Background

Though therapeutic techniques are developing rapidly nowadays, some human diseases such as cancer and AIDS are extremely difficult to effect a radical cure. Gene therapy cures diseases by using DNA that encodes a functional, therapeutic gene to replace a mutated gene. Therefore, gene therapy is efficient to cure diseases results from gene mutation such as cancer and chronic infectious diseases.

Two major methods are applied to gene therapy. One is viral vectors. The other is non-viral vectors. Viral vectors are efficient to transfer foreign gene into cells and gene is efficiently expressed. But, on the other hand, viral vectors are hard to target specific cells which will decrease the percentage of the target gene into target cells. Moreover, we can't sure that viral vectors are not infectious. It ceases to be a safe way for gene therapy. As a consequence, non-viral vectors gain increasing attention since the late 20th century. Several methods for gene therapy with non-viral vector are studied by researchers such as electroporation, gene gun, magnetofection and so on.

In our project, the novel modular DNA carrier protein is selected as one of the gene transfer vector. This nucleic acid transfer system was developed by Prof. Dr Wels from the Institute for Experimental Cancer Research, Tumor Biology Center and his cooperators in 1998.

Introduction

Our novel modular carrier protein is made up of three domains: Cell Binding Domain-specific binding to target cell, Translocation Domain- facilitating gene delivery via receptor-mediated endocytosis, DNA Binding Domain-specific binding to DNA containing UAS. Therefore, it can act as non-viral nucleic acid transfer system by delivering the carried target DNA to recognized the target cell via receptor-mediated endocytosis.

TEG

Novel DNA carrier protein TEG mimics the structure of Pseudomonas exotoxin A(ETA). TEG acts as a vector transferring specific target DNA into cells via receptor-mediated endocytosis. ETA consists of two subunits linked by disulfide bridge known as A-B toxin falling into three main structural and functional domains: the N-terminal receptor (R) binding domain I, translocation (T) domain II and the catalytic (C) domain III. Fig. 1 is the 3D structure of ETA.

Fig. 1 Structure of ETA

The N-terminal receptor binding domain is replaced by a human EGF(epidermal growth factor) receptor ligand TGF-a. EGF receptor is overexpressed by human carcinomas cells. TEG containing TGF-a recognizes the EGF receptor and then binds to the EGF receptor. As a result, cells overexpressing EGF receptor are specifically combined with TEG. Similarly, replacing the C-terminal enzymatic domain of the toxin with the DNA-binding domain of the yeast GAL4 transcription factor leads to interaction with plasmid DNA of TEG. Overall, TEG contains TGF-a at N-terminal following by translocation domain of ETA and GAL4 at C-terminal

GD5

GD5 is another novel DNA carrier protein mimics the structure of diphtheria toxin (DT). DNA can be transferred into cells by GD5 via receptor-mediated endocytosis. DT is composed of two disulfide bridges linked subunits divided into three main structural and functional domains. The structure and function of DT are similar to ETA. But DT with cell binding domain at C-terminal and catalytic doamin at N-terminal, which is the inverse of ETA. Fig. 2 shows the 3D structure of GD5.

Similarly to TEG, chimeric fusion protein GD5 is assembled with antibody fragment specific for the tumor-associated ErbB2 antigen, translocation domain of DT as an endosome escape activity and Gal4 as DNA binding domain. Accordingly, ErbB2 antigen single chain antibody fragment FRP5 is placed at C-terminal via DT translocation domain, and GAL4 at N-terminal.

Construction

DNA fragments encoding amino acids 1 to 50 of human TGF-a, amino acids 252 to 366 of Pseudomonas exotoxin A (translocation domain) and amino acids 2 to 147 of the yeast GAL4 protein (DNA-binding domain) were assembled into one single open reading frame. The resulting plasmid pWF47-TEG encodes under the control of the IPTG-inducible tac promoter. A cluster of six histidine residues are added between TGF-a and ETA to facilitate the purification of the fusion protein via Ni2+ affinity chromatography. An N-terminal E. coli ompA signal peptide, a synthetic FLAG epitope for detection,and a KDEL signal for intracellular routing and full activity of TEG are the rest part of the chimeric fusion protein. The plasmid is under the control of the IPTG inducible tac promoter. Fig. 3 shows Schematic representation of the TEG fusion gene.

Fig. 3 Schematic representation of the TEG fusion gene

The construction of DNA carrier protein GD5 is similar to TEG, but GD5 does not contains ompA and KDEL. Fig. 4 shows Schematic representation of the GD5 fusion gene.

Fig. 4 Schematic representation of the GD5 fusion gene

Mechanism

Purification

Transfection

Results

Prospects

References

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1242130/pdf/bcr1264.pdf von Minckwitz, G., Harder, S., Hövelmann, S., Jäger, E., Al-Batran, S. E., Loibl, S., ... & Wels, W. S. (2005). Phase I clinical study of the recombinant antibody toxin scFv (FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas. Breast Cancer Research, 7(5), R617.]

[http://www.freepatentsonline.com/6498233.html Wels, W., & Fominaya, J. (2002). Nucleic acid transfer system.]

[http://europepmc.org/abstract/MED/9614577 Fominaya, J., Uherek, C., & Wels, W. (1998). A chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the EGF receptor. Gene therapy, 5(4), 521-530.]

[http://www.jbc.org/content/273/15/8835.short Uherek, C., Fominaya, J., & Wels, W. (1998). A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery. Journal of Biological Chemistry, 273(15), 8835-8841.]