Gene therapies are experiencing a new era. CRISPR is current. CRISPR is trendy. CRISPR is on many researchers’ minds. But its sudden success has also triggered concerns about its safety. Can the proteome help solve these issues?
Although the landscape of genome editing has been evolving for nearly 50 years, it has only recently seen dramatic changes. After experiencing years of stagnation resulting from severe setbacks in clinical trials and a lack of efficiency, zinc-finger nucleases appeared on stage. This time, researchers were hampered by yet another problem: the patent landscape and design complexity. Licensed and commercialized for research by Sigma-Aldrich, the technology was costly which made it difficult to access as a general tool for researchers.
“At the time, zinc-finger nucleases were a fantastic adventure,” says Jens-Ole Bock, CEO of COBO Technologies. “It was exciting to know that we had a tool that could do specific changes to the genome. But zinc-finger nucleases were expensive, very complex, and difficult to access. There were some alternatives on the market for a few years, and then CRISPR-Cas9 appeared. Suddenly we had this really great genome editing tool that was cheap, with a simple mode of action, and available for everyone to work with.”
Why CRISPR safety has to be taken seriously
As a result of this democratization, research in the field of genome editing sped up dramatically and the CRISPR technology continues to evolve very fast. However, in 2018, a small number of publications raised concerns about the safety of CRISPR, revealing that the technology might cause severe side effects due to off-target mutations.
“With CRISPR we now have a very effective and easy system that everyone is using,” Bock says. “There are more than 5000 publications a year in CRISPR research and we are now moving towards its clinical application. This also means that we need to focus on CRISPR safety concerns. If we want to move to the clinic, we need to understand exactly what is going on inside the cells when we apply CRISPR. What is the target? What happens in the target area? Are there off-target events? Is there immunogenicity? How is the biology of the cells affected?”
In order to better anticipate the risks of genome editing tools, Bock and his colleagues founded COBO Technologies, a company that focuses exclusively on quality control of genome editing. The team has developed a platform called PIPPR® that can address concerns about CRISPR safety on a proteome level, together with SCIEX’s mass spectrometry SWATH® Acquisition technology. The PIPPR® platform offers a proteomic expression analysis solution for cells that have been genetically modified. Using SWATH® Acquisition researchers can then visualize their results and identify and quantify between 3000-5000 proteins in any cell line, whether of plant or animal origin.
Visualizing genetic modifications on a proteome level
“There is a growing need to understand how the proteome changes during different CRISPR applications,” explains Bock. “We need a powerful method that is robust, fast and sensitive, to both confirm expected changes and to check for unexpected changes in proteins and different pathways. PIPPR®, powered by SWATH® Acquisition, is the first platform to really address this and will make it possible for researchers to see and compare expression levels of more than 3000 proteins in their cell line projects. Using this detailed, large-scale proteome information, the efficacy and safety of genetic modifications can now be assessed.”
COBO Technologies’ customers work with genome editing tools and often want to understand how the cells’ biological makeup is modified when they have applied CRISPR. By looking at the expression of proteins in the edited cell line and comparing these to the wildtype cell line, the team can identify changes that may have occurred during genome editing.
The team at COBO Technologies has developed a full package service that enables customers to use validated reagents to extract proteins from their modified cells. Once extracted, the proteins are sent to COBO Technologies where they undergo a robust analysis using SWATH® Acquisition. Within four weeks, the customers will receive a full bioinformatic analysis of the cells’ proteins.
Comparing the proteomes of genetically modified cell lines
Some of the company’s customers use the PIPPR® platform and SWATH® Acquisition technology to compare the proteome of their modified cell line with that of the wild type cell line. This allows them to see what impact the genetic modification had at a proteome level.
Other customers are more interested in seeing the effect of different genome editing tools on their cell line and comparing these with each other. “They want to know whether there is a difference between the two, whether we get different proteomic profiles. Has anything changed? Is there toxicity in one, but not the other? Might we have immunogenicity issues with some CRISPR tools, but not with others? All of this information can be gained by looking at the proteome level, which is especially important when working with CRISPR safety,” says Bock.
“Since before the initial release in 2010, SWATH® Acquisition has been continually developed in collaboration with customers,” says Ferran Sánchez, Marketing Development Manager at SCIEX. “SWATH® Acquisition ultimately immortalizes a sample by creating a digital data record of all observable species. As a data-independent acquisition strategy, SWATH® Acquisition collects mass spectrometry (MS) and MS/MS information on every detectable peak leaving you with an option to re-interrogate your sample data should new questions arise tomorrow. The extra dimension of data interpretation achieved provides increased confidence in identification for both quantitative and qualitative discovery workflows.”
Moreover, the combination of the two technologies allows researchers to save time. “We are working in a research environment where time is a big thing,” says Bock. “Researchers are doing a lot of things and we need technology that is fast, robust, and efficient because CRISPR safety is becoming increasingly important as we move into clinical phases.”
“With SWATH® Acquisition, a single generic MS acquisition method is used all of the time,” explains Sánchez. “This means you select your compounds of interest from the digital map after you collect your comprehensive MS and MS/MS data on your sample. Should your data analysis present new questions, you can simply re-interrogate the data you’ve already collected rather than updating your acquisition method and re-analyzing your sample from step one. Once your SWATH® Acquisition method is optimized for a sample type, the same method can be used for analysis across many similar samples no matter what analytes you’re studying, saving tremendous time.”
The future of gene therapies and CRISPR safety
As basic genome editing moves closer from bench to bedside, more and more studies have to be done into the safety of CRISPR and other genome editing methods. Currently, interventions are used for diseases for which the genetic background is very clear and patient stratification is possible.
Furthermore, while most drugs today follow clear regulations, there has yet to be a standard laid down for CRISPR. This is especially important for CRISPR safety as the technology moves from research to clinical phases.
“We are at an extremely early stage,” says Bock. “At the moment, we are merely looking at what standards to define at the DNA level. But once we have defined these, we need to describe standards for the RNA level, and later the proteome level. We are at a very early stage regarding the safety and efficacy of CRISPR.”
Do you want to be at the forefront of CRISPR safety research? Get in touch with SCIEX at ferran.sanchez@sciex.com and learn how you can use SWATH® Acquisition to advance your studies. To request more information on the PIPPR® platform, contact COBO Technologies at jbock@cobotechnologies.com.
Images via Shutterstock.com, SCIEX, and COBO Technologies
Author: Larissa Warneck, Science Journalist at Labiotech.eu