MICA: A comprehensive genomic and functional approach to discover new ion channel targets for human pain treatment

Pain is common, affecting 1 in 6 adults. Pain is also often complex; the sudden pain of a cut, burn or fractured bone (nociceptive pain), or the longer term aching pain associated with cancer or chronic injuries (neuropathic pain) or, more commonly, a mixture of both types. It is currently impossible to tell how much pain a person is suffering or to compare this pain accurately between people. Furthermore it can be difficult to determine the origin of pain, and we know that once pain has started it can become self-perpetuating probably due to secondary changes in the spinal cord or brain. We also all have experienced the depression and debilitation pain can cause if it is either severe or long lasting. Finally, pain treatment if often successful for mild pain and short lived nociceptive pain; however treatments for chronic pain are far less effective and often lead to side effects.

We, and others, have previously shown that some members of a class of genes called ion channels are essential to produce pain. Ion channel genes code for proteins that sit on the surface of neurons and either detect pain and turn this into a voltage signal, or respond to other proteins that detect pain and amplify their signal. We found that an ion channel gene called SCN9A can control all nociceptive pain felt (causing a condition called Congenital Insensitivity to Pain in people with no working SCN9A gene) and that another ion channel gene HCN2 controls neuropathic pain (this time in mice studies, where the mice with no HCN2 gene did not feel any neuropathic pain). In both people with no SCN9A and mice with no HCN2 the lack of pain sensing was the only feature. This is very important and suggests that if you could design a drug to block SCN9A and/or HCN2 you would have a new type of analgesic without side effects.

We now want to look for further ion channels that are part of the mechanism that make people feel pain. We are taking a novel approach by using three groups of people who feel extremes of pain: one group are people with severe neuropathic pain who get referred to a specialized pain clinic in Cambridge after other approaches have failed. These people have multiple reasons why they first experienced pain but are distinguished by their persistent pain becoming more severe and stopping them working and functioning normally. This group is currently extremely difficult to treat and is a significant problem for our society and the NHS. Our second group consists of women who don't need pain relief during their first delivery - constituting only 1% of women in the Rosie Maternity Hospital in Cambridge. We don't know whether these women are stoical, just determined not to have any drugs, have a quick labour, or have a very high pain threshold (probably a mix), but most importantly some of these women have other family members who similarly didn't need analgesia during their first labour strongly suggesting that this may be a genetic trait. Our third group if of very rare families with extremes of pain sensing - either feeling no pain or very early onset of severe debilitating pain. It is from researching this group that we found SCN9A.

We will select the 300 people from these three cohorts with the most severe pain findings and sequence all of their ion channel genes. What we are looking for is changes in ion channel genes that will change the way they work. If we find an ion channel gene change in a patient, say with severe neuropathic pain, and then find that in the laboratory that the mutation alters the way the ion channel works, then that ion channel could be important for pain control in humans with neuropathic pain. We will need to perform further studies on each gene we identify to provide further proof of its role in pain. Each ion channel we identify has the potential to generate new analgesics leading to better pain control for our patients.

Objective one.
Ascertaining and phenotyping three cohorts of people with extreme pain phenotypes is detailed in the main application but will be performed by the investigators and with research ethics approval. All consented study participants will have a blood sample stored and QST performed.

Objective two.
We will perform next generation sequencing of all ion channel gene exons in 300 individuals with the most severe pain phenotypes. This will be done in the MRC sponsored East of England Sequencing and Bioinformatics Hub (EASIH). Currently exon capture is by Agilent SureSelect-All Exon 50mb, sequencing by Illumina machines, and alignment to the NCBI36 freeze of the human genome and potential pathogenic DNA change prediction by custom designed and Broad Institute software. This method currently results in all but 11 exons being captured - those will be sequenced at the Sanger Inst. Next generation methodology is rapidly evolving and we will reassess our approach in early 2012.

Objective three.
Each potentially pathogenic mutation will be confirmed to be present in genomic DNA (and where necessary sought in our control panels). For each we will make a wild type construct of the gene and one containing the mutation. Gene constructs will be tranfected into cells, usually HEK293, and subjected to patch clamp electrophysiological analysis. If required we will co-transfect essential accessory genes. We will confirm that the wild type performs as expected and then seek differences for the mutation. If no response is seen for the mutation we will assess whether the protein formation, trafficking and intra-cellular localization are perturbed.

Objective four.
Surety that we have identified new human pain genes will usually require further studies including, but not restricted to, discovering further mutations in each gene. Expression studies will be carried out to determine where in the pain pathway the gene/protein acts and in which cell types.

The beneficiaries of this research, should it prove successful, will be all people who suffer pain, both acute nociceptive pain and chronic neuropathic pain. This will arise from our identification of ion channel genes involved in pain sensation detected in our three extreme pain cohorts. Proof that mutations in these genes alters ion channel function, allied with further studies to determine mechanism, will identify these ion channel as new targets for analgesic production. A further benefit to patients, and their clinicians, would be the molecular dissection of who is at risk of particular types of pain and who will respond best to which analgesics (leading to faster pain relief without having to try out a number of analgesics, and the avoidance of side effects from unnecessary analgesics).

Doctors and health planners would benefit if our project were successful, by being able to target pain relief resources effecitively and efficiently. We expect that molecular profiling for pain will emerge from our work and that of others, analogous to antibiotic sensitivities allowing the correct choice of antibiotics for infection; this too will have beneficial health planning and delivery implications.

If successful the project will demonstrate that Britain is ideally placed to conduct world-class medical pharmaceutical research. The key points will be the large and diverse population size, high quality medical care and disease phenotyping, excellent resources within the NHS to facilitate translational research, world-leading biomedical research and an ability for clinicians, scientists and industry to work sucsessfully together.

The partnership with Neusentis/Pfzer that this MICA enables will contribute to: the Nation's health -through better, more effective and hence cheaper pain control, and the Nation's wealth - through the pharmaceutical industry work of Neusentis making and marketing new analgesics, and producing Intellectual Property Rights.

In three years of the proposed project, the beneficiaries would be: the MRC - for funding translational industrial collaborative research; Neusentis - by being able to tap into the clinical resources of the NHS and molecular genetic resources of the University of Cambridge; Addenbrookes Biomedical Campus - by continuing to perform translational research; the scietists employed throughout the proposal who will gain new and improved skills, experience and publications, and the clinicians and academic applicants of this project. Lastly we also know that out patients are excited by our research as they have already been volunteering in large numbers to enrol anticipating that pain may be more treatable in the foreseeable future.