What happens in VEGAS won’t stay in VEGAS, not if the eponymous system fulfills its purpose: expediting the directed evolution of DNA sequences in mammalian cells. VEGAS, which stands for Viral Evolution of Genetically Actuating Sequences, could make the development of scientific tools and new treatments less of a gamble. Specifically, it could avoid the development delays often seen with conventional forms of directed evolution, as well as the shortcomings particular to bacterial cell-based methods. VEGAS has already been used to evolve a transcription factor, a G-protein coupled receptor (GPCR), and llama-derived nanobodies toward specific in vivo activities. Each of these proof-of-concept campaigns were completed in less than a week.
The new approach, which comes from the University of North Carolina (UNC) School of Medicine, is comparatively quick, easy, and versatile way to carry out directed evolution, an artificial version of natural selection. It has been used for centuries, ever since people began selecting and breeding variants of animals and plants to possess desirable characteristics. In recent decades, directed evolution has been used in the laboratory, where biologists work to generate valuable molecules.
For example, biologists might arrange for a gene to be mutated randomly until a variant appears that has a desired property. If this sounds as though this kind of directed evolution generates long losing streaks punctuated by rare wins, it does. On the whole, directed evolution for biological molecules have been difficult to use and limited in their application.
The VEGAS system aims to generate more wins more quickly. To start, it works in mammalian cells, not bacterial cells. Although bacterial cells are often convenient platforms for directed molecular evolution campaigns, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Because VEGAS works in mammalian cells, it can be used to evolve new human, mouse, or other mammalian proteins that would be burdensome or impossible to generate with traditional bacterial cell-based methods.
Also, the VEGAS system uses the RNA alphavirus Sindbis as the carrier of the gene to be modified. The virus with its genetic cargo can infect cells in a culture dish and mutate quite rapidly.
Details about VEGAS appeared July 4 in the journal Cell, in an article entitled, “VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.” According to this article, VEGAS offers three major advantages for the directed evolution of biomedical tools and therapies.
- VEGAS evolves within the signaling framework of the host cell.
- VEGAS is wholly dependent on the host cell for transgene maturation
- VEGAS selection is constant and highly mutagenic, enabling it to overcome many of the pitfalls inherent to complex fitness landscapes
“We achieved 24-hr selection cycles surpassing 10−3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell,” the article’s authors wrote. “Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than one weeks’ time.”
In their experiments, the researchers set up conditions so that the only mutant genes that can thrive are the ones encoding proteins capable of accomplishing a desired function within the cells, such as activating a certain receptor, or switching on certain genes. One experiment was devoted to modifying a protein called a tetracycline transactivator (tTA), which works as a switch to activate genes and is a standard tool used in biology experiments. Normally, tTA stops working if it encounters the antibiotic tetracycline or closely related doxycycline, but the researchers evolved a new version with 22 mutations that allows tTA to keep working despite very high levels of doxycycline. The process took just seven days.
“To get a sense of how efficient that is, consider that a previously reported mammalian directed evolution method applied to the tetracycline transactivator took four months to yield just two mutations that conferred only partial insensitivity to doxycycline,” said the study’s lead author, Justin English, PhD, a postdoctoral research associate in the department of pharmacology at the UNC School of Medicine.
The scientists also applied VEGAS to a common type of cellular receptor called a GPCR. There are hundreds of different GPCRs on human cells, and many are targeted by modern drugs to treat a wide variety of conditions. Precisely how a given GPCR changes shape when it switches from being inactive to active is of great interest to researchers trying to create more precise treatments. English and colleagues used VEGAS to quickly mutate a little-studied GPCR called MRGPRX2 so that it would stay in an always-active state.
“Identifying the mutations that occurred during this rapid evolution helps us understand for the first time the key regions in the receptor protein involved in the transition to an active state,” English said.
In yet another demonstration, the team showed the potential of VEGAS to guide drug development more directly. They used VEGAS to rapidly evolve small biological molecules called nanobodies that could activate different GPCRs—including serotonin and dopamine receptors, which are found on brain cells and are targeted by many psychiatric drugs.
The team is now using VEGAS in an effort to develop highly efficient gene-editing tools, potentially for curing genetic diseases, and to engineer nanobodies that can neutralize cancer-causing genes.
“The scientific community has needed a tool like this for a long time,” said the study’s senior author, Bryan L. Roth, MD, PhD, the Michael Hooker distinguished professor in the department of pharmacology at the UNC School of Medicine. “We believe our technique will accelerate research and ultimately lead to better therapeutics for people suffering with many of the diseases for which we need much better treatments.”