ABSTRACT
A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.
Subject(s)
DNA/administration & dosage , Gene Expression , Vaccines, DNA/administration & dosage , Animals , Cells, Cultured , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Hepatitis B virus/immunology , Immunoglobulin G/metabolism , Interferon-gamma/metabolism , Interleukin-4/metabolism , Lasers , Mice , Mice, Inbred BALB C , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
Gene therapy holds a major promise. However, until now, this promise was fulfilled only in few cases, in rare genetic diseases. One very common clinical condition is anemia. Patients with anemia of chronic renal failure are treated with erythropoietin. The objective of this study was to develop a therapeutic platform for serum-secreted proteins like erythropoietin. We developed a tissue protein factory based on dermal cores (Biopump) harvested and implanted autologously. In this study, an adenovector was designed to express the human erythropoietin under the control of the cytomegalovirus (CMV) promoter. This vector transduced the harvested dermal cores ex vivo. The transduced cores were implanted, and erythropoietin and reticulocyte counts were measured. Dermal cores were harvested from 13 patients with chronic renal failure, and implantation was performed in 10. There were no significant drug-related side effects to this procedure. Erythropoietin serum levels increased significantly to therapeutic levels from day 1 after implantation reaching a peak during the first week of follow-up. The expression period was transient for up to 14 days. The rise of erythropoietin was followed by a transient significant increase in reticulocyte counts. The decrease of erythropoietin expression coincided with a significant dermal infiltrate of CD8 cytotoxic T cells. Antierythropoietin antibodies were not detected until day 90 following implantation. Implantation of dermal cores ex vivo transduced with human genes could eventually be used in the clinical setting to express therapeutic serum proteins. However, nonimmunogenic delivery system should be tested as gene vehicles.