Oligopeptides will help gene therapy
Gene Guides
Ivan Okhapkin, Moscow State University
An international team of scientists, which included researchers from Moscow State University, developed unique nanostructures for delivering therapeutic genes into cells.
Gene therapy is a relatively new field of medicine aimed at the treatment of hereditary diseases. The "success stories" here can still be counted on the fingers, but recently gene therapy has been developing rapidly, including schemes for the treatment of cancer. The essence of the method is in principle obvious – to introduce genetic material into the body that would "fix everything". But the devil is hiding in the details. How to introduce alien genetic information into an already formed genome? In some cases, nature itself suggests the solution, moreover, with the help of not the best of its representatives. The infamous HIV is a retrovirus – a particle capable of inserting its genome into human DNA by cunning manipulations, and thereby reproducing itself.
Scientists have copied this mechanism for a long time and invented the so–called retroviral vectors - genetically modified retroviruses carrying a gene that needs to be embedded in a cell. Alas, the technique has drawbacks. The transfer of genes from a vector to human DNA is not a physiologically fast process, in addition, it is often not possible to create a high concentration of viral particles. Until recently, the only approved drug that somehow accelerated everything was retronectin, a protein consisting of more than 500 amino acid residues. However, it is quite possible that everything will change soon. A short peptide of only 12 amino acid residues, overlapping retronectin in its capabilities, was developed by an international team of scientists, which included researchers from Moscow State University; their article was published in the authoritative journal Nature Nanotechnology (Yolamanova et al., Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses).
Scientists have discovered that the peptide, called EF-C, is capable of self-organization into rod-shaped nanostructures (fibrils) with a diameter of about 4 and a length of 100-400 nm, which bind to retroviral vectors and help them merge with the cell membrane, after which they can throw genetic material into the cell.
In experimental studies, the EF-C peptide accelerated the infection of cells with retroviruses and turned out to be at least 4 times more effective than other known peptide drugs, and at the same time low–toxic. In addition, it turned out to be much easier and more convenient to work with it than with analogues – EF-C combines with retroviruses in a conventional solution, and in the case of retronectin, specially prepared surfaces are required.
One of the most important questions of the whole work is what structural features help the fibrils to be so effective? This question was answered by Russian research participants who conducted computer modeling of self-assembly of fibrils on a supercomputer of Lomonosov Moscow State University.
The dimensions of the simulated systems were about 300 thousand atoms. Thanks to the use of parallel computing technologies using the domain decomposition method, calculations were carried out with the simultaneous use of 256 computing cores for each simulated system, which allowed calculations to be carried out at a rate of evolution of at least 30 ns per day. The simulation was carried out using the GROMACSv. 4.5 software package, the size of the simulated pit was 10x10x30 nm, which made it possible to model morphologically significant sections of fibrils with a length of 25 nm.
"When the acceleration of the delivery of retroviral vectors to eukaryotic cells was discovered, the staff of the Ulm University Medical Center, who discovered the effect, began to look for its molecular causes. To do this, it was necessary to make many measurements using a variety of methods, including what we did – molecular modeling of self–assembly of peptides," says Academician Alexey Khokhlov, Vice-rector of Moscow State University, who led this part of the study. – "The simulation explained the results obtained experimentally; then this explanation and the predictions of computer modeling, in turn, were verified by additional experiments."
On the Lomonosov supercomputer, scientists found out that fibrils twist into a spiral with a period length of 28 nm. "This is of key importance for their biological properties," says Alexey Shaitan, a researcher at the Faculty of Biology of Moscow State University.
The interaction of retroviral particles (negatively charged) with the cell membrane (also negatively charged) is possible if the repulsion between them is reduced. For example, positively charged polymer molecules are attached to viral particles – the resulting complexes are not repelled from the membranes. Such molecules wrap around viruses, tightly closing its charges, but thereby losing their own; the complex is mainly electroneutral as a result. "They wrap viruses like beads," comments Alexey Shaitan – How many charges are on the virus – about the same number of beads he clings to himself. It is now an uncharged particle that may or may not sit on the membrane, because the beads also close the glycoproteins of the virus, with which it interacts with external cellular receptors."
With the fibrillation, everything is different: according to computer modeling, it is twisted into a thick spiral, around the axis of which positive charges are located on all sides. Therefore, if one side of the spiral "clings" to the virus, then the other is always free and positively charged; this helps the complex to be attracted to the membrane, and eventually leads to a greater capture of viral particles by the cell. "It is clear that rigid fibrils do not wrap the virus yet – glycoproteins are free, and can interact with receptors on the cell surface," says Shaitan. His computer experiment also showed that the fibrils themselves are very stable in solution and can exist in this form for as long as they like, without breaking up into the original peptide molecules and not sticking together. "It's not a fact that we would be able to talk about prospects for clinical practice if the fibrils were unstable," Shaitan believes.
In a fragment of a drawing from an article in Nature Nanotechnology (a) – molecular models of peptide dimers and an elementary unit of fibrils, (b) – models of fibrils; at the top the color corresponds to the hydrophobicity of amino acid residues, at the bottom – positively charged positive areas are highlighted in blue, negatively charged – in red (VM).
It is noteworthy that the computer modeling methods used to solve the problem with fibrils were initially intended for other purposes.
"By a lucky coincidence, we have recently gained experience in studying the self-assembly of polypeptides into fibers in a project on modeling strong potentially electrically conductive fibers based on polypeptide-polythiophene block copolymers. We were able to immediately use these developments in a new task," Khokhlov notes. An article on potentially electrically conductive fibers was published in 2011 in the journal ACSNano. (Shaytan et al., Self-Assembling Nanofibers from Thiophene–Peptide Diblock Oligomers: A Combined Experimental and Computer Simulations Study).
Portal "Eternal youth" http://vechnayamolodost.ru18.02.2013