Biomedical subjects

J Glasspool-Malone

Publications and source records attributed to J Glasspool-Malone.

4 recordsLinked to original sources

PubMed2001

Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization.

Naked DNA injection with electropermeabilization (EP) is a promising method for nucleic acid vaccination (NAV) and in vivo gene therapy. Skin is an ideal target for NAV due to ease of administration and the accessibility of large numbers of antigen-presenting cells within the tissue. This study demonstrates that in vivo skin EP may be used to increase transgene expression up to an average of 83-fold relative to naked DNA injection (50 microg DNA per dose, P < 0.005). Transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. Transfected cells were also observed in lymph nodes draining electropermeabilized sites. A HBV sAg-coding plasmid was used to test skin EP-mediated NAV in a murine model. Analysis of humoral immune responses including immunoglobulin subclass profiles revealed strong enhancement of EP-mediated NAV relative to naked DNA injection, with a Th1-dominant, mixed-response pattern compared to immunization with HBV sAg protein that was exclusively Th2 (P = 0.02). Applications for these findings include NAV-based modulation of immune responses to pathogens, allergens, and tumor-associated antigens and the modification of tolerance.

Adipocytes↗

PubMed2000

Efficient nonviral cutaneous transfection.

Preclinical in vivo rodent, porcine, and primate experiments aimed at enhancing nonviral transgene delivery to skin have been performed. These investigations have identified a compound (aurintricarboxylic acid or ATA) that enhances transfection activity of "naked" plasmid and pulsed electrical fields (electroporation or EP) that synergistically boosts transgene expression to an average of 115-fold more than that observed with free DNA (P < 0.00009). When plasmid is intradermally injected with or without ATA, the transfected cells are typically restricted to the epidermis. However, when electroporation is added after the same injection, larger numbers of adipocytes and fibroblasts and numerous dendritic-like cells within the dermis and subdermal tissues are transfected. This advance creates new opportunities for cutaneous gene therapy and nucleic acid vaccine development.

Adipocytes↗

PubMed2000

Theory and in vivo application of electroporative gene delivery.

Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

Electroporation↗

PubMed1999

Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid.

Simple, nontoxic, and pharmaceutically defined methods for genetic modification of respiratory tissues may enable development of a variety of molecular medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmonary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccination. "Free," "direct," or "naked" plasmid administration is a simple, apparently safe, and pharmaceutically defined gene delivery method. Murine, macaque, and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transduction, and specifically to provide a theoretical framework for developing a category of adjuvants, nuclease inhibitors, that augment the transfection activity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose escalation studies were performed using lung homogenate assays to characterize transfection. Potential toxicity was assessed histologically. The data indicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expression. The effective dose for ATA enhancement of direct lung transfection was 0.5 microg/g mouse weight, and the LD50 was approximately 6 microg/g. These findings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory transfection technology.

Animals↗