Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein.

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.

Molecular and functional analysis of Shiga toxin-induced response patterns in human vascular endothelial cells.

Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells.