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

Mechanism of A-B toxin infection

A-B toxin infect human cell by binding specific cells and then translocate enzymatic domain into cells. They damage the cells by ADP-ribosylation-the transfer of ADP-ribose from NAD to a target protein, changes the behavior of the target protein. Here Figures. 5 shows the infectious mechanism of ETA.

Fig. 5 SMain entry route and mechanism of action of Pseudomonas exotoxin A

1. Following removal of a C terminal lysine residue by plasma carboxipeptidase, the toxin binds to its cell-surface receptor (CD91, also called α2MR/LRP)

2. The toxin is internalized mainly via clathrin-coated pits

3. In the early endosome (EE), the toxin undergoes conformational change and is cleaved by the protease furin in a furin sensitive loop, in domain II. The two cleavage products remain linked by the intradomain disulfide bond

4. Following reduction of the disulfide bond, the enzymatically active C terminal fragment, which is composed of domain III and about two thirds of domain II, is routed to the trans-Golgi network where it binds via its C terminally exposed REDL sequence to KDEL receptor and travels to the endoplasmic reticulum (ER)

5. In the ER, sequences in domain II mediate the retro‑translocation of the polypeptide via the Sec61p translocon into the cytoplasm

6. The catalytic domain inactivates eukaryotic translation elongation factor 2 (eEF2) by ADP‑ribosylation, which causes translation inhibition and consequently cell death.

Chimeric Fusion Protein Facilitates Gene Transfer

The translocation domain of the chimeric fusion protein has function and mechanism similarly to the parental toxin. Hence, TEG or GD5 facilitates endosome escape of protein-DNA complexes upon internalization into target cells. Because of this property,an acidic environment is needed on the transit. Acidotropic reagent chloroquine have an enhancement of the efficiency of chimeric protein DNA delivery via receptor-mediated endocytosis. Endosomal acidification is blocked in the presence of chloroquine. Chloroquine but also result in endosome destabilization and the release of internalized DNA by accumulating in intracellular vesicles and inducing osmotic swelling of the endosomes.

Plasmids DNA is negative charged, which will do harm to the cell membrane when being transferring into cells. Moreover, it is not easy for plasmids DNA binding to chimeric fusion protein if they are scattered. Thereby, poly-cation interacts to DNA could neutralize the negative charged of plasmids DNA. And it can also condense DNA. As a consequence, more Plasmids can interact with chimeric fusion protein. In our project, poly-l-lysine acts as compensation of excess negative charged and condensation of DNA.

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.]

[http://www.mdpi.com/2072-6651/2/11/2519/pdf Shapira, A., & Benhar, I. (2010). Toxin-based therapeutic approaches. Toxins, 2(11), 2519-2583.]

http://textbookofbacteriology.net/diphtheria_3.html