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Transposon-activated Genome-wide search for novel Nociceptors

Periodic Reporting for period 1 - TransGeNo (Transposon-activated Genome-wide search for novel Nociceptors)

Reporting period: 2020-04-20 to 2022-04-19

Chronic pain is a prevalent and debilitating condition, affecting approximately 30% of western societies. Our existing therapeutics carry severe risks, such as addiction. There is an urgent medical need to identify novel molecular targets in the sensory nervous system, to enable development of safer and more efficacious analgesics. Molecular pain receptors, found peripheral sensory nerves, present a uniquely promising target for such novel medications. These proteins provide the opportunity to inhibit pain at the very place its signal initiates, bypassing the need to modulate the central nervous system, and this promising both enhanced safety and efficacy.
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
Our approach is based on random genetic modification of hundreds of millions of target cells, such that expression of genes in the vicinity of these random mutations is induced. The law of large numbers thus results in the right, sought-after gene to be randomly induced in a very small subset of cells (Figure 1-p). These are identified by genetically encoded reporters, inducing fluorescence of a specific colour. These cells are selected, grown into clonal population and the mutations read and mapped onto the genome, thus identifying the correct gene.
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.
We have progressed beyond the state of the art in three important ways. We have developed a bioinformatics pipeline that is capable of statistically evaluating a landscape of genomic insertions, and after analysing insertion patterns in a group of samples identifying and separating common clusters and hot spots. This has allowed us to demonstrate definitive proof of concept for a novel genome-wide screening technology, by re-identifying two known molecular receptors of cold, as well as identifying two new subsets of cells that express unknown, novel molecular receptors of cold. These novel receptors represent a separate advancement beyond the state of the art, in addition to providing proof of concept for the screening technology, as they will further our understanding of the molecular mechanisms of mammalian somatosensation and present potential molecular targets for future analgesics. The latter carries the most important potential for socio-economic impact of our work, as currently existing analgesic therapy lacks tools that are both safe and efficacious. By identifying novel molecules involved in the pain pathway, we provide promising targets for molecules that do not need to cross the blood-brain barrier to affect the process of nociception, thereby promising increased safety. Chronic pain affects a large proportion of society, these targets are therefore of high clinical importance.
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