The Rossi Lab has developed a novel RNA-based technology that advances regenerative medicine and could have broader applications.  Reprogramming of adult cells into pluripotent stem cells holds enormous clinical potential for regenerative medicine and disease research.

But the use of induced pluripotent stem cells in the clinic is limited by the low efficiency of current reprogramming protocols and the need to employ viruses or DNA to reinstate the stem cell identity, which permanently alters the genome of the cells. Perhaps more importantly, safe and efficient ways of steering induced stem cells to form clinically useful cell types such as blood, neurons or muscle are lacking.

To surmount those hurdles, researchers at the Program in Molecular Medicine at Children's Hospital Boston and the Immune Disease Institute along with colleagues at the Harvard Stem Cell Institute have developed a novel non-mutagenic technique for reprogramming adult cells into pluripotent stem cells. In a paper appearing in the October issue of Cell Stem Cell (online on September 30), Derrick Rossi and colleagues report using synthetic RNA to drive the expression of stem cell-inducing proteins without irreversibly altering the cells' genetic material. The resulting stem cells recapitulate the functional and molecular properties of human embryonic stem cells, and are generated at much higher efficiencies than standard virus-based techniques.

Application of modified-RNA technology for the clinical translation of induced pluripotent stem (iPS) cells.

Caption: Application of modified-RNA technology for the clinical translation of induced pluripotent stem (iPS) cells.

Modified RNA can also be used to direct the fate of the pluripotent stem cells into tissue-specific cell types faster and in greater numbers than current techniques, the researchers show. "We believe that protein expression mediated by modified RNA has the potential to become a major and perhaps even central enabling technology for cell-based therapies and regenerative medicine," Rossi said.

The ground-breaking discovery in 2006 of a way to reprogram fully differentiated adult skin cells into pluripotent stem cells opened up a door to new clinical and research applications of stem cell technology. The stem cells are produced without destroying embryos, and because they are derived from a patient's own cells, cells and tissues generated from them can be transplanted back into patients with no risk of immune response and rejection. Getting differentiated cells to regress, or "reprogram" to a stem cell state requires introduction of four key proteins that work by reestablishing an embryonic stem cell-like state in the otherwise differentiated cells. The proteins are most often introduced using viruses, an approach that carries the risk of causing mutations in the reprogrammed cells, which could trigger cancers.

Rossi and colleagues thought to employ messenger RNA (mRNA) to drive expression of reprogramming factors, as a way of getting around the problem of mutagenesis. However, they found that cells responded to the foreign mRNA with a potent anti-viral reaction that destroyed the RNA and killed the cells themselves. To overcome this obstacle, the investigators spent a year developing synthetic, chemically modified RNAs that no longer trigger the anti-viral response. This permitted the modified mRNA to drive protein expression effectively for days and weeks in human cells without any adverse affects on the cells.

The researchers then tried treating cultured fibroblast cells derived from human skin with a cocktail of modified mRNAs encoding the four major reprogramming proteins. With daily treatment, the cells reverted to a state similar to human embryonic stem cells, based on extensive molecular and functional analysis. The RNA induction process was completed in about half the time required for standard virus-based techniques, and was up to 100 times more efficient.

In addition to inducing stem cells from mature skin fibroblasts, the modified RNA technology was also effective at redirecting stem cells to form other tissue types. The addition of a modified RNA encoding a factor important for muscle differentiation to the stem cells resulted in the production of functional muscle cells, the researchers showed. This provides a proof of concept that the RNA method could be useful for generating patient-specific cells of various types for use in regenerative therapies.

"If tissue engineering is to progress to the clinic, there is a pressing need for efficient, non-mutagenic strategies to redirect cell fate," Rossi said. "Our results show that this novel RNA technology can be used to generate patient-specific pluripotent stem cells, and can likewise be harnessed to direct the fate of such stem cells towards specialized cell types that have the potential be used clinically."

Rossi notes that their technology has potential reaching far beyond the stem-cell field. The modified mRNAs can be used to boost production of any needed protein in a cell, and therefore could be potentially utilized for treating any genetic disease in which a protein is missing, deficient or defective, such as cystic fibrosis. Whereas RNA interference (RNAi) technology is widely used to inhibit gene activity and protein production, a safe reverse technology hasn't existed until now. Rossi thinks the modified mRNA technology represents this missing technology and will thus be adopted by many labs. He has patented his findings and recently formed a company called ModeRNA Therapeutics that is dedicated to translation of these discoveries into clinical use.

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Luigi Warren, Philip D. Manos, Tim Ahfeldt, Yuin-Han Loh, Hu Li, Frank Lau, Wataru Ebina, Pankaj Mandal, Zachary D. Smith, Alexander Meissner, George Q. Daley, Andrew S. Brack, James J. Collins, Chad Cowan, Thorsten M. Schlaeger, Derrick J. Rossi.  "Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA." Cell Stem Cell, 2010 Nov 5;7(5):618-30. Epub 2010 Sep 30.