Biomedical subjects

Richard Harbottle

Publications and source records attributed to Richard Harbottle.

3 recordsLinked to original sources

PubMed2003

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.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

Active Transport, Cell Nucleus↗

PubMed2003

High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides.

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.

Amino Acid Sequence↗

PubMed2003

Integrins and angiogenesis: unlocking the route to gene therapy.

Angiogenesis is defined as the process of vascularization of a tissue, involving the development of new capillary blood vessels. Both the induction and inhibition of this process can have therapeutic benefits in various pathological conditions. Integrins are a structurally elaborate family of adhesion molecules; they participate in a wide range of biological processes, including angiogenesis. Endothelial cells are intimately involved in angiogenesis and are thought to mediate this function partially through the integrins on their cell membrane, which regulate cell-cell and cell-matrix contacts. Extensive research into elucidating the mechanisms involved in the angiogenesis process have led to the discovery of a growing number of genes encoding pro- and anti-angiogenic proteins. A variety of gene therapy approaches have been used to deliver many of these genes to induce or inhibit the angiogenesis process with varying levels of success. This review investigates whether targeting gene therapy vectors to integrin receptors found on endothelial cells is a viable means to improve the efficiency of the gene transfer process.

Adenoviridae↗