Advances in synthetic biology and through editing have led a growing industry to develop tailored cell lines for medical research. However, these engineered cell lines can be susceptible 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 the manufacturer’s intellectual property (IP) rights. The engineers demonstrated the method in a study published online on May 4 and in the print edition of May 6 The progress of science.
The patent-pending technology is the result of an interdisciplinary collaboration between UT Dallas faculty members. The corresponding authors of the study are Dr. Leonidas BlerisProfessor of biotechnology specializing in genetic engineering, and Dr. Yiorgos MakrisProfessor of electrical and computer technology who is an expert in electronics hardware security.
Adapted 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, an increase from $ 22.8 billion by 2021, according to a forecast from 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 (PUF) in the electronics industry. A PUF is a physical property that can act as a unique “fingerprint” for a semiconductor device such as a microprocessor. In semiconductors, PUFs are based on natural variations that occur during the manufacturing process and must meet three requirements: They must have a unique fingerprint, produce the same fingerprint every time they are measured, and be virtually impossible to replicate.
To apply that concept to engineered cells, the researchers developed a two-step process that takes advantage of a cell’s ability to repair damaged DNA, which consists of sequences of small molecules called nucleotides.
First, they embedded a barcode library with five nucleotides in a part of the cell’s genome called a safe harbor, where the modification will not damage the cell. However, bar codes alone do not meet the three characteristics of PUF. In the second step, the researchers used the gene editing tool CRISPR to cut the DNA near the barcode. That action forces the cell to repair its DNA using random nucleotides, a process known as non-homologous repair. During this repair process, the cell naturally inserts new nucleotides into the DNA and / or deletes others – together these are called indels (insertions / deletions). These random fixes, combined with the barcodes, create a unique pattern of nucleotides that can help differentiate the cell line from all others.
“The combination of bar coding with the inherent stochastic cellular error repair process results in a unique and irreproducible fingerprint,” said Bleris, who is also Cecil H. and Ida Green Professor of Systems Biology.
This first generation of CRISPR-engineered PUF allows researchers to confirm that the cells were produced by a specific company or lab, a process known as proof of origin. With further research, the engineers aim to develop a method for tracking the age of a specific copy of a cell line.
“Companies that develop cell lines make a huge investment,” Bleris said. “We need a way to distinguish between 1,000 copies of the same product. Although the products are identical, each of them has a unique identifier that cannot be replicated.”
Makris said that the business of developing engineered cells is so new that companies focus on making money from their investments rather than on security and descent certificates. He said the semiconductor industry was similar in the beginning until counterfeiting and manipulation incidents showed the need for security measures.
“We think this time maybe we can stay ahead of the curve and have that ability developed when the industry realizes they need it,” Makris said. “It will be too late when they realize they have been hacked and someone made money on their IP.”
Other authors of the study include Dr. Yi Li, a biotechnology researcher; Mohammad Mahdi Bidmeshki PhD’18, a former postdoctoral fellow in Makri’s lab; Taek Kang, PhD student in biomedical technology and Eugene McDermott Graduate Fellow; and Chance M. Nowak, a bioengineering student.
Reference: Li Y, Bidmeshki MM, Kang T, Nowak CM, Makris Y, Bleris L. Genetic physical unclonable functions in human cells. Sci Adv. 2022; 8 (18): eabm4106. doi: 10.1126 / sciadv.abm4106
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