The authors, from NYU Grossman School of Medicine, created a strain of mice with human genetic material for angiotensin-converting enzyme 2 (ACE2)—a receptor through which SARS-CoV-2 enters the host cells. When infected with the virus that causes COVID-19, these mice developed symptoms similar to those of young people infected with the virus. In prior mouse models, the animals died upon infection.
The team was able to create mice with more humanlike levels of ACE gene activity by first using yeast cells to assemble DNA sequences of up to 200,000 letters. They used their new delivery method, “mammalian switching antibiotic resistance markers progressively for integration” (mSwAP-In), to put the “naked” DNAs into mouse embryonic stem cells.
By overcoming the size limits imposed by past methods, the researchers report that mSwAP-In delivered a humanized mouse model of COVID-19 pathology by “overwriting” 72 kilobases (kb) of mouse Ace2 code with 180 kb of the human ACE2 gene and its regulatory DNA.
“That these mice survive creates the first animal model that mimics the form of COVID-19 seen in most people—down to the immune system cells activated and comparable symptoms,” said senior study author Jef D. Boeke, PhD, the Sol and Judith Bergstein director of the Institute for Systems Genetics at NYU Langone Health. “This has been a major missing piece in efforts to develop new drugs against this virus.”
“Given that mice have been the lead genetic model for decades,” added Boeke, “there are thousands of existing mouse lines that can now be crossbred with our humanized ACE2 mice to study how the body reacts differently to the virus in patients with diabetes or obesity, or as people age.”
The study involves a new method to edit DNA—“genome writing.” Boeke’s lab first developed this approach in yeast but more recently adapted it to the mammalian genetic code. The team used mSwAP-In to create a humanized mouse model of COVID-19 pathology by “overwriting” 72 kilobases (kb) of mouse Ace2 code with 180 kb of the human ACE2 gene and its regulatory DNA. To accomplish this cross-species swap, they cut into a key spot in the DNA code around the natural gene, swapped in a synthetic counterpart in steps, and with each addition, added a quality control mechanism so that only cells with the synthetic gene survived.
The research team then worked with Sang Y. Kim, PhD, at NYU Langone’s Rodent Genetic Engineering Laboratory, using a stem cell technique called tetraploid complementation to create a living mouse whose cells included the overwritten genes.
Whereas the ACE2 experiments had swapped in an unchanged version of a human gene, the synthetic, swapped-in Trp53 gene was designed to no longer include a combination of molecular code letters—cytosine (C) next to guanine (G)—known to be vulnerable to random cancer-causing changes. The researchers overwrote key CG “hot spots” with code containing a different DNA letter, adenine (A).
“The AG switch left the gene’s function intact, but lessened its vulnerability to mutation, with the swap predicted to lead to a ten- to fiftyfold lower mutation rate,” said first author Weimin Zhang, PhD, from Boeke’s lab. “Our goal is to demonstrate in a living test animal that this swap leads to fewer mutations and fewer resulting tumors, and those experiments are being planned.”