Scientists seeking a simple and gentle way to provide short-term gene therapy have a new tool: nanoparticles. In a paper published August 30 in Nature Communications, Dr. Matthias Stephan at Fred Hutchinson Cancer Research Center describes nanoparticles he has developed that can streamline the delivery of bundled genetic material to specific cells.
“What we’re doing is ‘hit-and-run’ gene therapy,” a strategy in which a brief change to certain cells can have a permanent therapeutic effect, said Stephan, an immunobioengineer who led the study.
Currently, scientists pursuing gene therapy must choose either targeted approaches that permanently alter cells’ DNA, or short-term approaches that are damaging to cells and can’t be restricted to a particular cell type. Now there is a third option.
“It’s a really cool technology,” co-author and Hutch colleague Dr. Hans-Peter Kiem said of the new nanoparticles. Kiem, who holds Fred Hutch’s Endowed Chair for Cell and Gene Therapy, is a bone marrow transplant specialist who is using gene therapy to improve treatment for glioblastoma, HIV, and genetic diseases like Fanconi anemia and hemoglobinopathies.
The nanoparticles’ ability to gently and temporarily provide gene-editing proteins to specific cells is “really the key” that makes the new approach stand out, he said. The approach may also be simple enough to — someday — make short-term gene therapy feasible around the world, even in areas with little or none of the specialized technology now needed to genetically engineer cells.
Our cells run on proteins, and our DNA carries the “recipes” for all our proteins in the form of genes. When a genetic engineer adds a new gene to a cell, the cell “reads” the gene and produces a new protein — and gets whatever capabilities that protein provides.
Scientists are already using this careful rejiggering of cells’ DNA to improve health. For example, pioneers in cellular immunotherapy aim to save the lives of cancer patients by giving them immune cells genetically rewired to destroy the cancer.
Until now, genetic engineers had to choose between two methods:
The first are long-term strategies, such as those used in cellular immunotherapy or stem-cell gene therapy. These rely on carefully constructed carrier molecules that only enter certain cells and, once inside, stitch new genes into those cells’ DNA. But that stitching endures through a cell’s lifetime — which poses a problem if the genes that scientists want to introduce could have negative long-term effects.
The second method, called electroporation, does not permanently alter cells’ DNA, but it still has significant drawbacks, said Kiem. Electroporation “is very rough on cells. And we can’t really target any particular cells,” he said.
In electroporation, electric currents open holes in cells’ outer membranes. Using this method, researchers like Kiem can slip in messenger RNA, a type of genetic material that works like messages written in disappearing ink: It carries protein-building instructions from genes to protein-building factories elsewhere in the cell and then quickly degrades. If researchers slip messenger RNA for a specific protein into cells using electroporation, those cells can only build that protein during the short period before the messenger RNA disintegrates.
During electroporation, however, the membrane of every cell exposed to the current becomes porous. If scientists need to be selective about which cells they modify, they must go through more complex steps to separate out those cells.
Now, Stephan has brought together the best of both methods in a gentle, targeted and transient technique. The nanoparticles are formed by bundling synthetic messenger RNA (modified to be safer and more stable than natural RNA) into miniscule packages surrounded by a biodegradable coat. The coat itself is studded with molecules that help the nanoparticles home in on exactly the right cell type.
The researchers showed that hours after the nanoparticles are taken up by target cells, the cells begin churning out proteins based on the new messenger RNA they’ve received. Then, within days, production ceases as the messenger RNA degrades.
And, noted Stephan, the new approach is simple: The nanoparticles come in a dried form that just needs a little water and a little mixing before they’re ready to be added to cells.
“They really let you fulfill all your wishes as a genetic engineer because you can pack in all your different [gene-therapy] components and further improve the therapeutic potential of your cell product without additional manufacturing steps,” he said.
To prove the potential of his nanoparticles, Stephan and his team tested three different applications, all aimed at making various experimental cancer therapies safer and more effective.
In particular, Stephan focused on using the nanoparticles to improve a type of cellular immunotherapy known as chimeric antigen receptor (CAR) T-cell therapy. In this therapy, immune cells known as T cells receive genes that encode a CAR, a lab-designed molecule that directs them to recognize and destroy cancer cells. Stephan showed how his ‘hit-and-run’ gene therapy could enhance the approach by removing a different gene from the T cells that might otherwise cause them to attack patients’ healthy tissue.
Using his nanoparticles, he delivered messenger RNA for DNA-snipping molecular scissors developed by Hutch colleague Dr. Barry Stoddard. The scissors then snipped out the unwanted gene, leaving the newly engineered CAR T cells to focus solely on the target tumor cells.
In a separate set of experiments, Stephan and his team showed they could also enhance CAR T cells by temporarily providing genes that gave those cells a specific set of characteristics. Hutch innovators in the field of cellular immunotherapy have shown that using a particular type of T cell, called a “central memory” T cell, is most effective against cancer; central memory T cells can survive long-term, “remembering” their target and generating fresh fleets of T cells if that target — in this case the tumor — resurfaces.
Stephan’s team demonstrated that by giving CAR T cells critical central memory genes for just a few days, they could permanently program the therapeutic cells to become central memory CAR T cells. And when the researchers tested their engineered cells in a mouse model of leukemia, they found that mice who received the nanoparticle-programmed central memory CAR T cells lived twice as long as mice given conventional CAR T cells — an incredible therapeutic advance gained with a single, simple step, noted Stephan.
Finally, he also teamed up with Kiem to see if the nanoparticles could improve Kiem’s gene therapy applications. Kiem studies blood stem cells, the source of all our blood and immune cells. He has developed a gene therapy method that can help improve brain tumor treatment. A molecule called benzylguanine helps make brain cancer cells susceptible to chemotherapy but unfortunately also damages blood cells. Kiem has shown that inserting a gene that protects against benzylguanine into patients’ blood stem cells allows patients to reap the chemotherapy-enhancing effects of benzylguanine without risking its toxic effects.
“The more of these gene-edited cells we have, the better their therapeutic effect,” Kiem said. But, he noted, blood stem cells need particularly gentle handling. As they divide, they can lose their ability to serve as a perpetual source of new blood and immune cells. On the other hand, forcing them to remain as stem cells long-term — by manipulating specific genes — can lead to cancer.
Kiem and Stephan found the nanoparticles were tailor-made for overcoming this hurdle. When they used the nanoparticles to provide a quick shot of a gene that preserved the cells’ stem-ness only while they multiplied, it resulted in double the usual number of stem cells, without the risk of cancer.
“These are just three examples out of many,” said Stephan, who is working with several companies focused on improving T-cell therapies for cancer. He expects that the simplicity and scalability of the approach will attract other collaborators as well.
Kiem also sees the wide array of potential applications. He hopes to use gene editing to address a host of diseases, from blood disorders arising from defective hemoglobin to HIV infection. He and Stoddard have teamed up to make cells HIV-resistant by using Stoddard’s molecular scissors to edit cells’ DNA. Stephan’s nanoparticles have the potential to make what is now a complex, multi-step process much simpler, allowing researchers to provide gene editing tools to just the right cells in a single step, said Kiem.
And because Stephan’s nanoparticles are dried, they don’t need special shipping or storage conditions. The simplicity of transporting and using the nanoparticles means that they could bring short-term gene therapy to areas of the world lacking high-tech cell-processing facilities — exactly where most HIV patients are, Kiem said. If someday their cure could be shipped in unrefrigerated vials, “that’s much more attractive, much more doable.”
Currently, the nanoparticles do their job in lab dishes outside the body, but it may also be possible that, after a great deal more tweaking and testing, they could one day carry out short-term gene therapy right inside a patient’s body.
“That would be fantastic,” said Kiem.
The study was funded by the Bezos family, a National Institutes of Health Cancer Consortium Cancer Center Support Grant, the National Science Foundation, and the American Cancer Society.
Note: Scientists at Fred Hutch played a role in developing these discoveries, and Fred Hutch and certain of its scientists may benefit financially from this work in the future.
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a PhD in immunology from the University of Washington, an MA in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at firstname.lastname@example.org.