Periodic Reporting for period 1 - TransGeNo (Transposon-activated Genome-wide search for novel Nociceptors)
Período documentado: 2020-04-20 hasta 2022-04-19
Reliably identifying novel molecular targets within the approximately 20,000 coding genes of our genome presents a logistical challenge. To address this, we developed and validated a novel strategy: TAGS (Transposon Activated Genome-wide Screening). This approach involves inducing mutations at random in cells, such that they activate genes in their vicinity. This is done in millions of cells, relying on the evolutionary strategy of the law of large numbers for the random mutations to, in rare cases, accidentally activate the right gene. The cells in which this happens are isolated from the pool of millions, and the mutations are then reverse engineered to identify the correct gene.
Our overall objectives were to
1) Develop the TAGS pipeline by creating a bioinformatics analysis module
2) Identify novel mechanically activated receptors
3) Identify novel receptors for the analgesic compound hydroxy-alpha-sanshool
We have successfully developed a bioinformatics analysis pipeline capable of comprehensive analysis of genome-wide insertional datasets produced by our screening methods. We demonstrated its use not only in our existing (control) dataset, but by deploying this custom method to a new screening library as well.
We have made significant progress in developing the necessary screening environment to successfully finish Objective 2. This required the building of a novel, upgraded mechanical stimulation device with improved throughput and higher reproducibility. To this end, we have designed a device based on the concept of an iris shutter, and molded custom PDMS dishes to fit this device. We have identified the most suitable cell line for this screening and are currently progressing with genome-wide screening.
Objective 3 was diverted to an alternative as delineated in our Risk Management Plan. This was due to limited availability of the compound, OH-alpha-Sanshool, due to limitations in shipment during the pandemic. We therefore conducted a screen to identify novel receptors for noxious cold. Our screen has clearly and unequivocally re-identified known temperature sensor molecules TRPM8 and TRPA1 in the cold temperature range. Importantly, our results indicate that at least one, but likely more novel cold receptors were also identified by our efforts. We are currently in the process of validating these targets and testing their physiological relevance. Overall, we have successfully demonstrated proof of concept for our novel platform technology by successfully identifying novel target molecules. A PCT patent application has been filed on the basis of this, as a first step to commercial exploitation of our results.