Ning-Sun Yang

Rapid transgene expression in lymphocyte and macrophage primary cultures after particle bombardment-mediated gene transfer.

A particle bombardment technique was used for gene transfer to human peripheral blood mononuclear cells, and murine splenocytes, thymocytes and peritoneal macrophages in primary culture. Significant expression of a luciferase marker gene was observed in these cell types within 8 h of gene transfer. Luciferase expression was readily detected in peritoneal macrophages 4 h after culture initiation and transfection. Same day determinations of transgene activity in fresh human peripheral blood mononuclear cell samples were feasible. Promoter preference and ballistic parameters were examined to optimize transgene expression. Up to 6% of bombarded human T lymphocytes expressed transgenic beta-galactosidase activity. These results demonstrate that particle bombardment is an effective means for gene transfer and provides an attractive approach for rapid, quantitative analysis of transgene expression in various leukocyte primary culture systems.

Gene gun-mediated IL-12 gene therapy induces antitumor effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy.

Using three murine tumor models, we compared the antitumor efficacy and certain physiological effects of an in vivo interleukin-12 (IL-12) gene therapy protocol and a systemic IL-12 protein therapy protocol. An IL-12 cDNA gene construct was administered in situ into skin tissue via gene gun delivery, and recombinant IL-12 protein was administered subcutaneously at a dose of 1 microgram/mouse/treatment. Both treatment regimes induced a comparable level of regression of established intradermal MethA sarcomas. In B16 melanoma and P815 mastocytoma models, antitumor efficacy of IL-12 protein therapy appeared to be slightly higher than that of IL-12 gene therapy; however, the protein therapy protocol in this comparative study resulted in a high level of mortality of mice. It was also demonstrated that IL-12 gene therapy, in contrast to the IL-12 protein therapy, was not associated with weight loss, splenomegaly, increased Ly6 antigen expression in the spleen, or visible signs of toxicity, such as fur ruffling and lethargy. Moreover, serum levels of interferon-gamma (IFN-gamma) induced in response to IL-12 gene therapy were 300-1000 times lower than those induced by the systemic IL-12 protein administration. Together, these results suggest that gene gunmediated in vivo delivery of IL-12 cDNA may be considered as a safer alternative to IL-12 protein therapy for certain human cancers.

Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer

SummaryThe relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.

Developing particle-mediated gene-transfer technology for research into gene therapy of cancer

Gene therapy aims to (1) introduce specific genes into a host to replace defective ones (replacement therapy); (2) suppress expression of certain undesirable genes (antisense therapy); or (3) provide additional biological activities (supplement therapy). Naked DNA and viral or non-viral vectors containing candidate genes for human gene therapy are being actively pursued by researchers in molecular medicine. New gene transfer technologies are rapidly developing and some have proved to be powerful tools for medical research. This review discusses the development and application of particle-mediated gene transfer technology in experimental systems and its potential clinical utilities.

Interleukin 12 Gene Transfer into Skin Distant from the Tumor Site Elicits Antimetastatic Effects Equivalent to Local Gene Transfer

We have reported that particle-mediated interleukin 12 (IL-12) gene transfer into the skin overlying the local tumor inhibits systemic metastases. To further characterize this effect, we compared the antitumor and antimetastatic effects of IL-12 cDNA delivered at the local tumor site versus at a site distant from the primary tumor, in a spontaneous metastasis model of LLC-F5 tumor. Local IL-12 gene delivery into the skin overlying the intradermal tumor (local IL-12 treatment) on days 7, 9, and 11 after tumor implantation resulted in the most suppression of the growth of the primary LLC-F5 tumor, whereas IL-12 gene transfer into the skin distant from the tumor (distant IL-12 treatment) was less effective. In contrast, both local IL-12 and distant IL-12 treatment, followed by tumor excision, inhibited lung metastases to a similar extent, resulting in significantly extended survival of test mice. The results of in vivo studies using depleting anti-asialo GM1 antibody and anti-CD4/anti-CD8 monoclonal antibodies, or neutralizing anti-interferon gamma (IFN-gamma) monoclonal antibody demonstrated that natural killer (NK) cells, CD8(+) T cells, and IFN-gamma contributed to the antimetastatic effects in both treatment groups. Furthermore, the levels of mRNA expression of vascular endothelial growth factor and matrix methalloproteinase 9 at the tumor microenvironment were suppressed after both local and distant IL-12 treatment. These results suggest that the current particle-mediated IL-12 gene delivery in the spontaneous LLC-F5 metastasis model can confer antimetastatic activities, irrespective of the gene transfection site, via a combination of several mechanisms involving CD8(+) T cells, NK cells, IFN-gamma, and antiangiogenesis.

Immunization by particle-mediated transfer of the granulocyte-macrophage colony-stimulating factor gene into autologous tumor cells in melanoma or sarcoma patients: Report of a phase I/IB study

The primary objective of this phase I study was to determine the safety of an autologous tumor vaccine given by intradermal injection of lethally irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected autologous melanoma and sarcoma cells. Secondary objectives included validation of the gene delivery technology (particle-mediated gene transfer), determining the host immune response to the tumor after vaccination, and monitoring patients for evidence of antitumor response. Sixteen patients were treated with either of two different doses of GM-CSF-treated tumor cells. One patient received treatment with both doses of tumor cells. No treatment-related local or systemic toxicity was noted in any patient. Patients administered 100% treated cells (i.e., with a preparation of tumor cells that had all been exposed to GM-CSF DNA transfection) had a more extensive lymphocytic infiltrate at the vaccine site than did patients given 10% treated cells (a preparation of tumor cells in which 10% had been exposed to GM-CSF transfection) or nontreated tumor. The generation of a systemic immune response to autologous tumor by a delayed-type hypersensitivity response to the intradermal placement of nontransfected tumor cells was noted in one patient. One patient had a transient partial response of metastatic tumor sites. The entire procedure, from tumor removal to vaccine placement, was accomplished in less than 6 hr in all patients. Four of 17 patient tumor preparations produced greater than 3.0 ng of GM-CSF per 10(6) cells per 24 hr in vitro. The one patient with greater than 30 ng of GM-CSF per 10(6) cells per 24 hr in vitro had positive DTH, a significant histologic inflammatory response, and clinically stable disease. This technique of gene transfer was safe and feasible, but resulted in clinically relevant levels of gene expression in only a minority of patients.

Particle‐Mediated Gene Delivery In Vivo and In Vitro

Particle‐mediated or “gene gun” technology has been developed as a nonviral method for gene transfer into various mammalian tissues. Gene delivery is achieved by physical force: a strong shock wave is generated that accelerates DNA‐coated gold particles to high speeds, providing them with the momentum needed to penetrate the targeted cells. This unit describes general procedures for in vivo and in vitro DNA and RNA transfections by particle‐mediated delivery. The and an alternate protocol address in vivo delivery to mouse skin. In vitro delivery to cryopreserved and adherent cells is also described.