Science Spotlight

Hit-and-run genome engineering improves cell-based therapies

From the Stephan Lab, Clinical Research Division

Cell-based therapeutics are on their way to becoming a standard of care for cancer and many other diseases. Two prominent examples include stem cell transplantation to replace damaged or diseased bone marrow and genetic engineering of immune cells to attack and kill cancer cells. Because “our knowledge of the genes and gene networks that regulate cellular functions has dramatically increased in recent years,” efforts to improve cell-based therapies by manipulating their gene expression are now possible, says Dr. Howell Moffett, a post-doctoral research fellow in the Stephan Laboratory (Clinical Research Division). “However, our ability to manipulate the functions of specific genes to improve cell-based therapy has lagged far behind” our understanding. In a study recently published in Nature Communications, Moffett and his colleagues in the Stephan lab, along with collaborators in the Kiem (Clinical Research) and Stoddard (Basic Sciences) labs, present their efforts to make genetic modification of cytoreagents a simple, affordable process.

A) schematic of T-cell reprogramming, B) Schematic of mRNA nanocarrier structure and composition
a) Schematic of how cultured T-cells can be programmed to express therapeutically relevant transgenes carried by mRNA nanoparticles. b) Design of targeted mRNA nanoparticles. The inset shows a transmission electron micrograph of a representative nanoparticle; scale bar, 50 nm. Also depicted is the synthetic mRNA encapsulated in the nanoparticle, which is engineered to encode therapeutically relevant proteins. Image from the article

The authors constructed particles—termed mRNA nanocarriers—that perform a “hit-and-run” when mixed with target cells, meaning that their mRNA cargo is taken up and immediately but transiently expressed. By designing the mRNA sequence to encode specific functions, the researchers can theoretically modify target cell gene expression in whatever manner they desire. Each nanocarrier contains four ingredients: i) mRNA encoding the desired effector function(s) that is synthesized using modified bases in order to avoid activation of the immune system, ii) a positively charged, biodegradable carrier matrix to protect the mRNA from degradation, iii) a negatively charged coating to reduce surface charge and off-target binding and iv) a surface-bound antibody to allow entry into target cells via receptor-mediated endocytosis.

The versatility of the mRNA nanocarrier approach was validated by applying it in three distinct contexts. The authors first focused on a recently developed therapy in which T-cells are engineered to express a chimeric antigen receptor (CAR) that targets and destroys cancer cells. Because CAR T-cells still express their original T-cell receptor (TCR), there is a risk of graft-versus-host disease occurring when they are introduced into the patient. In order to prevent this, Moffett et al designed an mRNA nanocarrier that encodes nucleases capable of removing the TCR from the genome of the CAR T-cells. The nanocarrier eliminated TCR expression in 60% of cells without negatively affecting incorporation of the CAR or the ability of the cells to proliferate. Importantly, this approach proved to be simpler and more effective than electroporation, a common method for introducing mRNA into target cells.

Second, the authors tested whether mRNA nanoparticles could be used to confer the favorable properties of central memory T-cells to CAR T-cells, a process that normally requires complex in vitro manipulations. They successfully showed that nanoparticles encoding the transcription factor FOXO1, which promotes the effector-to-memory transition in T-cells, induced persistent expression of the memory T-cell receptor CD62L and globally shifted gene expression toward a memory cell-like program. Importantly, CAR T-cells treated with the FOXO1-containing nanocarriers had significantly higher antitumor activity than untreated cells in a mouse model of lymphoma.

To demonstrate the generality of their approach, Moffett et al also tested whether mRNA nanocarriers could be used in hematopoetic stem cells (HSCs) to help maintain their self-renewal properties. Because permanent integration of self-renewal genes into HSC genomes has the potential to cause uncontrolled cell growth, the transient expression offered by mRNA particles is attractive in this context. The researchers constructed nanocarriers that target CD105, a marker of the most immature HSCs, and mRNA for the self-renewal gene Musashi-2. Treatment of HSCs with this nanocarrier elicited a transient spike in expression of Musashi-2, increased abundance of cell surface markers associated with an undifferentiated state and enhanced regenerative potential.

Together, the results of the mRNA nanocarrier project represent an important step toward the goal of improving cell-based therapies by optimizing their gene expression. Because mRNA nanocarriers need only to be mixed with target cells, this approach represents a simple, scalable and efficient alternative to existing methods such as electroporation, viral transduction, or use of cell-penetrating peptides. In the future, mRNA nanocarriers designed to target a wide variety of cell types may be able to decrease both the cost and difficulty of translating cell-based therapies from the lab bench to the clinic.



Moffett HF , Coon ME, Radtke S, Stephan SB, McKnight L, Lambert A, Stoddard BL, Kiem HP, Stephan MT. “Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers.” Nature Communications. 2017 Aug 30;8(1):389. doi: 10.1038/s41467-017-00505-8.

This research was supported by the Bezos Family Foundation, the National Institutes of Health, the Department of Defense, the National Science Foundation and the American Cancer Society.

Fred Hutch, HFM and MTS have filed a patent pertaining to mRNA delivery via targeted polymeric nanoparticles. The remaining authors declare no competing financial interests.