For more than three decades Michael Koob has been working out complicated puzzles using the tools of molecular biology and genetics.  Today his deliberative labors are paying off -- with untold implications for the study of human disease and the development of drug therapies and vaccines.   Koob has figured out how to replace entire genes of laboratory mice with their human counterparts, transporting huge segments of human DNA to their proper corresponding location in mouse chromosomes.   Now he is applying his genetic puzzle-solving ingenuity to  the scourge of the COVID-19 pandemic.

An LMP associate professor, Koob launched his molecular investigations while a graduate student at the University of Wisconsin in Madison, where he earned a PhD in molecular and cellular biology in 1990.  His graduate adviser was the legendary molecular geneticist Waclaw Szybalski.  Koob and Szybalski pioneered a technique they called “Achilles' heel cleavage” that cuts DNA in a single targeted location, which enabled them to create large DNA segments.  Koob joined the LMP faculty in 1995.  He brought with him those early insights about how to use molecular tools to manipulate DNA in human and animal cells and thereby answer questions about health and disease.

Now Koob has set his sights on COVID-19, the disease caused by coronavirus SARS-CoV-2 infection.  SARS-CoV-2 respiratory viruses enter human lung tissue via a cell-surface receptor molecule called angiotensin-converting enzyme 2 or ACE2.  Once in the lung the virus multiplies and travels throughout the organ, in some patients causing Acute Respiratory Distress Syndrome (ARDS), which can be fatal. 

But there’s a problem in using mice to understand SARS-CoV-2 infection and COVID-19 disease progression.  “In the mouse, the ACE2 receptor doesn’t bind the virus, so mice don’t get infected and show the respiratory symptoms we see in people,” Koob said.  But what if mice expressed the human gene for the ACE2 receptor instead of their own?  That would potentially enable investigators to track COVID-19 pathology beginning with infection and viral replication in airway epithelial cells all the way to lower lung zones where the virus often settles, consolidates, and can cause viral pneumonia.  That mouse model is under construction in Koob’s laboratory.

Infection at the entry point would make the mouse model work for COVID-19, and full human ACE2 receptor gene substitution for the mouse version should make infection possible, Koob said.  “The internal viral replication will be maintained between the mouse and humans.  So this should model the infection route, disease progression in the lungs, everything like that.  It’s really just basic cell biology.  If you want to mimic what happens in a person the most important thing really is to get the cell types correct.  If the right cells are ACE2 receptor-positive, then you can mimic what happens in people.” 

Other research groups have transferred only a small part of the ACE2 receptor DNA gene sequence into mice, creating transgenic animals but ones that do not mimic the potentially lethal lung pathology of a SARS-CoV-2 infection and COVID-19, such as ARDS.  Koob’s team will replace the entire mouse ACE2 receptor gene with the entire human ACE2 receptor gene plus associate regulatory sequences—transferring in all some 70,000 DNA sequences to the precise location on the mouse chromosome where its own ACE2 receptor gene once resided.  “The mouse gene will be gone, and the human gene will be there,” Koob said.   “It now becomes a human ACE2 receptor gene in a true sense.  The sequence of tissues that become positive for ACE2 receptor expression should be recapitulated.” 

When a human gene is put in the same spot where the mouse gene once resided, genomic regulatory factors come into play that are appropriate for that gene, Koob said. “There’s a global regulatory context to take into account in animals that have a common ancestor, which all mammals do.  Mice and humans are fairly close on the evolutionary tree.  So there’s global regulation if we put it in the right spot.”  The “right spot” transfer of the human gene construct is into a mouse embryonic stem cell, which Koob then puts into a blastocyst or early mouse embryo.  Selective breeding yields mice with the human gene in all cells and tissues.

A search of the database yields more than 400 studies when the terms “COVID-19” and “lung therapy” are combined.  Small molecule drugs, therapeutic antibodies and antivirals, immunotherapies, stem cells and natural killer cells, steroids, and laser and radiotherapies are among the lung injury therapies currently being investigated.  A validated, reliable, and clinically informative mouse model for testing COVID-19 lung injury therapies would be invaluable, as it would be for future coronavirus vaccine trials. 

Koob anticipates his human ACE2 receptor gene mouse strain will be ready by this fall.  He will send it by courier to Jackson Laboratory (JAX) in Bar Harbor, Maine to join more than 11,000 strains of mice that JAX distributes to researchers around the world.  JAX will breed the mice over several months while Koob and LMP professors Steve Jameson and Kris Hogquist and Department of Medicine assistant professor Tyler Bold, all at the Center for Immunology, conduct characterization and SARS-CoV-2 infection studies of the mice in a Level 3 biosafety facility.  JAX is currently distributing Koob’s full gene replacement mouse strain that carries the human microtubule-associated protein tau, which is responsible for the neurofibrillary tangles in the brain associated with Alzheimer's disease and other dementias.  Koob is making full gene replacement mouse models of other neurodegenerative diseases.

“Our philosophy is to make our mouse strains available to the research community in an expedited way,” Koob said.  “I contacted JAX about this ACE2 receptor gene replacement mouse.  They’re very happy to collaborate with us because they don’t have anything like this.  And we’re making it available to researchers without restrictions.”

With Koob and his laboratory scientist Kellie Benzow as inventors, the University has filed a patent on “Methods of full gene replacement and transgenic non-human cells comprising full human genes.”   

It’s been a long time since Koob collaborated with his graduate adviser Waclaw Szybalski, now a 98-year-old professor emeritus.  Together their research careers encompass the history of molecular biology going back to the early 1950s with the discovery of the DNA double helix.  Szybalski was born in 1921 just after a pandemic virus infected an estimated one-third of the Earth’s population and killed tens of millions of people.  A century later, with another pandemic raging, the timing couldn’t be better for his student to exercise his manifest molecular inventiveness. 

William Hoffman, Genome Writers Guild "flash talk" --  Meet Mr. Jingles