Bacterial injection system delivers proteins in mice and human cells

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have harnessed a natural bacterial system to develop a new protein delivery approach that works in human cells and animals. The technology, described today in Nature, can be programmed to deliver a variety of proteins, including ones for gene editing, to different cell types. The system could potentially be a safe and efficient way to deliver gene therapies and cancer therapies.

Led by Broad core institute member and McGovern Institute investigator Feng Zhang, the team took advantage of a tiny syringe-like injection structure, produced by a bacterium, that naturally binds to insect cells and injects a protein payload into them. The researchers used the artificial intelligence tool AlphaFold to engineer these syringe structures to deliver a range of useful proteins to both human cells and cells in live mice.

“This is a really beautiful example of how protein engineering can alter the biological activity of a natural system,” said Joseph Kreitz, the study’s first author and a graduate student in Zhang’s lab. “I think it substantiates protein engineering as a useful tool in bioengineering and the development of new therapeutic systems.”

“Delivery of therapeutic molecules is a major bottleneck for medicine, and we will need a deep bench of options to get these powerful new therapies into the right cells in the body,” added Zhang. “By learning from how nature transports proteins, we were able to develop a new platform that can help address this gap.”

Zhang is senior author on the study and is also the James and Patricia Poitras Professor of Neuroscience at MIT and an investigator at the Howard Hughes Medical Institute.

Injection via contraction

Symbiotic bacteria use the roughly 100-nanometer-long syringe-like machines to inject proteins into host cells to help adjust the biology of their surroundings and enhance their survival. These machines, called extracellular contractile injection systems (eCISs), consist of a rigid tube inside a sheath that contracts, driving a spike on the end of the tube through the cell membrane. This forces protein cargo inside the tube to enter the cell.

On the outside of one end of the eCIS are tail fibers that recognize specific receptors on the cell surface and latch on. Previous research has shown that eCISs can naturally target insect and mouse cells, but Kreitz thought it might be possible to modify them to deliver proteins to human cells by reengineering the tail fibers to bind to different receptors.

Using AlphaFold, which predicts a protein’s structure from its amino acid sequence, the researchers redesigned tail fibers of an eCIS produced by Photorhabdus bacteria to bind to human cells. By reengineering another part of the complex, the scientists tricked the syringe into delivering a protein of their choosing, in some cases with remarkably high efficiency.

The team made eCISs that targeted cancer cells expressing the EGF receptor and showed that they killed almost 100 percent of the cells, but did not affect cells without the receptor. Though efficiency depends in part on the receptor the system is designed to target, Kreitz says that the findings demonstrate the promise of the system with thoughtful engineering.

The researchers also used an eCIS to deliver proteins to the brain in live mice — where it didn’t provoke a detectable immune response, suggesting that eCISs could one day be used to safely deliver gene therapies to humans.

Packaging proteins

Kreitz says the eCIS system is versatile, and the team has already used it to deliver a range of cargos including base editor proteins (which can make single-letter changes to DNA), proteins that are toxic to cancer cells, and Cas9, a large DNA-cutting enzyme used in many gene editing systems.

In the future, Kreitz says researchers could engineer other components of the eCIS system to tune other properties, or to deliver other cargos such as DNA or RNA. He also wants to better understand the function of these systems in nature.

“We and others have shown that this type of system is incredibly diverse across the biosphere, but they are not very well characterized,” Kreitz said. “And we believe this type of system plays really important roles in biology that are yet to be explored.”

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This work was supported in part by the National Institutes of Health, Howard Hughes Medical Institute, Poitras Center for Psychiatric Disorders Research at MIT, Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center at MIT, K. Lisa Yang Brain-Body Center at MIT, Broad Institute Programmable Therapeutics Gift Donors, Pershing Square Foundation, W. Ackman, N. Oxman, J. and P. Poitras, BT Charitable Foundation, C. and L. Asness, the Phillips family, D. Cheng, and R. Metcalfe.

About Broad Institute of MIT and Harvard
Broad Institute of MIT and Harvard was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods and data openly to the entire scientific community.
 
Founded by MIT, Harvard, Harvard-affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide.
 
About McGovern Institute for Brain Research at MIT
The McGovern Institute is an inclusive and collaborative community of MIT scientists, engineers, and support staff who work together to unravel the mysteries of the brain. Our researchers are committed to meeting two of the greatest challenges of modern science: understanding how the brain works and discovering new ways to prevent or treat brain disorders. To address this scientific challenge, we study the brain at many levels and collaborate with academic, clinical, and industry partners around the world.
 
The McGovern Institute was established in 2000 by technology entrepreneur Lore Harp McGovern and the late Patrick J. McGovern, former chairman of International Data Group (IDG). Our director is Robert Desimone, the Doris and Don Berkey Professor of Neuroscience at MIT and former head of intramural research at the National Institute of Mental Health. The McGovern Institute has grown from six founding faculty members to more than 20 distinguished investigators including one Nobel laureate and six members of the National Academy of Sciences.