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N S Yang

Publications and source records attributed to N S Yang.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Animals↗
PubMed1996

Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization.

The use of mRNA to transfer genetic information into mammalian somatic cells in vivo or ex vivo may be advantageous in a number of gene therapy protocols. Success in utilizing in vivo RNA delivery for transgene expression has been extremely limited, partially due to RNA instability and to the lack of an efficient intracellular delivery mechanism applicable to a wide variety of tissue or organ systems. We report here that a particle-mediated gene delivery technology can be used to effectively deliver RNA molecules into a number of mammalian somatic tissue types. Expression from RNA transcripts of three reporter genes, firefly luciferase, human growth hormone and human alpha-1 antitrypsin, was detected in monolayer and suspension cell cultures bombarded in vitro, and in in vivo bombarded rat liver tissues, and mouse liver and epidermal tissues. Gene gun treatment of mouse epidermis in vivo with human alpha-1 antitrypsin messenger RNA elicited a strong, consistent antibody response which showed an increased titer with subsequent boosts. Results from this study point to future opportunities of applying RNA delivery techniques for transgenic studies, genetic vaccination, and gene therapy.

Animals↗
PubMed1996

Selected strategies to augment polynucleotide immunization.

We sought to amplify the immune response to polynucleotide immunization through co-delivery of complementary DNA (cDNA) encoding a cytokine or co-stimulatory molecule to enhance antigen presentation. In the context of intramuscular immunization, we examined co-delivery of cDNAs for B7-1 and human carcinoembryonic antigen (CEA) within separate plasmids or a dual plasmid with two independent expression cassettes. Intramuscular delivery of the dual expression plasmid produced anti-CEA antibody responses and antitumor effects superior to those generated by plasmid DNA encoding CEA alone. However, co-delivery of cDNAs encoding B7-1 and CEA in the form of two separate plasmids produced no augmentation. The importance of single plasmid delivery suggests the effectiveness of this strategy is contingent upon co-expression of B7-1 and CEA within the same cell. The success of cutaneous polynucleotide immunization by particle bombardment is thought to derive largely from the presence of Langerhans cells within the skin. We hypothesized that co-delivery of plasmid DNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF) by particle bombardment would enhance the antigen presenting capacity of Langerhans cells at the inoculation site similar to its effects in vitro. Augmentation of CEA-specific lymphoblastic transformation and antibody response was observed when plasmid GM-CSF (pGM-CSF) was administered 3 days prior to each dose of plasmid DNA encoding CEA. These strategies for augmentation of immune response to polynucleotide immunization should be applicable to a wide variety of antigenic targets including infectious agents and other tumor-associated antigens.

Animals↗
PubMed1995

Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues.

To increase transgenic production of granulocyte-macrophage colony-stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM-CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20-fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM-AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t 1/2 = 95 minutes) than hGM-AUUUA mRNA (t 1/2 = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.

Animals↗
PubMed1995

In vivo cytokine gene transfer by gene gun reduces tumor growth in mice.

Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumor-bearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor alpha and interferon gamma genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon gamma genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon gamma were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

Animals↗
PubMed1995

Transcriptional regulatory and response mapping of the rat Ha-ras upstream sequence using primary mammary epithelial cells.

The rat Ha-ras upstream sequence (-2876 to +986 bp relative to the most 5' transcriptional start site) was transcriptionally mapped at a gross level. Ha-ras upstream sequence and 5' unidirectional deletion reporter constructs were transfected via particle bombardment into primary cultures of rat mammary epithelial cells. Analyses of Ha-ras reporter expression show that a fragment extending from -2876 to -2110 bp contains a positive regulatory sequence. The majority of Ha-ras expression is attributed to this sequence, since its deletion results in a 4-fold decrease in expression. The Ha-ras gene was also assessed for autoregulation using similar co-transfection experiments. Wild-type and activated (codon 12 G-->A transition) Ha-ras expression vectors, transcriptionally driven by Ha-ras upstream sequence, were co-transfected with Ha-ras upstream sequence deletion reporter constructs. The activated Ha-ras gene product induced its own transcription 2-fold, targeting the regulatory region between -2119 and -313 bp.

Animals↗
PubMed1995

Expression of a tumor-reactive antibody-interleukin 2 fusion protein after in vivo particle-mediated gene delivery.

We have used a particle-mediated gene transfer method to analyze the posttransfection expression pattern of an antibody-cytokine fusion protein (FP) in vivo. The FP, denoted CC49-IL2, consists of a single-chain antibody containing the antigen recognition domain from the murine monoclonal antibody CC49 (recognizing the tumor-associated antigen TAG-72), a human IgG1 constant heavy chain, and human interleukin-2 (IL-2). This FP can bind to TAG-72-expressing tumor cells and exhibits IL-2 activity. To induce systemic levels of this FP in vivo, we have transferred the FP gene into murine epidermal cells by direct delivery of DNA-coated gold particles using a transcutaneous "gene gun." After the pericutaneous delivery of the FP gene via gold particles, production of the exogenous FP was detected at the epidermal target site. The FP produced in vivo at the site of gene delivery has cytokine activity and antigen recognition capabilities similar to those present in CC49-IL2 FP purified from hybridoma culture supernatants in vitro. FP was also detectable in the serum from test animals treated with particle-mediated gene transfer. Time course experiments indicated that serum levels of FP reached a peak level within 8 hours after DNA delivery, whereas the epidermal target tissue levels continued to increase for 24 hours before plateauing. Our results indicate that exogenous protein levels consistent with immunotherapeutic effects of the FP can be readily achieved at the skin tissue site of gene delivery, with the potential for achieving therapeutic levels systemically.

Animals↗
PubMed1994

Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.

Antigens, CD↗
PubMed1994

Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells.

Because AIDS has been refractory to traditional pharmacologic interventions, alternative approaches have been developed. Although the introduction of specific antiviral genes into T leukemia cells can provide relative resistance to human immunodeficiency virus (HIV) replication, the testing of such genes against primary viral isolates in human CD4+ lymphocytes has been limited, and safety questions remain regarding gene delivery into cells from HIV-infected patients. In this report, we evaluate the efficacy of a transdominant mutant protein, Rev M10, against cloned and primary HIV isolates in human peripheral blood lymphocytes and describe different methods of gene transfer into peripheral blood lymphocytes from HIV-infected individuals. We show that gold microparticles can mediate stable Rev M10 gene transfer into these cells. Introduction of Rev M10 by these techniques conferred resistance to HIV infection in vitro to cloned and clinical isolates. Nonviral delivery of HIV protective genes will facilitate the development of gene therapy for AIDS and the analysis of viral and cellular gene expression in human T lymphocytes.

Base Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment.

The particle bombardment method of gene transfer provides an alternative approach for analysis of in vivo promoter activity and transgene expression. Transient expression of the firefly luciferase gene from five viral and five cellular promoters was assessed after in vivo gene transfer using this method. The relative strengths of these promoters were quantitatively determined in five different rat tissues: skin epidermis, dermis, muscle, liver, and pancreas. Cytomegalovirus immediate early enhancer/promoter activity was consistently the highest in each tissue, whereas other promoters displayed tissue-specific preferences. In liver, the mouse phosphoenolpyruvate carboxykinase and metallothionein promoters were stimulated in vivo by inducing agents at 1 and 5 days posttransfection. In dermis, sustained luciferase activity was observed for over 1.5 years after gene delivery. In vivo transgene expression was also detected in bombarded mouse, rabbit, and rhesus monkey tissues. These results suggest that particle bombardment provides an effective system for studies of in vivo gene transfer and gene therapy.

Animals↗
PubMed1993

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

The 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 approximately CMV approximately SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.

3T3 Cells↗
PubMed1993

Particle bombardment-mediated gene transfer and expression in rat brain tissues.

We have previously demonstrated that the particle bombardment method for gene transfer (Accell) provides a new means for transfection of various cell types in culture. In this study we evaluate its application to rat brain systems. Using a luciferase (luc) gene as a reporter, we obtained high levels of transient gene expression in primary cultures of fetal brain tissue. Reduced but significant levels were also detected in adult brain primary cultures. Both neuron and glial cells were transfected using this technique. The transient gene expression level obtained with Accell was at least 100-fold higher than that obtained with three other gene transfer methods. The relative strengths of four cellular and seven viral promoters were also evaluated in these cultures. In vivo gene expression was studied using freshly excised and bombarded fetal brain tissues which were immediately transplanted into caudate or intracortical brain tissues of adult host animals. Assays showed that luciferase activity was present in transplants for up to two months following gene transfer. In vitro and in vivo expression of a rat tyrosine hydroxylase (TH) gene, a candidate gene for treatment of Parkinson's disease, was also detected in this rat brain system. Our results suggest that the particle bombardment gene transfer technology can be employed as an effective method for ex vivo gene transfer into brain tissues.

Animals↗
PubMed1993

Differential expression of Rel family members in human T-cell leukemia virus type I-infected cells: transcriptional activation of c-rel by Tax protein.

The Tax protein of the human T-cell leukemia virus type I (HTLV-I) has been shown to induce nuclear expression of Rel family NF-kappa B-binding proteins. However, under different experimental conditions, different J. H. Kim, Y. Daitoku, and W. G. Greene, J. Virol. 65:6892-6899, 1991). In this study, using specific immunological reagents capable of distinguishing individual members of the Rel family proteins, we show that only c-Rel, not NF-kappa B p50 or p65, is induced in HTLV-I-infected cells. Preferential c-rel induction by HTLV-I infection was detected at the protein and RNA levels as well as in the nuclear NF-kappa B-binding form. Induced c-rel expression was also detected in cells stably transfected with tax cDNA, further correlating the c-rel induction with viral Tax expression. An increase in c-rel mRNA was detected within 3 h after induction of Tax expression, suggesting that this effect is at least partially regulated at the level of transcription. Furthermore, using a particle bombardment method for gene cotransfection, we show that Tax can transcriptionally activate the c-rel promoter in a T-cell line, Jurkat.

Base Sequence↗
PubMed1992

Gene transfer into mammalian somatic cells in vivo.

Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.

Animals↗
PubMed1990

Maize sucrose synthase-1 promoter directs phloem cell-specific expression of Gus gene in transgenic tobacco plants.

Tobacco plants were transformed by using a chimeric gene construction, in which a corn sucrose synthase-1 gene (Sh) promoter was used to direct expression of the beta-glucuronidase (Gus) reporter gene. Expression of Sh-Gus activity in these plants was found to be cell type specfifc. GUS activity was detected only in the phloem cells but not in any other cell types of vegetative tissues. In addition, Sh-Gus expression was found to be anaerobically inducible in tobacco roots. Sh-Gus was also expressed at high levels in the endosperm tissue of maturing tobacco seeds. We thus demonstrated that the corn Sh promoter can direct cell-type-specific and inducible expression in a heterologous dicotyledonous plant.

Journal Article↗
PubMed1990

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.

Animals↗