Optimized In Vivo Transfer of Small Interfering RNA Targeting Dermal Tissue Using In Vivo Surface Electroporation

Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner

The Enzymology of Transcriptional Regulation: the Work of E. Peter Geiduschek

Construction and Properties of a Cell-free System for Bacteriophage T4 Late RNA Synthesis (Rabussay, D., and Geiduschek, E. P. (1979) J. Biol. Chem. 254, 339–349

Restriction Endonuclease Activity Induced by PBCV-1 Virus Infection of a \u3ci\u3eChlorella\u3c/i\u3e-Like Green Alga

An enzyme was isolated from a eucaryotic, Chlorella-lie green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5\u27 to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells

Enhancement of antibody and cellular immune responses to malaria DNA vaccines by in vivo electroporation

We evaluated the effectiveness of in vivo electroporation (EP) for the enhancement of immune responses induced byDNAplasmids encoding the pre-erythrocytic Plasmodium yoelii antigens PyCSP and PyHEP17 administered intramuscularly and intradermally to mice. EP resulted in a 16- and 2-fold enhancement of antibody responses to PyCSP and PyHEP17, respectively. Immunization with 5 g of DNA via EP was equivalent to 50 g of DNA via conventional needle, thus reducing by 10-fold the required dose to produce a given effect. Moreover, IFN- responses were increased by approximately 2-fold. Data demonstrate the potential of EP to enhance immune responses to DNA vaccines against infectious agents