A faster and easier approach to genetically reprogram cells, compared to current viral and electroporation methods is being explored by scientists based at the Fred Hutchinson Cancer Research Center. According to these scientists, nanoparticles can be used to deliver transient gene changes to targeted cells, making therapies for a variety of diseases—including cancer, diabetes, and HIV/AIDS—faster and cheaper to develop, and more customizable.
Details appeared August 30 in in the journal Nature Communications, in an article entitled “Hit-and-Run Programming of Therapeutic Cytoreagents Using mRNA Nanocarriers.” The article describes the development of targeted mRNA nanocarriers that can be mixed with cells to reprogram them via transient transfection. The article goes on to describe three examples to make the case that the approach is simple and generalizable.
“First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells,” the article’ authors wrote. “Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties.”
Essentially, nanoparticles carried a gene-editing tool to T cells of the immune system that snipped out their natural T-cell receptors, and then was paired with genes encoding a chimeric antigen receptor, or CAR, a synthetic molecule designed to attack cancer.
Next, nanoparticles were targeted to blood stem cells and equipped with mRNA that enabled the stem cells to multiply and replace blood cancer cells with healthy cells when used in bone marrow transplants.
Finally, nanoparticles were targeted to CAR T cells and equipped with Foxo1 mRNA, which signals the anticancer T cells to develop into a type of "memory" cell that is more aggressive and destroys tumor cells more effectively and maintains antitumor activity longer.
"Our goal is to streamline the manufacture of cell-based therapies," said lead author Matthias Stephan, M.D., Ph.D., a faculty member in the Fred Hutch Clinical Research Division and an expert in developing biomaterials. "In this study, we created a product where you just add it to cultured cells and that's it—no additional manufacturing steps."
Dr. Stephan and his colleagues developed a nanoparticle delivery system to extend the therapeutic potential of mRNA, which delivers molecular instructions from DNA to cells in the body, directing them to make proteins to prevent or fight disease.
The researchers' approach was designed to zero in on specific cell types—T cells of the immune system and blood stem cells—and deliver mRNA directly to the cells, triggering short-term gene expression. It's called "hit-and-run" genetic programming because the transient effect of mRNA does not change the DNA, but it is enough to make a permanent impact on the cells' therapeutic potential.
Other attempts to engineer mRNA into disease-fighting cells have been tricky. The large messenger molecule degrades quickly before it can have an effect, and the body's immune system recognizes it as foreign—not coming from DNA in the nucleus of the cell—and destroys it.
Stephan and his Fred Hutch collaborators devised a workaround to those hurdles.
"We developed a nanocarrier that binds and condenses synthetic mRNA and protects it from degradation," Dr. Stephan explained. The researchers surrounded the nanoparticle with a negatively charged envelope with a targeting ligand attached to the surface so that the particle selectively homes in and binds to a particular cell type.
The cells swallow up the tiny carrier, which can be loaded with different types of man-made mRNA. "If you know from the scientific literature that a signaling pathway works in synergy, you could co-deliver mRNA in a single nanoparticle," Dr. Stephan elaborated. "Every cell that takes up the nanoparticle can express both."
The approach involves mixing the freeze-dried nanoparticles with water and a sample of cells. Within four hours, cells start showing signs that the editing has taken effect. Boosters can be given if needed. Made from a dissolving biomaterial, the nanoparticles are removed from the body like other cell waste.
"Just add water to our freeze-dried product," Dr. Stephan emphasized. Since it's built on existing technologies and doesn't require knowledge of nanotechnology, he intends for it to be an off-the-shelf way for cell-therapy engineers to develop new approaches to treating a variety of diseases.
The approach could replace labor-intensive electroporation, a multistep cell-manufacturing technique that requires specialized equipment and clean rooms. All the handling ends up destroying many of the cells, which limits the amount that can be used in treatments for patients.
Gentler to cells, the nanoparticle system developed by the Fred Hutch team showed that up to 60 times more cells survive the process compared with electroporation. This is a critical feature for ensuring enough cells are viable when transferred to patients.
"You can imagine taking the nanoparticles and injecting them into a patient; then you don't have to culture cells at all anymore," he asserted.
Dr. Stephan has tested the technology in cultured cells in the lab, but it's not yet available as a treatment. He is looking for commercial partners to move the technology toward additional applications and into clinical trials where it could be developed into a therapy.