Pharmacology

Garrett Rettig

Mentor: Kevin Rice, PhD

Year Entered Into Program: 2003

PhD Institution: University of Iowa, 2008

Research Description

The focus of the Rice lab is to design novel peptide-DNA conjugates to mediate non-viral gene delivery and gene expression in vivo. A major barrier in non-viral gene delivery is targeting plasmid DNA (pDNA) to the nucleus. The T antigen of SV40 is widely known to possess a short, lysine-rich peptide that is sufficient to mediate nuclear localization. Therefore, we are pursuing methods to incorporate a peptide containing a known nuclear localizing sequence (NLS) into our gene formulations.
We have synthesized and characterized a sequence of nine basic amino acids with an N-terminal photo-labile linker. The photochemical labeling of pDNA by the NLS has been confirmed by agarose gel electrophoresis and HPLC-based methods. Our hypothesis is that pDNA labeled with NLS will boost gene expression by facilitating nuclear trafficking. We have done gene transfer studies to compare the transfection efficiency mediated by NLS-pDNA relative to plasmid labeled with a control nuclear localizing sequence (cNLS-pDNA). We are working toward optimizing in vitro gene transfer efficiency by varying both the ratio of NLS to pDNA and the duration of transfection. The NLS-labeled pDNA has shown greater than 10-fold increase in transgene expression compared to controls. Confocal microscopy images support the conclusion that the increase in transgene expression is the result of increased nuclear localization of NLS-pDNA when compared to the control peptide.
Preliminary in vivo studies also indicate that NLS-pDNA enhances gene expression compared to controls. We have incorporated intramuscular dosing of the plasmid encoding luciferase with whole-animal bioluminescent imaging. This assay is used to quantify photons emitted from skeletal muscle as an indirect measurement of transgene expression. Additionally, we have synthesized a series of 12 NLS-containing peptides that can be photochemically linked to pDNA. The linker sequence has been varied in order to address optimal presentation of the NLS to its receptor in the nuclear membrane. Further gene transfer studies will be carried out in vitro and in vivo with this series of peptides. 

Award(s)

  • Recipient of AFPE Pre-Doctoral Fellowship, 2005-2006