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An accidental discovery becomes a tool for researchers: peptide facilitates gene transfer in the laboratory

It was pure chance that led to Frank Kirchhoff’s and Jan Münch’s discovery of a highly interesting peptide. The two virologists from Ulm are involved in AIDS research and a while ago their interest was aroused by a protein derivative just twelve amino acids long.

The results of subsequent interdisciplinary investigations into the protein derivative with the Ulm chemists Christoph Meier and Tanja Weil were so convincing that cooperation partners from Hanover, Germany, decided to commercialise the peptide as a laboratory tool for biomedical research. It also appears that nanotechnologists have expressed an interest in the so-called nanofibrils and are hoping to be able to put them to good use in materials science applications.

Prof. Jan Münch - AIDS researcher from Ulm © University of Ulm

It all started when Münch and his colleagues from the Institute of Molecular Virology at Ulm University decided to investigate the interactions between the AIDS virus (HIV-1) and cell receptors in greater detail. They specifically focused on the effect peptides of the HIV-1 envelope protein gp120 have on the entry of the virus into cells.

They discovered a peptide that increased infection rates 34-fold. They also found that the newly discovered peptide had much higher activity rates than the semen-derived enhancer of viral infection (SEVI), which Kirchhoff and Münch discovered several years ago in cooperation with colleagues led by Wolf-Georg Forssmann at the Hanover Medical School. SEVIs are protein fragments from human sperm that form rod-like structures known as fibrils. 

Fast, effective and cheap

Atomic force microscope image of nanofibrils. © Münch

An aqueous solution that became turbid upon the addition of the newly discovered peptide reminded Münch immediately of SEVI. His suspicions were confirmed: the newly discovered protein fragment formed fibrils just like the previously discovered SEVI peptide. In addition, it also had properties that made it worth analysing in greater detail. On the one hand, the newly discovered peptide enhanced infectivity more efficiently than SEVI and other known infection enhancers. Moreover, the fibrils formed within a few seconds of exposure to the new peptide. In contrast, SEVI peptides only formed fibrils when the solution was agitated for a number of hours. 

Why is a substance that enhances the infectivity of a potentially deadly virus so interesting? HIV-1 belongs to a family of viruses known as retroviruses. Genetically modified, harmless retroviruses are used as vectors for introducing genes into cells in many laboratories around the world. The process in which a retroviral shuttle is used to introduce genes into cells is called transduction. However, transduction rates are often very low, which is why many experiments cannot be carried out or need to be repeated. In order to overcome these limitations, researchers use transduction enhancers such as polybrene, protamine sulphate or diethylaminoethyl (DEAE)-dextran.

Direct comparison reveals obvious advantages

The peptide enhances retroviral transduction, e.g. in human fibroblasts such as shown here. © Münch

These substances increase the gene transfer rate of retroviral vectors; however, they are often not very efficient and in higher doses they are also toxic. The compound RetroNectin significantly enhances retroviral transduction into hard-to-infect mammalian cells and is frequently used in stem cell research to transfer genes into haematopoietic stem cells. However, it costs up to 300 euros per milligramme and also requires the application of a rather complicated and time-consuming protocol. The Ulm researchers’ discovery is far easier and more comfortable to use: after the cells are sown, the fibrils are added to the virus and the mixture applied to the cells. 

Münch and his colleagues have published their work in Nature Nanotechnology in which they reported on the production of many different retroviral vectors used in research and clinical application and checked whether the peptide fibrils were able to enhance retroviral gene transfer into different cell lines and primary cells – with resounding success. Even experiments with macrophages, which are known to be hard to infect, showed that the peptide was excellently suited as a biomedical research tool. The researchers found that their artificial nanofibrils increased the normal transduction rate from two to nearly fifty percent. 

An electrostatic bond with adhesive effect

Refined molecular model of the peptide: cysteine (grey), asparagine (blue), pyrrolysine (red), serine (yellow). © Münch

In cooperation with colleagues from Russia, Spain, USA and England, the Ulm scientists also clarified how the nanofibrils enhance the infection with HIV-1 and transfection using retroviral vectors. They found that the fibrils increased the adhesion rate of the viral particles to the cell surface. The larger number of cells also increases the probability of virus entry into the cells. Münch and his colleagues have also discovered the reason for this: they found that a kind of electrostatic bond is responsible for this highly effective interaction. And since the virus membrane is no more than the cytoplasmic membrane of the cell, the fibrils not only bind very well to the virus, but also very effectively to the cell. The scientists also believe that some hydrophobic interactions facilitate the adhesion of the viral particles. 

Can these nanofibrils be made to measure?

Dr. Christoph Meier, co-author of the Nature Technology paper; he and Münch are continuing their research on the peptide. © University of Ulm

Meanwhile, the scientists are trying to gain an understanding as to why the identified peptide is so efficient and quick in forming nanofibrils. In a project funded by the Volkswagen Foundation, the researchers are working on the production of customised nanofibrils that allow the targeted transfer of genes. Their goal is to transduce and kill only specific target cells, tumour cells for example. This work is carried out with colleagues from the Department of Organic Chemistry (Christoph Meier and Tanja Weil). This research could potentially also serve as a starting point for further research in other disciplines. “The results are of major interest for materials scientists who are always looking for the smallest possible building blocks that form a highly organised superstructure,” said Tanja Weil. In future, the researchers will market the nanofibrils they have identified under the brand name Protransduzin.

Reference 
Münch, J. et al.: Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses, Nature Nanotechnology, 20.1.2013, DOI: 10.1038/NNANO.2012.248

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