Way of Detecting PMP22 Gene Activity May Advance CMT1A Therapies
A new way to rapidly identify compounds with the ability of lower production of PMP22, a protein that is overproduced in Charcot-Marie-Tooth disease type 1A (CMT1A), has been developed, and could be helpful in spotting molecules with a potential to treat this disease.
The approach takes advantage of gene editing and light-emitting technology to detect a specific process, known as transcription, by which a gene’s DNA sequence is converted into messenger RNA (mRNA), which serves as a template for making proteins.
It also might be applied to others diseases that could benefit from modulators of gene activity, the research team reported.
The study, “Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for PMP22 Gene-Dosage Disorder Drug Discovery,” was published in the journal ACS Pharmacology and Translational Science.
CMT1A, the most common form of CMT1, is caused by the presence of an extra copy of the PMP22 gene. This gene encodes for the PMP22 protein, an important component of the myelin sheath produced by Schwann cells. Overexpressed, the PMP22 protein cannot be processed correctly and is less functional, affecting the structure and working of myelin — the protective and insulating covering of nerve cells projections (axons). Problems with myelin, in turn, impair the transmission of electrical signals by nerves, leading to muscle weakness and atrophy.
Researchers with the National Center for Advancing Translational Sciences (NCATS), in collaboration with scientists at the University of Wisconsin and the National Human Genome Research Institute, developed a test, or assay, in aid efforts in discover CMT1A therapies.
The assay was created with the ultimate goal of pinpointing compounds, from among a large group of candidates, that show effectiveness in lowering PMP22 production.
In previous work, the team genetically engineered cells to produce a reporter enzyme that emits light whenever the PMP22 gene is transcribed. However, an investigational therapy could affect the light-producing reporter, and test assessments be inaccurate.
“Standard approaches to rapidly measure gene transcription in thousands of compounds can lead to large numbers of samples being identified as having positive results, when they are actually negative,” James Inglese, PhD, the study’s senior author, said in a press release.
To address this concern, the scientists inserted two different reporters into the PMP22 gene in rat Schwann cells. Adding the second reporter, which they called the “coincidence reporter” approach, reduced the probability of false positive results, because the likelihood of a compound affecting both reporters is much lower.
Researchers also found a way to measure PMP22 proteins, using gene editing and a reporter to detect PMP22 protein levels.
Next, quantitative high-throughput screening of 42,577 compounds in NCATS’ chemical, using the coincidence-reporter approach, identified 181 molecules that reduced PMP22 transcription without harming cells. Gene editing plus a reporter then showed that 16 of these 181 compounds not only reduced transcription, but also lowered PMP22 protein production.
According to the team, the coincidence-reporter approach for measuring gene activity could aid in searching compounds that might treat disorders other than CMT1A. “The coincidence reporter is a particularly effective tool in those diseases where a compound is needed that would suppress or activate a disease-related gene,” Inglese said.
“Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational [protein-making] regulation of PMP22 … which could be efficiently incorporated into drug discovery campaigns for CMT1A,” the investigators wrote.