Using a single-cell experimental approach, James Eberwine, PhD, professor of Pharmacology, has been able to literally "reprogram" one type of cell into another, such as transforming a neuron into an astrocyte. Similar to the manner in which stem cells differentiate themselves into various cell types or even the process by which viruses invade and take over a cell, it's done by injecting the cell with the mRNA of another, which causes the cell to transform itself into the new type. This was the first demonstrated use of RNA as a therapeutic, heralding the development of the field of “RNA therapeutics.”
Rat neuron with a micropipette inserting mRNAs directly onto the cell. After laser photoporation the mRNA goes into the cell and the TIPeR-induced changes in cell phenotype are initiated.
The approach Eberwine uses, called TIPeR (Transcriptome-induced phenotype remodeling), is more direct than the process of differentiating cells from stem cells, since it doesn't require the intermediate steps of first creating pluripotent cells and then changing them into the final desired cell type.
The TIPeR methodology can be applied to essentially any cell type putting between 2 and 10,000’s of RNAs into any cell in any abundance. If that cell has a nucleus then that cell’s phenotype can be converted from one cell type to another or from disease to potentially non-diseased.
Other experimental procedures developed in Eberwine's lab allow them to study and manipulate RNA and proteins, and their movement within an individual cell. Aside from new insights into the many roles of RNA in cell biology, his lab’s focus may point the way toward targeted cell-based therapies for neurodegenerative diseases and other conditions.
In 2012, Eberwine's lab was awarded a $10 million grant from the National Institutes of Health to examine the varying functions of mRNA within nerve cells at the single-cell level and how these cells can be "reprogrammed" using TIPeR and other techniques.
Earlier in 2005, Eberwine discovered that mRNA splicing wasn't limited solely to the cell nucleus as had been previously believed, but could also occur elsewhere, such as in the dendrites of nerve cells. Using new and highly sensitive techniques that allowed cellular activity to be observed and influenced, Eberwine next found that some introns – once thought of as "junk" DNA cut out of mRNA strands during splicing – actually have an important function outside of the nucleus. These introns help to establish the proper number and formation of important electrical pathways known as BKCa channels within the dendrite. Eberwine and his team believe that this intron mechanism may play a critical role in the regulation of neuronal mRNA.
One example Eberwine uncovered involves an intron called i17a. In order for the BKCa channel protein to do its work, it requires a stretch of nucleic acids called the STREX exon, which mRNA translates into a proper protein. The i17a intron immediately precedes STREX in the BCKa mRNA. But if i17a isn't first removed from the BCKa mRNA during splicing in the cytoplasm, the resulting protein will contain very little STREX, which impairs the BCKa channel and affects proper functioning of the neuron. Learning how to control cytoplasmic mRNA splicing could result in new therapeutic approaches for diseases involving neuronal misfiring, such as epilepsy.
Eberwine’s lab also established links between the role of nerve cell mRNAs, their resultant proteins with memory and learning. In 2013, his lab scrutinized dendritic protein synthesis in live nerve cells, using laser fluorescence techniques to observe the spatial and temporal complexity of the protein translation in dendrites. This process permits the chemical flexibility of synapses that make learning possible and the retention of memories once they're established.
Understanding the dynamics of normal processes will help to develop ways to correct them when they go wrong in neurological and psychiatric illnesses.