antigens other than growth factor receptors. The versatility of this vector was demonstrated further by infection of primary T cells with vectors targeted with antibodies to CD44 (resting and activated T cells) and biotinylated IL-2 (activated T cells only). Taken together, directly biotinylated adenovirus vectors represent a versatile and efficient method for redirection of virus infection to specific cells.
Asunto(s)
Adenovirus Humanos/genética , Antígenos CD/fisiología , Vectores Genéticos , Proteínas Proto-Oncogénicas c-kit/fisiología , Factor de Células Madre/genética , Linfocitos T/fisiología , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígenos CD34/genética , Antígenos CD34/fisiología , Biotinilación , Línea Celular , Células Cultivadas , Citomegalovirus , Proteínas Fluorescentes Verdes , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/fisiología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Recombinantes de Fusión/biosíntesis , Recombinación Genética , TransfecciónRESUMEN
The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.