Using human IPSC derived nociceptors as a cellular model to investigate and therapeutically target Nav1.7

Chronic pain is a major health problem affecting 1 in 5 of the general population and unfortunately current treatments are inadequate. This is because they only work in a minority of patients and they are also associated with side effects. Moreover, a significant number of clinical trials testing new drugs to treat pain have failed, despite these drugs showing encouraging results in animal models. This may partly be due to differences in the structure and function of some of these drug targets between human and rodent species. We therefore need to understand more about the fundamental biology of pain processing in humans and find intermediate steps to test new drugs in a human cellular model. We will use human induced pluripotent stem cells (iPSCs) which are cells that have been taken from healthy volunteers (and in some cases patients) and reprogrammed so that they can be grown in the laboratory We can then use these cells to generate human nociceptors. Nociceptors are the sensory neurons which sense tissue injury giving rise to the sensation of pain. We will use these cells to investigate the role of and test drugs targeting the ion channel Nav1.7, which is an important target for developing new drugs to treat pain. Ion channels are important for regulating the excitability of neurons and this channel in particular is a key drug target because data from rodents suggest that it is needed for the generation of electrical signals in nociceptors. Nav1.7 shows selective expression within sensory neurons (so targeting this ion channel should avoid side effects in other regions of the body such as the heart and brain). Finally, patients with mutations in the gene encoding this ion channel are born unable to perceive pain. We will use gene technology so that we can produce a 'tagged' version of human Nav1.7 in order to determine how this protein is trafficked to the different specialised regions of human nociceptors. We will also determine which other proteins can bind to hNav1.7 and whether these proteins may modulate its function or alter its trafficking. We will determine whether the new drugs being developed to target hNav1.7 can alter the excitability of human nociceptors and whether this is mediated through effects on hNav1.7. Ultimately, we hope to learn more about the fundamental role of Nav1.7 and its interactors in regulating the excitability of human nociceptors and validate human iPSC derived nociceptors as a platform for drug discovery.

Chronic pain is a major health problem and an important area of research and development for the UK pharma industry. Current analgesic treatments are inadequate and a number of drug trials have failed even in agents demonstrating good efficacy in preclinical pain models. Human induced pluripotent stem cells differentiated into nociceptors could provide an intermediate step in analgesic drug discovery by presenting human ion channels in their normal cellular context and doing so in scalable fashion. In collaboration with Astra Zeneca, we will use human iPSCs to study the expression, trafficking, function and pharmacology of the voltage gated sodium channel Nav1.7 in human nociceptors. This is an important analgesic drug target because: (1) loss of function mutations in Nav1.7 lead to congenital insensitivity to pain, (2) Nav1.7 is thought to have a key role in the electrogenesis of action potentials in nociceptors (but may also modulate synaptic transmission and endogenous opioid production) and (3) shows relatively selective expression in peripheral neurons. We will use genome engineering to tag endogenous hNav1.7 in human iPSC lines in order to study its expression in human nociceptors as well as its trafficking to specialised cellular compartments such as terminals, the axon, node of Ranvier and soma membrane. We will investigate the regulation of such trafficking and also define the interactome of hNav1.7. We will study the effects of known and newly developed peptide and small molecule selective blockers of hNav1.7 (and its interactors) on the excitability of iPSC derived nociceptors. Availability of knockout lines will enable us to test selectivity and in addition, we will compare findings in human iPSC derived nociceptors to rodent nociceptors as a form of cross validation. Our aims are to provide insight into the fundamental biology of Nav1.7 and to establish whether iPSC derived nociceptors provide a useful platform for analgesic drug development.

The pharmaceutical industry and neuroscience
The pharmaceutical industry has a major interest both in improving human cellular models of neurological disorders and in finding new treatments for pain. Chronic pain is extremely common and becoming more so with an aging population and the increased prevalence of diabetes. It is therefore a very large drug market. The current opioid crisis highlights the inadequacies of current treatment options. Not only Astra Zeneca (the partners in this grant) but also Biogen, Lilly, Pfizer and GSK have ongoing programmes relating to chronic pain. Many smaller biotechnology companies and SMEs also have interests in pain and neurobiology and are exploring the use of new cellular platforms. Preclinical data and human genetics provide evidence that Nav1.7 is a key target for novel therapeutics. Successful targeting of Nav1.7 would also provide important proof of principle regarding selective blockers of voltage gated sodium channels, which could extend to other disorders of hyper-excitability such as epilepsy. These findings may ultimately facilitate clinical trial development/design as it will enable comparison of efficacy of drugs targeting Nav1.7 on human neuronal function in vitro, with emerging data on pharmacokinetics in vivo. The training and interaction between Astra Zeneca and Oxford will facilitate bilateral interaction in terms of both the drug development process in the pain field and using new technologies such as iPSC-derived neurons.

Using iPSC-derived neuronal models in academia and industry
The development of human iPSC models in this application will facilitate other groups also developing iPSC models for the purpose of aiding drug discovery and providing insight into human neurological disease. Neurons are post-mitotic and access to live human neural tissue is scarce, thus iPSC derived models are a significant advance. iPSCs also are amenable to gene editing, which enables modulation of expression and tagging of proteins of interest. Our comparison of this human iPSC derived neuronal platform versus using rodent cells in the context of Nav1.7 will be an important step in the validation of these models. In neuroscience in general, and pain specifically, there has been a legacy of failed clinical trials, even using agents that showed efficacy in preclinical models. Efficacy of a drug in a human iPSC derived neuronal model provides an important intermediate step in the drug development process.

Understanding the role of Nav1.7
Academics benefitting from this research will include pain scientists, stem cell biologists, pharmacologists and neurobiologists. Understanding how Nav1.7 regulates the excitability of nociceptors is of major interest to the pain field. We will advance knowledge regarding the regulation of Nav1.7 expression and trafficking. We will make data on Nav1.7 interactors publicly available in an accessible format in order to encourage its adoption by other researchers. In the context of nerve or tissue injury the function of this key ion channel is enhanced and so findings may have wider relevance for neural plasticity.

Societal impact of pain:
Patients and wider society may benefit from these outputs. Pain affects 1 in 5 Europeans with a major negative impact on quality of life and function at work. To take the USA as an example, the total costs associated with persistent pain in adults, is now estimated at $560-635 billion. These costs are reported to exceed those estimated for heart disease, cancer and diabetes. Unfortunately, in the majority of patients chronic pain remains inadequately treated due to both poor efficacy and tolerability of current analgesics. The current opioid crisis in which excessive prescribing often in the context of chronic pain has led to substance misuse and lasting harm illustrates these problems. Nav1.7 is an important analgesic drug target which will not have addictive potential which is a significant advantage.