Advances in synthetic biology and genome editing have led a growing industry to develop personalized cell lines for medical research. However, these modified cell lines may be vulnerable to misidentification, cross-contamination, and illegal replication.
A team of researchers from the University of Texas at Dallas has developed a first-of-its-kind method to create a unique identifier for each copy of a cell line to allow users to verify its authenticity and protect intellectual property (IP ) from the manufacturer. The engineers demonstrated the method in a study published online May 4 and in the May 6 print edition of Science Advances.
The patent-pending technology is the result of an interdisciplinary collaboration among faculty members at UT Dallas. The corresponding co-authors of the study are Dr. Leonidas Bleris, professor of bioengineering specializing in genetic engineering, and Dr. Yiorgos Makris, professor of electrical and computer engineering, an expert in electronic equipment security.
Personalized cell lines are used in the development of vaccines and targeted therapies for a range of diseases. The global cell culture market is expected to reach $41.3 billion by 2026, from $22.8 billion in 2021, according to forecasts by market research firm MarketsandMarkets.
UT Dallas engineers’ research to develop unique identifiers for genetically modified cells was inspired by what are known as physically unclonable functions (PUFs) in the electronics industry. A PUF is a physical characteristic that can serve as a unique “fingerprint” for a semiconductor device such as a microprocessor. In semiconductors, PUFs are based on the natural variations that occur during the manufacturing process and must meet three requirements: they must have a unique footprint, produce the same footprint each time they are measured, and be virtually impossible to replicate.
To apply this concept to engineered cells, the researchers developed a two-step process that takes advantage of a cell’s ability to repair damaged DNA, which is made up of sequences of small molecules called nucleotides.
First, they embedded a five-nucleotide barcode library into a part of the cell’s genome called the safe harbor, where the modification will not harm the cell. However, barcodes alone do not satisfy all three properties of PUFs. In the second step, the researchers used the gene-editing tool CRISPR to cut the DNA close to the barcode. This action forces the cell to repair its DNA using random nucleotides, a process called non-homologous error repair. During this repair process, the cell naturally inserts new nucleotides into the DNA and/or deletes others – collectively these are called indels (insertions/deletions). These random patches, in combination with the barcodes, create a unique pattern of nucleotides that can help distinguish the cell line from any other.
“The combination of the barcode with the inherently stochastic cellular error-repair process results in a unique, non-reproducible fingerprint,” said Bleris, who is also the Cecil H. and Ida Green Professor of Systems Biology Sciences. .
This first generation of CRISPR-designed PUFs allows researchers to confirm that cells were produced by a particular company or lab, a process called attestation of provenance. Along with further research, the engineers aim to develop a method to track the age of a specific copy of a cell line.
“Companies developing cell lines are making a huge investment,” Bleris said. “We need a way to differentiate between 1,000 copies of the same product. Even if the products are identical, each of them has a unique identifier, which cannot be replicated.
Makris said the tech cell development business is so new that companies are focusing on monetizing their investments rather than security and provenance attestation. He said the semiconductor industry was the same in the beginning until incidents of counterfeiting and tampering highlighted the need for security measures.
“We think this time maybe we can be ahead of the curve and have that capability developed just as the industry realizes it needs it,” Makris said. “It will be too late when they realize they have been hacked and someone has monetized their IP.”
Other study authors include Dr. Yi Li, a bioengineering research scientist; Mohammad Mahdi Bidmeshki PhD’18, former postdoctoral researcher in the Makris lab; Taek Kang, biomedical engineering doctoral student and Eugene McDermott Graduate Fellow; and Chance M. Nowak, graduate student in biological engineering.
The research was funded by the National Science Foundation, the UT Dallas Office of Research and Innovation’s Interdisciplinary Research Seeds Program, and the TxACE Collaborative Initiative, which supports collaborations of researchers from the Texas Analog Center of Excellence. (TxACE) with non-TxACE faculty members.
- Yi Li, Mohammad Mahdi Bidmeshki, Taek Kang, Chance M. Nowak, Yorgos Makris, Leonidas Bleris. Unclonable genetic physical functions in human cells. Scientific Advances, 2022; 8 (18) DOI: 10.1126/sciadv.abm4106
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