Proteins necessary for reprogramming human fibroblasts into induced pluripotent stem (IPS) cells have been produced in the fibroblasts via introducing protein-coding messenger RNA (mRNA), MIT researchers say today in the open access journal PLoS One. Click here for the article in PDF.
The cells produced seem like induced pluripotent stem cells; however, they have yet to be differentiated into other cells types to prove their pluripotency. That work is ongoing, say the MIT researchers, Mehmet Fatih Yanik, an associate professor of electrical and biological engineering and Matthew Angel, an electrical engineering graduate student.
The study’s key accomplishment is to show that mRNA can be efficiently delivered into cells and produce the reprogramming proteins. Another significant benefit is that the technology eliminates the need to inhibit p53, a major tumor-suppressing gene.
Yanik and Angel said they overcame the immune response to introduced mRNA, or “long RNA,” by using a method patterned on how RNA viruses inhibit the immune response in the cells they infect. Their paper is titled: “Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins.”
MIT researchers used RNA to induce these fibroblast cells to express four genes necessary to reprogram cells to an immature state. Credit: Yanik Laboratory, MIT
They wrote in the article:
“Although the innate immune response to exogenous RNA is initiated and regulated by intra- and extracellular signaling networks containing a great deal of redundancy, RNA viruses have evolved methods of disrupting these pathways by destroying or inhibiting specific immune-related proteins to enable persistent infection. We hypothesized that mimicking viral immunoinhibition by co-transfecting cells with an siRNA cocktail designed to directly knock down expression of immune-related proteins could desensitize cells to exogenous RNA, and thus enable repeated long-RNA transfection. . . ”
“Here we have shown that combined knockdown of Ifnb1, Eif2ak2, and Stat2 rescues human fibroblasts from the innate immune response triggered by frequent transfection with protein-encoding RNA, and enables sustained, high-level expression of active proteins.”
The RNA world has been abuzz over siRNA, or Short Interfering RNA, for the last several years. These short RNA snippets have the ability to silence specific genes, and are being researched by various biotech companies for their therapeutic potential. Antisense pioneer Isis Pharmaceuticals Inc. is among those working with siRNA technology.
On July 14, Silence Therapeutics and AstraZeneca extended their siRNA drug development partnership.
Safer approach
IPS cells are typically produced by using a viral vector to introduce genes that produce reprogramming proteins. However, this can disrupt the genome’s integrity, so it’s undesirable for producing cells for transplantation into humans. So researchers are trying to find other methods to induce pluripotency, such as by using plasmids.
IPS cells have previously been made by protein transduction, or transferring the reprogramming proteins into the cell through the cell membrane. But that method is inefficient, the researchers said. By placing the mRNA that codes for the reprogramming proteins inside the cells, Yanik and Angel say they’ve caused the proteins to be made efficiently.
The mRNA for the proteins can be introduced multiple times, which would kill non-immunosuppressed fibroblasts, the researchers wrote.
As another benefit of their approach, Yanik and Angel write that their method of knocking down the immune-related proteins eliminates the need to knock down p53, a tumor-suppressor gene. The authors wrote that this “may facilitate the use of long-RNA transfection in therapeutic applications as p53 is crucial for the maintenance of genomic integrity.”
