Method for the treatment of endometriosis

Abstract

The present disclosure relates to a chemogenetic system and method treating endometriosis using a gene therapy vector to deliver a therapeutic payload to specific cells (nociceptive neurons). Overexpression of the payload increases the sensitivity of the cells to an activator, thereby enabling lower doses of activator drugs to be administered. The disclosure also related to vectors to deliver a nucleotide sequence to the target cells to enable overexpression thereof.

Description

[0001] METHOD

[0002] Field of the Invention

[0003] The present invention relates to a system and vector for, and methods of, treating endometriosis. The invention beneficially reduces side effects associated with neuromodulatory drugs, increases efficacy of neuromodulatory drugs in transduced neurons, and reduces excitability of transduced neurons innervating endometriotic lesions. The invention also enables endometriotic lesions to be druggable by certain neuromodulatory drugs that previously did not significantly affect neurons innervating them.

[0004] to the Invention

[0005] Pain is a fundamental sensory experience that serves as a protective mechanism, alerting us to harmful and noxious stimuli, and prompting action to avoid injury. The central neural circuits that mediate pain are highly complex and involve multiple regions of the central nervous system. However, the peripheral component of the pain circuit is relatively simple and well understood.

[0006] Pain is sensed through a specialised type of peripheral neuron called a nociceptor or nociceptive neuron. These neurons fire in response to tissue damage or inflammation and relay this information to the dorsal spinal cord (see figure 16). There are different types of nociceptive sensory neurons that innervate different tissue types and relay different types of signals. Although nociceptive neurons fire in response to underlying tissue damage or injury, there are pain syndromes that are associated with malfunctioning nociceptive neurons. In these conditions (for example peripheral sensitisation), nociceptive neurons can show aberrant growth and increased innervation of tissues, as well as molecular changes that lowers the activation threshold of these neurons - meaning that they signal pain inappropriately. Although there are many described mechanisms that underlie alterations in nociceptor biology, ultimately, they lead to overactive electrical activity in the neurons. These types of changes can be considered analogous to the hyperactivity of motor neurons.

[0007] Endometriosis is a chronic condition where endometrial tissue that lines the uterus grows outside the uterus resulting in many different symptoms including severe pelvic pain, severe period pain, pain during sex, urinary / bowel symptoms, heavy periods, and infertility. This is a highly prevalent disorder affecting around 10% of reproductive age women (around 190 million cases, globally). Endometriosis results in a significant deterioration in quality of life, is a substantial economic burden (costing the US and EU healthcare systems billions of dollars annually) and can lead to increased stress, anxiety, and depression.

[0008] Endometriotic lesions are densely innervated by pain sensing neurons / nociceptors (Figure 17). This innervation is the basis of endometriosis pain, which can be exacerbated over time with peripheral and central sensitisation. In a similar mechanism to BP / IC peripheral sensitisation can cause nociceptive afferent hyperactivity and pain. Recent work has indicated that sensory neuron innervation, immune response and lesion growth form a feedback loop maintaining lesion growth, with ablation of sensory neurons reducing lesion size and number (Fattori et aL, 2023).

[0009] Pharmaceutical therapies have been developed aimed at ameliorating the abnormal neural activity typically by modulating ion channels that carry excitatory and inhibitory currents. However, drug delivery to the nervous system is difficult and existing therapies often have undesirable side effects due to low specificity.

[0010] Thus, although neuromodulatory drugs developed for neurological disorders have shown some therapeutic benefit, this has been limited by off-target effects on non-dysfunctional neurons and other systems expressing the target (cardiovascular, respiratory, gastrointestinal etc).

[0011] Potassium (K+) channels are membrane proteins that allow rapid and selective flow of K+ions across the cell membrane, and thus generate electrical signals in cells. Voltage-gated K+channels (Kv channels), present in all excitable animal cells, open and close upon changes in the transmembrane potential. Kv channels are one of the key components in generation and propagation of electrical impulses in nervous system. Upon changes in transmembrane potential, these channels open and allow passive flow of ions from the cell to restore the resting membrane potential. Activation of Kv channels is therefore considered inhibitory to action potential generation.

[0012] There are 40 human voltage-gated potassium channel genes belonging to 12 subfamilies (Kvl-Kvl2). The subfamilies are typically restricted to specific locations or cell / tissue types and are observed in excitable and non-excitable tissues. Remarkable diversity of Kv channels may be achieved due to the mix and match of Kv channel subunits. Within each of the Kvl, Kv2, Kv3, Kv4 and Kv7 families, homomeric and heteromeric channels may form with a range of functional properties. Kv2 family members may also assemble with Kv5, Kv6, Kv8 or Kv9 family members with more restricted expression patterns in the nervous system and smooth muscles.

[0013] Voltage-gated potassium channels, in neurons, are targeted to various subcellular compartments, and channels of different subunit compositions may be present in different subpopulations of neurons. The tetrameric structure of Kv channels is made of two functionally and structurally independent domains: an ion conduction pore, and voltage-sensor domains. The ion conduction pore is made of four subunits which are arranged symmetrically around the conduction pathway. Voltage-sensor domains are positioned at the periphery of the channel and consist of four transmembrane segments (S1-S4). Structural rearrangement of the voltage-sensor domains in response to changes in the membrane potential, and in particular S4, which includes positively charged amino acids at every third position, results in conformational changes in the conduction pore, which could open or occlude the ion conduction pathway. The mechanism of voltage-gating is not fully understood.

[0014] The Kv7 family of low threshold voltage-gated potassium (K+) channels consists of five members (Kv7.1-7.5) encoded for by the KCNQ1-5 genes, respectively. Kv7 channels assemble as tetramers of identical or compatible a subunits, with each a subunit consisting of six transmembrane segments (S1-S6) and cytoplasmic N- and C-termini. Kv7 is widely expressed in both the CNS and other tissues, with Kv7.2 and 7.3, in particular, being known to control excitability of neurons.

[0015] Kv7.2 and 7.3 channels normally form heteromers with each other to increase conductance and are rarely expressed as low conductance homomers. Kv7.2 and 7.3 are expressed in most neuronal subtypes and tend to be localised to the initial segment of the axon where action potentials (spikes) are initiated. Kv7.3 and Kv7.2 conductances are therefore considered particularly potent inhibitors of action potential generation and activity in the same neuron as well as its targets (for example, motor neuron inhibition reduces muscle activity).

[0016] Neuromodulatory drugs targeting Kv7 channels have been developed to treat disorders associated with neuronal hyperexcitability. Retigabine is an anticonvulsant that acts mainly as a Kv7.2-Kv7.5 potassium channel opener, and GABA positive allosteric modulator at higher concentrations. The term "channel opener" refers to a left shift in the voltage dependence for channel opening, towards more negative potentials. In the presence of Retigabine, Kv7 channels open at more negative potentials, thus contributing to membrane hyperpolarisation and voltage stabilisation.

[0017] It has also been shown that Retigabine stabilises the open Kv7.2 / 7.3 (heteromeric) channel, making deactivation slower with little change in voltage dependence of deactivation. The overall effect is greater potassium conductance, hyperpolarisation of the membrane potential, and therefore inhibition of action potential generation. This effect of Retigabine is observed at concentrations below 10 pM, in vitro (Corbin-Leftwich et al 2016; Villalba-Galea 2020). Orally administered Retigabine at 600-1200 mg per day, which corresponds to a mean plasma concentration of 0.83 pM (Gunthorpe et al., 2012), has been shown to have up to 45% clinical efficacy for seizure reduction in humans with epilepsy (Brodie et al., 2010; French et al., 2011). Of note, while the EC50 of Retigabine is 0.6 pM for Kv7.3 channels, most channels in the nervous system are Kv7.2 / 7.3 heteromers, for which the EC50 is 1.6 pM. For some patients, these doses resulted in eye and skin discolouration that led to restriction of the indication. Retigabine was later voluntarily withdrawn for commercial reasons (Brickel et al., 2020).

[0018] There is continued pharmaceutical interest in Kv7 as a therapeutic target. At least 3 activators with increased potency, increased selectivity for Kv7.2 / 7.3 and a lack of cosmetic side effects are in clinical trials and have shown favourable outcomes. However, due to the ubiquity of Kv7.2 / Kv7.3 expression in the nervous system, neuronal side effects (somnolence, fatigue etc) are currently unavoidable. Although new Kv7.2 / 7.3 activators have improved safety profiles, neural side effects can only be reduced by increasing selectivity for dysfunctional neurons only.

[0019] Chemogenetics is a method by which proteins are engineered to interact with previously unrecognised small molecule chemical actuators. Since the 1990s, a large number of chemogenetic platforms have been developed that have been particularly useful for neuroscientists wishing to modulate neural activity in specific populations. Several classes of proteins have been engineered in this way, including kinases, non-kinase enzymes, ligand-gated ion channels and G-protein-coupled receptors (GPCRs). The most widely used of these are known as Designer Receptors Activated by Designer Drugs (DREADDs). DREADDS are engineered GPCRs that respond to synthetic ligands that cross the blood brain barrier (such as clozapine-N-oxide and olanzapine), but not their natural ligand, acetylcholine.

[0020] In use, a viral vector inserts the gene that encodes the DREADD protein into the cell to be studied. Various viral serotypes, promoters, and administration routes can be used to help select the target cells. Once transduced, the infected cell takes two or three weeks to express the engineered receptor protein which can then be activated using a DREADD ligand. Thus, activity can be modulated specifically in the targeted neurons with minimal influence on off target cells. Described herein is a method similar to that employed by DREADDs, in which an ion channel responsive to specific drugs is expressed in neurons in order to modulate activity. Importantly, the method flips the existing concept of designing specifically paired, de novo receptor and drug and repurposes wild type and mutant ion channels to reduce or eliminate side effects of drugs acting on the channels. The method overexpresses wild type and / or mutated channels.

[0021] Furthermore, the method described beneficially uses channels that can be either opened (agonists) or closed (antagonists) depending on the drug used. In direct contrast, DREADD activation induces unidirectional excitability changes (either excitatory or inhibitory).

[0022] Herein, we propose that, by administering a Kv7.3 genetic payload to nociceptive neurons associated with endometriosis (including those innervating endometriotic lesions or tissues / organs underlying lesions), expression of Kv7.3-containing channels will be increased, permitting a lower dosage of a Kv7 channel opener to be administered, thereby reducing nociceptor hyperactivity and reducing pain. It is anticipated that reduction in hyperactivity of endometriotic nociceptors may also reduce lesion size and number.

[0023] Summary of the Invention

[0024] The present disclosure relates to a chemogenetic system and method that allows modulation of activity in targeted neurons, thus increasing drug efficacy while reducing side effects. At least in part, due to the ability to use a lower dose. In some cases, the increased drug efficacy opens up the possibility of using that drug to treat previously inaccessible conditions. The method involves using gene therapy methodology via a delivery vector (e.g. AAV) to overexpress (upregulate) a therapeutic ion channel in transduced targeted nociceptive neurons that innervate endometriotic lesions, so that the channel's conductance (and therefore neuronal activity) can be modulated by low doses of specific activating and inhibiting drugs than would have been required in untreated (non-transduced) neurons. That is, the method specifically increases the sensitivity of transduced neurons to neuromodulatory drugs.

[0025] We have shown that AAV-mediated overexpression (upregulation) of the voltage gated potassium channel Kv7.3 (encoded by KCNQ3) enables a Kv7 activator, such as retigabine, to significantly reduce excitability of human neurons at the individual neuron and population level. At low concentrations (0.3-3pM), retigabine reduced action potential number in individual hIPSC-derived sensory and motor neurons. At the same doses, retigabine has minimal effect on control neurons expressing green fluorescent protein only.

[0026] Using multielectrode arrays, we have shown that retigabine was also effective at selectively reducing spontaneous bursting activity from a network of thousands of motor neurons overexpressing Kv7.3, but not GFP-expressing neurons. The in vitro results translated to a mouse model of spasticity, in which hyperactive motor neurons cause excessive muscle contractions (muscle spasms). In this model, overexpression of Kv7.3 in motor neurons via an intramuscular injection of an AAV-Kv7 construct enabled a low dose of retigabine (2mg / kg) to reduce the intensity and duration of muscle spasms. However, the same dose did not significantly reduce spasticity in mice injected with an AAV-GFP construct. The reduction in excitability of motor neurons was confirmed as the mechanism for the reduction in spasticity as 3pM retigabine reduced AP firing in Kv7.3 expressing mouse motor neurons, but not those expressing GFP. Additionally, we have shown that introducing a point mutation at amino acid residue 315 (Zaika et al., 2008; Gomez-Posada et al., 2010) of the Kv7.3 protein reduces motor neuron excitability at baseline in vitro and in vivo and further increases the effect sizes of retigabine at the tested concentrations in vitro. With in vitro patch clamp recordings of sensory neurons we have observed simar effect sizes of retigabine in sensory neurons overexpressing Kv7.3 and application of retigabine as we see in motor neurons under the same condition.

[0027] Without wishing to be bound by theory, the present inventors have observed that the expression or overexpression of Kv7.3 in nociceptive neurons will improve the susceptibility of those neurons to neuromodulatory drugs as described herein and as observed in motor neurons.

[0028] The therapy will use AAV to express or overexpress Kv7.3 ion channel subunits and variations thereof, either as homomers or Kv7.3-containing heteromers, in hyperexcitable or sensitised nociceptive neurons that innervate endometriotic lesions of patients with endometriosis, and use (low doses of) channel modulators to normalise activity.

[0029] According to a first aspect there is provided a system for use in the treatment of endometriosis, the system comprising:

[0030] a gene therapy vector comprising:

[0031] a. a capsid that selectively transduces target nociceptive neurons, and b. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ. ID No. 9 to SEQ ID No. 15,

[0032] wherein, when the gene therapy vector transduces the target nociceptive neurons, expression of the Kv7.3 ion channel subunit is upregulated and functional Kv7.3-containing ion channels are formed; and a neuromodulatory drug that targets the Kv7.3-containing ion channels.

[0033] Advantageously, drugs that may not have previously been considered, or available, as a therapeutic in the treatment of endometriosis may become useful due to the expression or overexpression of Kv7.3 by nociceptive neurons innervating endometriotic lesions. For example, historically retigabine and its derivatives would not have been considered as a therapy for endometriosis. However, using the disclosed system and method unlocks the possibility of treating endometriosis and / or symptoms associated with endometriosis with neuromodulatory drugs.

[0034] In one embodiment, expression or overexpression of Kv7.3 by target nociceptive neurons innervating endometriotic lesions makes target transduced neurons susceptible to treatment by a neuromodulatory drug.

[0035] According to a second aspect there is provided a method of treating endometriosis comprising the steps:

[0036] a. administering to a subject in need thereof, a therapeutically effective amount of a gene therapy vector comprising:

[0037] i. a capsid that selectively transduces target nociceptive neurons, and ii. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No. 9 to SEQ ID No. 15,

[0038] such that the gene therapy vector transduces the target nociceptive neurons to produce transduced nociceptive neurons;

[0039] b. upregulating expression of the Kv7.3 ion channel subunit in the transduced nociceptive neurons, such that the transduced nociceptive neurons form functional Kv7.3-containing ion channels; and

[0040] c. administering to the subject a neuromodulatory drug known to target Kv7.3-containing ion channels, wherein the neuromodulatory drug is administered at a dose lower than the dose that would be required to elicit an equivalent response in nociceptive neurons that are not transduced by the gene therapy vector

[0041] Advantageously, by increasing the number of functional Kv7.3-containing ion channels expressed by the transduced neurons, the sensitivity of the transduced neuron to a Kv channel-targeting neuromodulatory drug is increased, effectively increasing the efficacy of the drug in those neurons. This means that a lower dose of the drug can be administered to obtain the same therapeutic effect or to achieve a therapeutic effect where that would not previously have occurred. This, beneficially, means that the occurrence of side effects can be reduced, as can off-target effects.

[0042] This methodology allows target nociceptive neurons (for example, in an endometriotic lesion) to be sensitised to a neuromodulatory drug whilst other (non-transduced) neurons are not sensitised.

[0043] In one embodiment, expression or overexpression of Kv7.3 by target nociceptive neurons makes target transduced neurons susceptible to treatment by a neuromodulatory drug.

[0044] In one embodiment the neuromodulatory drug is selected from Retigabine / Ezogabine and derivatives thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine.

[0045] Side effects of neuromodulatory drugs can range from minor to serious and can affect a subject's quality of life. For example, for Retigabine the NIH lists the most common adverse effects, occurring in greater than 10% of subjects in the clinical trials, include abnormal gait, confusion, dizziness, fatigue, headache, nausea, somnolence, speech disorder, tremor, urinary tract infection, and blurred vision (Harris and Murphy, 2011).

[0046] In some cases, side effects (adverse effects) can lead to drugs being removed from the market because the dosage needed leads to unacceptable adverse effects. If the dose can be lowered, adverse effects can be reduced and use of the drug can potentially be restored.

[0047] A third aspect provides a gene therapy vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No.9 to SEQ ID No.15 and a capsid that selectively transduces target nociceptive neurons wherein, when the gene therapy vector transduces the target nociceptive neuron, expression of the Kv7.3 ion channel subunit is upregulated and functional Kv7.3-containing ion channels are formed.

[0048] Advantageously, delivery of the nucleotide sequence to a population of specific target nociceptive neurons innervating endometriotic lesions (including tissues or organs underlying lesions) results in the nucleotide payload only being transduced into the neurons that exhibit increased excitability. These neurons then express or overexpress the ion channels, presenting more targets for the neuromodulatory drugs which, in turn, allows a lower dosage of the drug to be effective, resulting in fewer side effects. Targeting neurons in this way also means that off target activity is less likely.

[0049] In one embodiment the upregulation of the Kv7.3 ion channel subunit expression reduces excitability of the target nociceptive neuron in the presence of the neuromodulatory drug.

[0050] In one embodiment the upregulation of the Kv7.3 ion channel subunit expression by the target nociceptive neuron reduces activity of cells innervated by axon terminals of the target nociceptive neuron in the presence of the neuromodulatory drug.

[0051] In a further aspect there is provided a gene therapy vector comprising an AAV capsid that selectively transduces target nociceptive neurons, and a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No.9 to SEQ ID No.15, for use in the treatment of endometriosis.

[0052] In one embodiment the Kv7.3-containing ion channel assembles as a Kv7.3 homomer, a Kv7.2 / Kv7.3 heteromer, or a Kv7.3 / Kv7.5 heteromer.

[0053] As discussed above, in some cases the upregulation of Kv7.3 may make endometriosis treatable with new therapeutics, such as neuromodulatory drugs like retigabine and its derivatives, thereby opening up entirely new therapeutic options.

[0054] In another aspect there is provided the use of a gene therapy vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No.9 to SEQ ID No.15 in the preparation of a medicament for use in the treatment of endometriosis or symptoms associated with endometriosis.

[0055] In a further aspect there is provided a therapeutic system comprising a gene therapy vector comprising a capsid that selectively transduces target nociceptive neurons, and a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No.9 to SEQ ID No.15, and a neuromodulatory drug that targets Kv7.3-containing ion channels.

[0056] In one embodiment there is provided use of the gene therapy vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No. 9 to SEQ ID No.15 in the treatment of endometriosis or symptoms associated with endometriosis.

[0057] Advantageously, delivery of the Kv7.3 nucleotide sequence to a population of specific target nociceptive neurons results in the nucleotide payload only being transduced into those neurons which may, for example, be neurons that exhibit increased excitability. These neurons then overexpress the Kv7.3 ion channels, presenting more targets for the neuromodulatory drugs which, in turn, allows a lower dosage of the drug to be effective, resulting in fewer side effects. Targeting neurons in this way also means that off target activity is less likely.

[0058] In a further aspect there is provided the use of a neuromodulatory drug selected from the group consisting of: retigabine or a derivative thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine in the treatment of endometriosis or symptoms associated with endometriosis.

[0059] Brief Description of the Drawings

[0060] For a better understanding of the invention and to show how the same may be carried into effect, there will now be described by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:

[0061] Figure 1 shows the AAV constructs used to transduce neurons with either Kv7.3 (upper construct) or Kv7.3 A315T (lower construct). Note that a GFP sequence was included downstream of Kv7.3 and Kv7.3 A315T via T2A linkers, meaning GFP fluorescence is indicative of Kv7.3 expression.

[0062] Figure 2 shows that AAV transduction of hIPSC neurons leads to expression of proteins encoded by transgenes packaged into the AAV constructs (A). Specifically, in the presented example, hIPSC neurons were transduced with AAVs to induce expression of either green fluorescent protein (AAV-GFP) or human Kv7.3 channels and GFP (AAV-Kv7.3).

[0063] Figure 2 also shows the endpoints assessed using patch clamp electrophysiology at baseline (expression of Kv7.3 or GFP without activator drug (B), and following exposure to activator (retigabine, C). The endpoints were: rheobase (minimum current input to evoke 1 action potential), number of action potentials at 3x rheobase, and resting membrane potential. In the presence of retigabine, the excitability of the neurons is reduced as shown by an increase in the rheobase, a decrease in the number of action potentials at 3x rheobase and a hyperpolarisation of the resting membrane potential. Figure 3 shows that baseline overexpression of Kv7.3 does not significantly alter the excitability of hIPSC sensory neurons compared to control neurons transduced with AAV -GFP. Panel (A) shows the mean number of action potentials generated at each current input value for AAV-GFP (dashed line) and AAV-Kv7.3 (solid line) transduced neurons. Shaded areas show 95% confidence intervals. Excitability levels were determined to be similar due to the extremely small effect sizes measured for APs / 500ms (B), RMP (C) and Rheobase (D). In these Cummings estimation plots, individual points represent number of action potentials generated at 3x rheobase for neurons transduced with AAV-GFP (light grey) or AAV-Kv7.3 (dark grey). On the right hand side of the plot, Hedges G effect sizes and 95 % confidence intervals are plotted for bootstrap resampled (1000 repeats) data.

[0064] Figure 4 shows that retigabine delivered at concentrations of 0.3 pM, 1 pM, and 3 pM had limited effect on excitability of hIPSC sensory neurons transduced with AAV-GFP as determined by measuring the number of action potentials at 3x rheobase. However, large and very large effect sizes were measured at the same concentrations for neurons transduced with AAV-Kv7.3. In each of the estimation plots (A, B and C), the upper graphs are line plots representing the change in AP number between recordings made before and after exposure to retigabine. Lower plots show the Hedges G effect sizes and confidence intervals.

[0065] Figure 5 shows that retigabine delivered at concentrations of 0.3 pM, 1 pM, and 3 pM had limited effects on excitability of hIPSC sensory neurons transduced with the AAV-GFP as determined by measuring the rheobase (minimum current to generate 1 action potential). However, large effect sizes were measured at the same concentrations for neurons transduced with AAV-Kv7.3. Figure 6 shows that retigabine delivered at concentrations of 0.3 pM, 1 pM, and 3 pM had small and medium effects on excitability of hIPSC sensory neurons transduced with the AAV-GFP as determined by measuring the resting membrane potential (RMP). However, large and very large effect sizes were measured at the same concentrations for neurons transduced with AAV-Kv7.3.

[0066] Figure 7 shows that baseline expression of KV7.3A315T (dashed line) significantly reduces the excitability of hIPSC sensory neurons. Excitability levels were shown to be decreased due to the inability of AAV-KV7.3A315T transduced neurons to generate more than 3 action potentials (A, B). RMP was also hyperpolarised at baseline (C) but Rheobase was not substantially changed (D).

[0067] Figure 8 shows that retigabine delivered at concentrations of 0.3 pM, 1 pM, and 3 pM caused very large increases in the rheobase of AAV- KV7.3A315T transduced neurons. Effect sizes were at least 2-3 times larger for AAV-KV7.3A315T as compared with AAV-GFP. Note the differences in scale for both rheobase (primary axis), and Paired Hedges g effect size (secondary axis)

[0068] Figure 9 shows that retigabine delivered at concentrations of 0.3 pM, 1 pM, and 3 pM caused very large effects on hyperpolarisations of the RMP in hIPSC sensory neurons transduced with AAV- KV7.3A315T. Effect sizes were also at least 2-3 times larger than hIPSC neurons transduced with AAV-GFP.

[0069] Figure 10 shows Kv7.3 overexpression in hIPSC motor neurons by western blot (A) and ELISA (B). Note the strong bands at around 80 kDa.

[0070] Figure 11 shows that overexpression of Kv7.3 in hIPSC derived motor neurons induces a small baseline reduction in the frequency-current relationship (A-B), however action potential firing at 3 x rheobase (C) and rheobase (D) were unaffected. (E-H) Kv7.3 overexpression increased the sensitivity of hIPSC MNs to retigabine. Concentrations under 3μM had no effect on the excitability of AAV-GFP transduced neurons but reduced AP firing by up to 80% in AAV-Kv7.3 transduced neurons. Retigabine efficacy was also increased by Kv7.3 overexpression as demonstrated by the increase in the maximal effects on AP frequency (G), and Fl slope (H).

[0071] Figure 12 shows that Kv7.3 overexpression increases the sensitivity of MNs to RTG at the population level, as measured using a multielectrode array (MEA) plate. hIPSC MNs are grown in a well plate with electrodes etched into the base so that spontaneous activity can be recorded. 50% of the wells are treated with AAV-GFP and 50% with AAV-Kv7.3. After sufficient transduction time, spontaneous activity is recorded at baseline (0 pM) and then increasing concentrations of RTG. RTG does not affect the viability of motor neurons, assessed using electrode impedance (A). However, MNs treated with RTG show greater sensitivity to RTG as evidenced by the leftward shift in the dose response for retigabine and spontaneous action potential burst frequency (B), burst duration (C) or total number of spikes. E-F show representative examples of activity recorded in wells treated with AAV-GFP (E) or AAV-Kv7.3 (F) after adding 1.8 μM (RTG). Vertical lines in top panels show network burst frequency. Below are raster plots of action potential firing recorded by each of the 16 electrodes (rows). G-H show representative examples of individual bursts from MNs in wells treated with AAV-GFP (G) or AAV-Kv7.3 (H). Each row is spiking activity recorded by a single electrode. The right of each plot is a heat legend indicating the change in firing frequency. Figure 13 shows that in a neonatal mouse model of spasticity, induced by spinal cord injury, Kv7.3 can be successfully over expressed in motor neurons following intramuscular injection of AAV-Kv7.3. (A) shows a Kv7.3 western blot of spinal cords taken 2 weeks after injection and spinal cord injury. Note the strong bands at the expected molecular weight for Kv7.3 in the spinal cord samples from AAV-Kv7.3 treated mice but not the AAV-GFP treated mice. (B) shows Kv7.3 expression specifically in MNs using immunohistochemistry. (C-D) show changes in the duration of muscle spasms after administration of 2mg / kg RTG in AAV-GFP treated and AAV-Kv7.3 treated mice. E-F shows the change spasm intensity (number of action potentials per spasm) after administration of 2mg / kg RTG in AAV-GFP treated and AAV-Kv7.3 treated mice. G-H are representative electromyography (EMG) traces showing the effect of 2mg / kg RTG in AAV-GFP treated and AAV-Kv7.3 treated mice. Top traces are pre RTG and lower traces are 20 minutes after RTG.

[0072] Figure 14 shows that Kv7.3 overexpression in motor neurons via an intramuscular injection of AAV-Kv7.3 does not significantly affect the baseline electrophysiological properties of motor neurons in a neonatal mouse model of spasticity. Recordings were made from spinal cord slices taken from mice with spasticity. (A) shows that the excitability of MNs was not reduced as the number of action potentials evoked at 2 x rheobase not different between AAV-GFP controls and AAV-SRx-C490 transduced MNs. (B) shows that the threshold for evoking a single action potential was not different in mice AAV-SRx-C490 transduced MNs. (C) shows that the resting membrane potential (RMP) was not different between the groups. (D) shows that MN size was not different between the groups. (E-H) shows that 3 pM retigabine significantly reduced excitability in MNs from AAV-SRxC490 treated mice but not AAV-GFP treated mice, as assessed by the change in number of action potentials at 2 x threshold and the rheobase.

[0073] Figure 15 shows that motor neurons in mice with spasticity treated with AAV-GFP fire repetitive trains of action potentials (A), however motor neurons from mice injected with a viral construct to overexpress a mutated form of Kv7 (A315T) are incapable of firing repetitively. This suggests a significant and permanent reduction in excitability of these motor neurons.

[0074] Figure 16 shows a schematic of basic nociceptor biology.

[0075] Figure 17 shows a schematic of endometriosis showing endometriotic lesions (1).

[0076] Figure 18 shows an MEA round and shows the effects of an alternative compound (compound 2) on bursting at baseline (OuM), O.luM, 0.2uM. 3 wells were transduced with AAV-GFP and 1 well with AAV Kv7.3 (WT).

[0077] Detailed Description

[0078] A system for use in the treatment of endometriosis as employed here refers to a chemogenetic therapeutic system which is a 2-step therapy involving a (gene therapy) vector to deliver Kv7.3-encoding nucleic acid sequence to target nociceptive neurons and a neuromodulatory drug which acts as an activator of Kv7.3-containing ion channels. The system may be provided as a kit of parts (therapeutic system) comprising the vector and the neuromodulatory drug. The system is suitable for use in the treatment of endometriosis. Treating or treatment as employed herein refers to the reversal of a condition, amelioration or relief of symptoms associated with a condition or prevention of further development / worsening of a condition. The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the vector may be administered a day or more before the neuromodulatory drug is administered to the subject.

[0079] As employed herein a gene therapy vector (vector) refers to any suitable gene delivery vector such as those known in the art. Although AAV capsids are described herein, it will be appreciated that other vectors may be suitable and used without deviating from the scope of the present invention. Including, but not limited to, non-AAV viral vectors (including adeno-associated virus, adenovirus and lentivirus). Furthermore, non-viral methods (such as naked DNA or chemically assisted delivery methods) are envisioned.

[0080] Typically, the gene therapy will be used to treat endometriosis. One of the major symptoms of endometriosis is pain, caused by the endometriotic lesions that are densely innervated with nociceptive neurons. Many forms of pain are a reaction to noxious stimuli such as tissue damage, which is sensed by nociceptive neurons which send electrical impulses to the CNS, conveying the painful stimulus. In some circumstances these nociceptive neurons become hypersensitised or hyperexcitable, and convey pain signals to the CNS with no, or minimal, underlying tissue damage. Therefore, by reducing the excitability of these neurons, pain may be reduced as these neurons are no longer conveying a pain signal to the CNS. In endometriosis as described above, Fattori et al (2023) have suggested that the feedback cycle between sensory neuron innervation, immune response and lesion growth helps maintain lesion growth. They showed that ablation of sensory neurons reduces lesion size and number. It is the present inventors' belief that reducing pain using the gene therapy described herein may also be beneficial in reducing lesion size and / or number.

[0081] Where the vector is a viral vector, it comprises a capsid and a payload. In the present gene therapy vectors, the payload is a therapeutic payload encoding Kv7.3.

[0082] A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The proteins making up the capsid are called capsid proteins or capsid peptides.

[0083] In one embodiment the gene therapy vector is an AAV vector. That is, the capsid as an AAV capsid. AAV capsid (capsid) as employed herein refers to the capsid of adeno-associated viruses. Herein, AAV capsids are comprised of AAV capsid peptides (capsid peptides) assembled into a functional capsid. It is known that the AAV capsid is capable of remarkable selectivity due to its make up (the AAV capsid is composed of a mixture of VP1, VP2, and VP3 totalling 60 monomers arranged in icosahedral symmetry in a ratio of 1:1:10) and post-translational modifications. AAV capsid proteins contain 12 hypervariable surface regions, but the genome, in general, presents highly conserved replication and structural genes across serotypes. Serotype as employed herein refers to standard nomenclature, AAV1, AAV2 etc, of the wild-type capsid prior to mutation (by insertion) as disclosed herein. In the wild, multiple serotypes of AAV have been identified each with unique sequences of capsid gene, and hence distinct tropisms, although wild serotypes tend to be able to infect multiple tissue and cell types. These serotypes are denoted by numbers: AAV1, AAV2, etc. It has been shown that modification of capsid sequences via DNA recombination methods can generate non-native sequences with tailored properties and tropism directed towards (or against) specific cells or tissues, and that evade the immune system (Vandenberghe et al., 2009).

[0084] In one embodiment the AAV capsid is an AAV1, AAV2, or AAV6 serotype capsid.

[0085] As employed herein the target neurons are sensory (nociceptive) neurons. Specifically, those innervating endometriotic lesions.

[0086] Selectively transduces as employed herein refers to the ability of the AAV capsid to selectively transduce one cell type in preference to another. The specificity may be neurons in preference to muscle cells, for example. Alternatively, it may be motor neurons in preference to sensory neurons, for example. In some cases, selective transduction may refer to a specific part of a neuron, e.g. the axon.

[0087] As employed herein increased tropism is synonymous with selective transduction and refers to the property shared by the disclosed targeted gene therapy viral vectors whereby the capsid can infect / transduce target nociceptive neurons more than their equivalent WT capsid. Viral tropism is the ability of different viruses to infect different cellular types, ultimately to produce a successful infection. Improved specificity / selectivity of transduction may refer to one or more of the following:

[0088] Increasing, improving or introducing tropism to target nociceptive neurons, and Detargeting non-target-neuron cell types

[0089] The effectiveness of a capsid to transduce / infect specific neurons can be determined by counting the number of neurons that express the viral DNA, such as GFP. For example, multiple target neurons innervate the same targets, e.g. endometriotic lesion, and the proportion of these neurons that have been transduced by the viral vector (particle) can be counted. The effectiveness of a viral particle to infect neurons or specific parts of neurons can also be determined by DNA sequencing or RT- PCR to look at the "copy number" of the viral DNA that is in the neural cell. This would give an estimate of how many times the same cell was infected with the viral particle. Other methodologies may be apparent to the skilled person. Alternative methods might employ NGS (next generation sequencing) or qPCR.

[0090] The viral capsids employed herein have specific tropism for sensory neurons and selectively transduce sensory neurons. Capsids employed herein display increased sensory neuron transduction relative to WT AAVs of the same serotype. Capsids employed herein display increased transduction of sensory neurons in vitro and in vivo. Capsids employed herein preferably display limited transduction of cell types that are not sensory neurons.

[0091] The capsid employed herein can selectively transduce sensory neurons rather than surrounding lesion (endometriotic) tissue and may selectively transduce specific parts of sensory neurons, such as the axon. In some embodiments the capsid may also be detargeted from infecting or transducing other (non-sensory neurons) cell types. Such properties are beneficial to avoid undesirable off-target effects during gene therapy or chemogenetics therapy.

[0092] Detargeted refers to the reduction or removal of the virus' ability to be transported to and / or to infect other cell types. In the present disclosure, it is undesirable for the capsids to travel to and infect cells other than target nociceptive neurons. For example, if endometriotic lesion tissue were transduced that would reduce the amount of vector available to transduce the neurons innervating that lesion and may lead to off-target effects. In general, detargeting is beneficial in reducing the body's immune response to the virus.

[0093] Overexpressing or overexpression as employed herein is synonymous with upregulation of a gene and intended to refer to an increase in the expression of a gene relative to a cell (neuron) that has not been transfected or transduced. To be effective, the neuron should be expressing higher levels of assembled (functional) Kv7.3-containing ion channels, either as Kv7.3 homomers or as heteromers, for example Kv7.2 / 7.3 or Kv7.3 / 7.5 heteromers.

[0094] Overexpression or upregulation occurs when expression levels are at least 25% higher relative to a cell (neuron) that has not been transfected or transduced.

[0095] Expressing may be used to refer to the expression of a gene which is not normally expressed in the neuron. The expression is still considered to be upregulation, although the baseline for comparison is zero, or near-zero expression.

[0096] As employed herein neuromodulatory drug refers to a voltage gated potassium channel-targeting drug. Known drugs include, but are not limited to, Retigabine and derivatives thereof (see Musella et al., 2022 for examples), BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, linopirdine. Such drugs can be considered to target Kv7.3-containing ion channels.

[0097] In one embodiment the neuromodulatory drug is a channel opener.

[0098] In one embodiment the neuromodulatory drug is a channel blocker.

[0099] In one embodiment the neuromodulatory drug is selected from: Retigabine or a derivative thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, linopirdine.

[0100] "Increasing the susceptibility of a target nociceptive neuron to a neuromodulatory drug" as employed herein means that the target nociceptive neuron is made to express more of a receptor (target) of a neuromodulatory drug, in this case Kv7.3-containing ion channels. Where the nociceptor may not have previously bound (enough of) the drug for the drug to have a significant effect on it, the increased Kv7.3 expression renders the nociceptor susceptible to the drug. The drugability of the nociceptor is thereby increased.

[0101] Innervates or innervating as employed herein means that the neuron is functionally communicating with the endometriotic lesion, either directly or indirectly. Endometriosis is a chronic condition where endometrial tissue that lines the uterus grows outside the uterus resulting in many different symptoms including severe pelvic pain, severe period pain, pain during sex, urinary / bowel symptoms, heavy periods, and infertility. This is a highly prevalent disorder affecting around 10% of reproductive age women (around 190 million cases, globally). Endometriosis results in a significant deterioration in quality of life, is a substantial economic burden (costing the US and EU healthcare systems billions of dollars annually) and can lead to increased stress, anxiety, and depression.

[0102] Endometriotic lesions as employed herein refers to the lesions characteristic of endometriosis. The lesions are densely innervated by pain sensing neurons / nociceptors (Figure 17). This innervation is the basis of endometriosis pain, which can be exacerbated over time with peripheral and central sensitisation. This sensitisation can cause nociceptive afferent hyperactivity and pain. Recent work has indicated that sensory neuron innervation, immune response and lesion growth form a feedback loop maintaining lesion growth, with ablation of sensory neurons reducing lesion size and number (Fattori et al., 2023). Endometriotic lesions also extends to the tissue and organs that underlie the lesions. These may also be innervated and lead to pain.

[0103] In one embodiment endometriotic lesion includes tissue and / or organs underlying an endometriotic lesion.

[0104] As employed herein introducing a nucleotide sequence refers to the transduction or transfection of a cell, such as a neuron, with one or more nucleotide sequences to either supplement existing genetic material with more of what is already present, or to provide new nucleotide sequences.

[0105] Kv7.3 ion channel subunit is a monomer of a voltage-gated potassium channel which assembled to form a tetrameric function ion channel. Kv7.3 can assemble as a homomer or as a heteromer with other Kv7 subunits, including Kv7.2 and Kv7.5, or other auxiliary subunits (e.g. KCNE).

[0106] Kv7.3 ion channels as employed herein is intended to refer to both Kv7.3 homomer and Kv7.3-containing heteromers.

[0107] As employed herein a nucleotide sequence encoding a Kv7.3 ion channel subunit refers to a nucleotide sequence (RNA or DNA including cDNA) encoding the Kv7.3 ion channel subunit.

[0108] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.1 or a variant of SEQ ID No.1 encoding the same amino acid sequence encoded by SEQ ID No.1.

[0109] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.2 or a variant of SEQ ID No.2 encoding the same amino acid sequence encoded by SEQ ID No.2.

[0110] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.3 or a variant of SEQ ID No.3 encoding the same amino acid sequence encoded by SEQ ID No.3.

[0111] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.4 or a variant of SEQ ID No.4 encoding the same amino acid sequence encoded by SEQ ID No.4. In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.5 or a variant of SEQ ID No.5 encoding the same amino acid sequence encoded by SEQ ID No.5.

[0112] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.6 or a variant of SEQ ID No.6 encoding the same amino acid sequence encoded by SEQ ID No.6.

[0113] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.7 or a variant of SEQ ID No.7 encoding the same amino acid sequence encoded by SEQ ID No.7.

[0114] In one embodiment the nucleotide sequence comprises the sequence of SEQ ID No.8 or a variant of SEQ ID No.8 encoding the same amino acid sequence encoded by SEQ ID No.8.

[0115] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.9.

[0116] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.10.

[0117] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.11.

[0118] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.12.

[0119] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.13.

[0120] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.14.

[0121] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.15.

[0122] In one embodiment the Kv7.3 ion channel subunit comprises or consists of the amino acid sequence according to SEQ ID No.16.

[0123] In one embodiment the nucleotide sequence encodes the Kv7.3 ion channel subunit according to any one of SEQ ID 9 to 16.

[0124] In certain embodiments the Kv7.3 ion channel subunit may comprise a mutation at amino acid residue 315 of SEQ ID No.9. For example, A315T, A315S, A315V, A315C, A315N, or A315Q.

[0125] In one embodiment the ion channel subunit is wild type Kv7.3.

[0126] In one embodiment the ion channel subunit is not wild type Kv7.3.

[0127] In one embodiment the Kv7.3 ion channel sequence is A315T. As indicated by SEQ ID No. 2 (nucleic acid and SEQ ID No.10 (amino acid). Functional ion channel as employed herein means that the ion channel subunit has assembled into a tetramer, either as a homomer or as a heteromer, and is located at the neuron membrane in a position suitable to act as a voltage-gated potassium channel.

[0128] As employed herein, the term neuron includes a neuron and a portion or portions thereof (e.g. the neuron cell body, an axon and / or a dendrite). The term neuron denotes nervous system cells that are electrically active and include a cell body (or soma) and up to two types of extensions or projections: dendrites, by which the majority of neuronal signals are conveyed to the cell body, and axons, by which the majority of neuronal signals are conveyed from the cell body to effector cells. Neurons can convey information from tissues and organs into the central nervous system (afferent or sensory neurons) and transmit signals from the central nervous systems to effector cells (efferent or motor neurons). Other neurons, designated interneurons, connect neurons within the central nervous system (the brain and spinal cord). Yet more neurons, designated "projection" neurons, extend their axons from one region of the nervous system to another.

[0129] As employed herein the target nociceptive neuron is a neuron which innervates an endometrial lesion or the tissue and / or organs underlying the endometriotic lesion, including but not limited to, the fallopian tubes, uterus, ovaries, bladder or bowel.

[0130] As employed herein "administering a lower dose" refers to the subsequent exposure of a neuron that is overexpressing the introduced gene to a lower dose of a drug than would normally be administered to a patient in order to be therapeutically effective. For example, to treat epilepsy, Retigabine was typically administered at a dose of 600-1200 mg per day, which corresponds to a mean plasma concentration of 0.83 pM. Using the current method, we have shown that concentrations lower than 0.5 pM are effective in human neurons in vitro. Without wishing to be bound by theory, the present inventors consider that this would enable an effective dose lower than 0.5 pM to be administered in vivo.

[0131] In some cases, the prior dose required to affect the neurons herein would have been so high that the neuromodulatory drug was not considered to be a therapeutic for such targets.

[0132] In one embodiment the therapeutically effective dose of the neuromodulatory drug is reduced by up to 100%. For example, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 95%, 90% or 95%.

[0133] Whilst not wishing to be bound by theory, the inventors believe that overexpression of Kv7.3 may have a baseline effect that reduces excitability of neurons even in the absence of a neuromodulatory drug. Thus, in some embodiment administration of a neuromodulatory drug is not required. For example, Kv7.3 A315T is highly effective at membrane insertion and has higher conductivity than WT Kv7.3, therefore may be able to act at a therapeutic level without the requirement for administration of an additional neuromodulatory drug.

[0134] It is expected that the reduction in therapeutically effective dose is sufficient to reduce side effects of the neuromodulatory drug to clinically acceptable levels. Clinically acceptable levels will be understood by those skilled in the art.

[0135] As employed herein "increasing drug efficacy" refers to the increased therapeutic effect of a given drug dose in transduced neurons that are overexpressing the transgene relative to non-transduced neurons. As employed herein "reducing the side effects" refers to the lowering or elimination of undesirable side effects related to administration of a drug to a subject at a given dose.

[0136] For example, the neuromodulatory drug retigabine (ezogabine), which was marketed as an anticonvulsant, was found to cause drowsiness, dizziness, tinnitus and vertigo, confusion, and slurred speech at therapeutic doses (600-1200 mg). Less common side effects included tremor, memory loss, gait disturbances, and double vision. In 2013 the FDA warned the public that ezogabine can cause blue skin discoloration and eye abnormalities characterised by pigment changes in the retina. Whilst not wishing to be bound by theory, the inventors consider that increasing Kv7.3 expression in hyperexcitable neurons associated with epileptic seizures would be expected to shift the therapeutic range for retigabine to include lower doses, thereby reducing or eliminating above side effects. That is, the therapeutic index for retigabine would be increased. Indeed, trials including lower doses of retigabine (300-1200 mg) reported fewer dermatological, ophthalmological, and neurological adverse effects (Lerche et al., 2015; Brickel et al., 2020).

[0137] As employed herein "increasing the efficacy" means an increase in the ability to produce the desired result. This may be achieved by improving the response to a drug rather than improving the effectiveness of the drug. Improved efficacy means that the same level of response can be achieved using less of the drug.

[0138] "Increasing the susceptibility of a target (nociceptive) neuron to a neuromodulatory drug" as employed herein means that the target neuron is made to express more of a receptor (target) of a neuromodulatory drug, in this case Kv7.3-containing ion channels. Where the neuron may not have previously bound (enough of) the drug for the drug to have a significant effect on it, the increased Kv7.3 expression renders the neuron susceptible to the drug. The drugability of the neuron is thereby increased.

[0139] Therefore, increasing the efficacy means that a lower dosage can be used.

[0140] The excitability of neurons is determined by the frequency of action potentials generated in response to a given stimulus. Increased excitability is indicated by one or more of the measures described below. Increased excitability as employed herein is defined as a neuron displaying at least one of the following:

[0141] a. An increase (depolarisation) of a neuron's resting voltage (resting membrane potential-RMP) such that it is closer to the threshold for generation of an action potential.

[0142] b. A decrease in the stimulus magnitude required to cause neurons to generate action potentials (rheobase).

[0143] c. An increase in the number of action potentials generated by a given stimulus (frequency) compared to a neuron with "normal" levels of excitability.

[0144] d. Persistent generation of action potentials beyond the cessation of a stimulus responsible for initiating firing.

[0145] e. An increase in the frequency of action potentials in a population of neurons (2-100,000) recorded using an array of microelectrodes (microelectrode array-MEA) f. An increase in the number or duration of action potential bursts recorded by an MEA, defined as at least 5 consecutive action potentials with intervals of 100ms or less between each action potential.

[0146] For example, a hyperexcitable neuron may have a RMP of -55 mV which is roughly 15 mV more positive than is expected of neurons, and closer to the threshold for action potential generation (~ -40 mV). If a stimulus magnitude of 50 picoamperes (pA) causes a normal neuron to generate an action potential (rheobase = 50 pA), 25 pA may be sufficient in a hyperexcitable neuron (rheobase = 25 pA). Similarly, if a stimulus magnitude of 100 pA causes a normal neuron to generate 10 action potentials in 1 second (frequency= 10Hz), a hyperexcitable neuron may generate 20 at the same stimulus magnitude (frequency= 20Hz).

[0147] Additionally, action potential firing is often sustained beyond the cessation of the stimulus in hyperexcitable neurons but not normal neurons. This may be the case in endometriotic lesions, whereby the neurons fire more action potentials for longer time periods, and often in response to a lower stimulus.

[0148] Typically, the gene therapy vector is an AAV vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit. The ion channel subunit may be a wild type as encoded by SEQ ID No.1 or a mutant as encoded by SEQ ID No.2, or equivalent sequence thereof that encodes the amino acid sequences of SEQ ID No.9 or SEQ ID No.10, respectively.

[0149] AAV viral particles, as employed herein, are adeno-associated virus viral particles comprising a single stranded DNA genome packaged within a viral envelope that is capable of transducing a cell, e.g. a target nociceptive neuron innervating an endometriotic lesion. In particular, the AAV viral particles employed herein include the nucleotide sequence according to SEQ ID No.1 or SEQ ID No.2.

[0150] AAVs are small viruses belonging to the genus dependoparvovirus containing a single strand of DNA, up to ~4.9 Kb. The AAV genome contains three capsids proteins VP1, VP2 and VP3, all of which are translated from one mRNA via alternate splicing. In the wild, multiple serotypes of AAV have been identified each with unique sequences of capsid gene, and hence distinct tropisms, although wild serotypes tend to be able to infect multiple tissue and cell types. These serotypes are denoted by numbers: AAV1, AAV2, etc. It has been shown that modification of capsid sequences via DNA recombination methods can generate non-native sequences with tailored properties and tropism directed towards (or against) particular cells or tissues, and that evade the immune system (Vandenberghe et al., 2009). As employed herein, the AAV viral particles selectively transduce target nociceptive neurons and the specific nociceptive neurons to be transduced are those innervating one or more endometriotic lesions.

[0151] The AAV capsid employed herein can selectively transduce a particular cell type such as neurons rather than muscle tissue and may selectively transduce target nociceptive neurons over other neurons, even specific parts of neurons, such as the axon. Herein, selective transduction refers to the selective transduction of nociceptor neurons innervating one or more endometriotic lesions.

[0152] In use, the AAV capsid encapsulating the nucleotide sequence according to any one of SEQ ID Nos: 1 to 8 (the viral particle) is delivered to a subject, for example, by injection into an endometriotic lesion, the viral particle infects target nociceptive neurons which become transduced neurons. The transduced neurons overexpress the amino acid sequences encoded by the nucleotide sequences, which assemble as functional Kv7.3-containing ion channels (either homomers or heteromers). In general, the time from delivery of the viral particles to the subject to overexpression of the function ion channels is approximately 3 weeks or more. Once a suitable period of time has elapsed for the transduced neurons to over express the ion channels, the subject can be treated with a neuromodulatory drug, such a Retigabine, at a lower dose that would be required to evoke the equivalent reaction in a subject not receiving the Kv7.3.

[0153] A subject as employed herein means a human or non-human mammal. Typically, the subject has endometriosis and is experiencing pain as a result. In some cases, the therapy may be prophylactic and symptoms may not be experienced.

[0154] In some embodiments the subject has symptoms of endometriosis, characterised by pain that may be linked to neuronal hyperexcitability which may or may not be associated with dysfunction of Kv7.3 ion channels.

[0155] In one embodiment the pain experienced due to endometriosis is associated with increased excitability of nociceptive neurons.

[0156] Lower dose as employed herein refers to a dose lower than that considered to be efficacious in a subject (or in the lower portion of an efficacious range), and therefore does not cause, or reduces severity of, side effects whilst maintaining therapeutic efficacy.

[0157] Therapeutically effective amount as employed herein means an amount of the compound, which is effective in treating the named disorder or condition.

[0158] In some embodiments the overexpressed Kv7.3 channels may assemble as heteromers, such as Kv7.2 / 7.3 or Kv7.3 / 7.5 heteromers. The subunit proportions of these heteromers may differ and allow refinement of the method described herein.

[0159] Similarly, in some embodiments the proportion of wild type to mutant Kv7.3 subunits may differ to allow refinement of the method. This could be true of both heteromers and homomers.

[0160] In some embodiments the overexpressed Kv7.3 channels may assemble as homomers.

[0161] Homomeric Kv7.3 channels may be minimally conducting such that they have minimal effect on baseline excitability of the transduced neuron, but still allow the neuromodulatory drug to act on the transduced neuron at a lower dose.

[0162] Rheobase as employed herein refers to the minimum current injection value required to depolarise neuronal membrane potential sufficiently to generate a single action potential.

[0163] Resting membrane potential as employed herein refers to the potential voltage difference recorded between the intracellular and extracellular neuronal compartments in the absence of any current injection. This voltage value is recorded using electrophysiology techniques such as whole cell patch clamp. As employed herein "shifts the resting membrane potential (RMP) of the target nociceptive neuron away from threshold" means that the voltage value in the neurons resting state is more negative (hyperpolarised) and therefore a greater stimulus magnitude is required to depolarise the voltage to a level at which action potentials are generated. For example, a typical RMP of a neuron may be -65 mV, meaning 500 pA of current (stimulus) is needed to depolarise the neuron sufficiently to reach the threshold membrane potential for generating action potentials (-45 mV). If the RMP becomes more negative ( -75 mV), then it is further away from threshold and therefore requires more current injection to reach threshold and generate action potentials.

[0164] Reduces the excitability of the target nociceptive neuron as employed herein means at least one of the following:

[0165] a. Hyperpolarising of a neuron's resting membrane potential such that it is further away from the threshold for generation of an action potential.

[0166] b. Increasing the stimulus magnitude required to cause neurons to generate action potentials (Rheobase).

[0167] c. Decreasing the number of action potentials generated by a given electrical or chemical stimulus (AP Frequency).

[0168] d. Reducing or eliminating persistent generation of action potentials beyond the cessation of a stimulus responsible for initiating firing.

[0169] e. Reducing the frequency of spontaneous action potentials in the absence of a stimulus.

[0170] f. Reducing the duration or frequency of spontaneous action potential bursts (bursts are defined as at least 5 consecutive action potentials with intervals of 100 ms or less).

[0171] Reductions in excitability can be measured using whole cell patch clamp electrophysiology in current clamp mode by injecting rectangular current pulses of increasing magnitude and recording the change in voltage and frequency of action potential generation during the duration of the current pulse. Resting membrane potential is measured under stable conditions, while there is no current injection (e.g. the voltage does not vary by more than (1-2 mV). Rheobase is measured as the minimum current input of a designated duration necessary to generate at least one action potential. Action potential frequency is measured as the number of action potentials generated over a specified time period, usually equal to the duration of a current pulse injected into a neuron.

[0172] Reductions in excitability can also be measured using a microelectrode array (MEA) consisting of a well plate with several electrode contacts located at the surface of the base of each well. A population of neurons is grown in contact with the electrodes in each well such that their activity can be recorded as extracellular action potentials (spikes). Neurons grown on MEAs produce spontaneous action potentials often referred to as spikes. Five or more consecutive spikes recorded with intervals of 100 ms or less are referred to as bursts. Spontaneous spike frequency and bursting characteristics (burst frequency, duration etc) may be used to determine neuronal excitability.

[0173] In some embodiments the upregulation of the Kv7.3 ion channel subunit in the transduced neuron reduces activity of cells innervated by axon terminals of the same transduced neuron. The invention also comprises methods of treatment which involves injecting AAV viral vectors comprising the nucleotide sequence of SEQ. ID No.l or SEQ ID No.2 into the endometriotic lesion; these AAV viral vectors can then transduce the target nociceptive neurons.

[0174] Expression of the nucleotide sequence leads to overexpression of Kv7.3 ion channels in target transduced neurons. The subject can then be treated with a neuromodulatory drug at a lower dose than would previously have been necessary to obtain a therapeutic effect. The lower dose, in turn, causes fewer side effects and has reduced toxicity. Furthermore, previously unavailable drugs become useful in the treatment of a new target. The goal of curing, alleviating symptoms, and / or improving the quality of life of patients with endometriosis is achieved without undesirable (or with fewer, less severe) side effects.

[0175] Prohibitively high side effect drugs may become usable by employing the method disclosed herein, simply by permitting a lower dose to be efficacious in specific cells.

[0176] In some embodiments, the method involves the gene therapy vector or AAV viral particle retrogradely infecting neurons for the purposes of delivering genetic material to neurons, with the purpose of treating endometriosis.

[0177] In the case of A315T, a channel with high membrane insertion rate and conductance levels, it is likely that only low expression levels are needed to be effective. This means the virus can be delivered at lower quantities, reducing the probability of immunogenicity and reducing costs.

[0178] As employed herein, symptoms associated with endometriosis refers to pain cause by, or linked to, endometriotic lesions.

[0179] In the context of this specification "comprising" is to be interpreted as "including".

[0180] Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

[0181] Where technically appropriate, embodiments of the invention may be combined.

[0182] Embodiments are described herein as comprising certain features / elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features / elements.

[0183] Approximately as employed herein is intended to mean ±10%.

[0184] Technical references such as patents and applications are incorporated herein by reference.

[0185] Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

[0186] Examples

[0187] Materials and methods

[0188] Cell culture Human iPSC sensory neuron progenitor cells (Axol Bioscience) were thawed and plated at low density on a monolayer of rat cortical astrocytes on poly D lysine coated glass coverslips. Cultures were grown in NbActiv4 (Neurobasal / B 27 supplemented with MAXIMIZER, GDNF, NGF, BDNF, and NT 3) to promote the maturation of the cells to a differentiated neuronal phenotype.

[0189] AAVs

[0190] pAAV-CMV-EGFP, pAAV-CMV-hKCNQ3-T2A-EGFP, pAAV-CMV-hKCNQ3(A315T)-T2A-EGFP, were added to wells containing neuronal cultures at 7 days in vitro and patch clamp recordings were made either 1 (DIV15-16) or 2 weeks later (DIV21-23).

[0191] Patch clamp electrophysiology

[0192] Standard patch clamp methods were employed. The external recording solution composition was 140 mM NaCI, 25 mM KCI, 2mM CaCI2, 3 mM MgCI2, 10 mM glucose, 10 mM HEPES, pH 7.3. The internal recording solution composition was 120 mM K gluconate, 20 mM KCI, 3 mM MgCI, 2.5 mM EGTA, 0.5 mM CaCI, 2.4 mM Na2- ATP, 0.3 mM Li GTP, 10 mM HEPES, pH 7.3. Upon establishing the whole cell configuration, passive membrane properties for every cell (capacitance and resistance) were measured in voltage clamp mode. All excitability measurements were made in current clamp.

[0193] Multielectrode array recordings

[0194] Neurons were grown as above in 24 well plates containing 16 electrodes per well, such that neurons were in direct contact with individual electrodes. Spontaneous firing activity was then recorded after 30 days. Recordings were made at baseline (no drug) and then at increasing concentrations of the activator drug.

[0195] Mouse spasticity model

[0196] Neonatal mice (Post natal day 0) received a complete spinal cord transection between the 9th-10ththoracic segment to induce spasticity. In the same surgery, AAVs were injected into both gastrocnemius muscles.

[0197] EMG recording

[0198] 10-14 days after spinal cord injury, EMG electrodes were inserted into the injected muscles to record electrical activity before and after intraperitoneal administration of an activator drug (retigabine). Different doses were tested on different days.

[0199] Patch clamp electrophysiology from mouse spinal cord slices

[0200] The spinal cord was harvested under terminal anaesthesia and sliced at 300 pm on a vibratome. Patch clamp recordings were then made from motor neurons expressing GFP as described above (patch clamp electrophysiology). Statements

[0201] 1. A method of increasing the susceptibility of a target nociceptive neuron to a neuromodulatory drug that targets Kv7.3 channels, comprising the steps:

[0202] a. introducing a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 to the target nociceptive neuron via a vector, and

[0203] b. expressing or overexpressing the Kv7.3 ion channel subunit in the target nociceptive neuron such that it forms functional ion channels,

[0204] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0205] 2. A method of increasing the efficacy of a neuromodulatory drug administered for the treatment of endometriosis comprising the steps:

[0206] a. introducing a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 to a target nociceptive neuron via a vector,

[0207] b. expressing or overexpressing the Kv7.3 ion channel subunit in the target nociceptive neuron such that it forms functional ion channels, and

[0208] c. administering a lower dose of the neuromodulatory drug,

[0209] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0210] 3. A method of reducing the side effects of a neuromodulatory drug administered for the treatment of endometriosis comprising the steps:

[0211] a. introducing a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 to a target nociceptive neuron via a vector,

[0212] b. expressing or overexpressing the Kv7.3 ion channel subunit in the target nociceptive neuron such that it forms functional ion channels, and

[0213] c. administering a lower dose of the neuromodulatory drug,

[0214] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0215] 4. A method of increasing the therapeutic index of a neuromodulatory drug administered for the treatment of endometriosis comprising the steps:

[0216] a. introducing a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 to a target nociceptive neuron via a vector, b. expressing or overexpressing the Kv7.3 ion channel subunit in the target nociceptive neuron such that it forms functional ion channels, and

[0217] c. administering the neuromodulatory drug,

[0218] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0219] 5. A method of reducing the excitability of a target nociceptive neuron comprising the steps: a. introducing a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 to the target nociceptive neuron via a vector,

[0220] b. expressing or overexpressing the Kv7.3 ion channel subunit in the target nociceptive neuron such that it forms functional ion channels, and

[0221] c. optionally administering a neuromodulatory drug,

[0222] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0223] 6. A method of treating endometriosis or symptoms associated with endometriosis comprising the steps:

[0224] a. administering to a subject in need thereof, a therapeutically effective amount of the gene therapy vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16,

[0225] b. upregulating expression of the Kv7.3 ion channel subunit in a target nociceptive neuron such that it forms functional ion channels, and

[0226] c. administering a lower dose of a neuromodulatory drug known to target Kv7.3 ion channels, wherein the target nociceptive neuron innervates an endometriotic lesion.

[0227] 7. A gene therapy vector comprising a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16 wherein, when the vector transduces a target nociceptive neuron innervating an endometriotic lesion, expression of the Kv7.3 ion channel subunit is upregulated and functional ion channels are formed.

[0228] 8. A gene therapy vector according to statement 6, wherein the vector selectively transduces the target nociceptive neuron innervating an endometriotic lesion.

[0229] 9. An AAV viral particle gene therapy vector comprising:

[0230] a. an AAV capsid that selectively transduces a target nociceptive neuron, and

[0231] b. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16, wherein, when the vector transduces the target nociceptive neuron, expression of the Kv7.3 ion channel subunit is upregulated and functional ion channels are formed,

[0232] wherein the target nociceptive neuron innervates an endometriotic lesion.

[0233] 10. A gene therapy vector comprising an AAV capsid that selectively transduces a target nociceptive neuron, and a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16, for use in upregulating expression of the Kv7.3 ion channel subunit by the target nociceptive neuron which innervates an endometriotic lesion such that it forms functional ion channels in a subject with endometriosis.

[0234] 11. A gene therapy vector comprising an AAV capsid that selectively transduces a target nociceptive neuron, and a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the sequence of any of SEQ ID No.l to SEQ ID No.8 or a nucleotide sequence encoding the Kv7.3 ion channel subunit amino acid sequence of any of SEQ ID No.9 to SEQ ID No.16, for use in treating a subject with endometriosis, wherein, once the vector has transduced the target nociceptive neuron which innervates an endometriotic lesion, expression of the Kv7.3 ion channel by the target nociceptive neuron innervating an endometriotic lesion is upregulated, such that a neuromodulatory drug can be effectively administered to the subject.

[0235] 12. The method according to any one of statements 1 to 6 or the gene therapy vector according to statement 11, wherein the neuromodulatory drug is selected from Retigabine / Ezogabine and derivatives thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123 and linopirdine.

[0236] 13. The method or the gene therapy vector according to any preceding statement, wherein the Kv7.3 ion channel is expressed as a Kv7.3 homomer, a Kv7.2 / 7.3 heteromer, or a Kv7.3 / 7.5 heteromer.

[0237] 14. The method or the gene therapy vector according to any preceding statement, wherein the upregulation of the Kv7.3 ion channel subunit reduces the excitability of the target nociceptive neuron.

[0238] 15. The method or the gene therapy vector according to any preceding statement, wherein the upregulation of the Kv7.3 ion channel subunit by the target nociceptive neuron reduces activity of cells innervated by axon terminals of the target nociceptive neuron.

[0239] 16. The method of statement 16 or 17 wherein the reduction of the excitability of the target nociceptive neuron, or the reduction in activity of cells innervated by axon terminals of the target nociceptive neuron, occurs or is increased in the presence of the neuromodulatory drug.

[0240] 17. Use of the gene therapy vector according to statement 7 to 11 in the preparation of a medicament for use in the treatment of endometriosis or symptoms associated with endometriosis. 18. Use of the gene therapy vector according to statement 7 to 11 in the treatment of endometriosis or symptoms associated with endometriosis.

[0241] 19. Use of a neuromodulatory drug selected from the group consisting of: retigabine or a derivative thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine, in the treatment of endometriosis or symptoms associated with endometriosis. References

[0242] Gomez-Posada, J. C. et al. A Pore Residue of the KCNQ3 Potassium M-Channel Subunit Controls Surface Expression. Journal of Neuroscience 30, 9316-9323 (2010).

[0243] Zaika, O., Hernandez, C. C., Bal, M., Tolstykh, G. P. & Shapiro, M. S. Determinants within the Turret and Pore-Loop Domains of KCNQ3 K+ Channels Governing Functional Activity. Biophys J 95, 5121-5137 (2008).

[0244] Musella, S. et al. Beyond Retigabine: Design, Synthesis, and Pharmacological Characterization of a Potent and Chemically Stable Neuronal Kv7 Channel Activator with Anticonvulsant Activity. J Med Chem 65, 11340-11364 (2022).

[0245] Corbin-Leftwich, A. et al. Retigabine holds KV7 channels open and stabilizes the resting potential.

[0246] Journal of General Physiology 147, 229-241 (2016).

[0247] Gunthorpe, M., Large, C., Epilepsia, R. S.- & 2012, undefined. The mechanism of action of retigabine (ezogabine), a first-in-class K+ channel opener for the treatment of epilepsy. Wiley Online Library 53, 412-424 (2012).

[0248] French, J. A. et al. Randomized, double-blind, placebo-controlled trial of ezogabine (retigabine) in partial epilepsy. Neurology 76, 1555-1563 (2011).

[0249] Villalba-Galea, C. A. Modulation of KV7 Channel Deactivation by PI(4,5)P2. Front Pharmacol 11, (2020). Brickel, N. et al. Safety of retigabine in adults with partial-onset seizures after long-term exposure: focus on unexpected ophthalmological and dermatological events. Epilepsy & Behavior 102, 106580 (2020). Lerche, H. et al. Efficacy and safety of ezogabine / retigabine as adjunctive therapy to specified single antiepileptic medications in an open-label study of adults with partial-onset seizures. Seizure 30, 93-100 (2015).

[0250] Harris, J. A. and Murphy, J. A. Retigabine (ezogabine) as add-on therapy for partial-onset seizures: an update for clinicians. Ther Adv Chronic Dis. 2011 Nov; 2(6): 371-6.

[0251] Sequences

[0252] SEQ ID No. 1 - wild type Kv7.3

[0253] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGGCCACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0254] SEQ ID No. 2 - Kv7.3 A315T

[0255] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315T ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGACCACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0256] SEQ ID No. 3 - Kv7.3 A315S

[0257] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315S ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGTCCACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0258] SEQ ID No. 4 - Kv7.3 A315V

[0259] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315V ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGGTCACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0260] SEQ ID No. 5 - Kv7.3 A315C > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315C ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGTGCACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0261] SEQ ID No. 6 - Kv7.3 A315N

[0262] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315N ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGAACACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0263] SEQ ID No. 7 - Kv7.3 A315Q.

[0264] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315Q ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGCAGACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0265] SEQ ID No. 8 - Kv7.3 A315Y

[0266] > ENA| AAC96101 | AAC96101. 1 Homo sapiens (human) potassium channel A315Y ATGGGGCTCAAGGCGCGCAGGGCGGCGGGGGCGGCTGGCGGCGGCGGCGACGGGGGCGGC GGAGGCGGCGGGGCGGCTAACCCAGCCGGAGGGGACGCGGCGGCGGCCGGCGACGAGGAG CGGAAAGTGGGGCTGGCGCCCGGCGACGTGGAGCAAGTCACCTTGGCGCTCGGGGCCGGA GCCGACAAAGACGGGACCCTGCTGCTGGAGGGCGGCGGCCGCGACGAGGGGCAGCGGAGG ACCCCGCAGGGCATCGGGCTCCTGGCCAAGACCCCGCTGAGCCGCCCAGTCAAGAGAAAC AACGCCAAGTACCGGCGCATCCAAACTTTGATCTACGACGCCCTGGAGAGACCGCGGGGC TGGGCGCTGCTTTACCACGCGTTGGTGTTCCTGATTGTCCTGGGGTGCTTGATTCTGGCT GTCCTGACCACATTCAAGGAGTATGAGACTGTCTCGGGAGACTGGCTTCTGTTACTGGAG ACATTTGCTATTTTCATCTTTGGAGCCGAGTTTGCTTTGAGGATCTGGGCTGCTGGATGT TGCTGCCGATACAAAGGCTGGCGGGGCCGACTGAAGTTTGCCAGGAAGCCCCTGTGCATG TTGGACATCTTTGTGCTGATTGCCTCTGTGCCAGTGGTTGCTGTGGGAAACCAAGGCAAT GTTCTGGCCACCTCCCTGCGAAGCCTGCGCTTCCTGCAGATCCTGCGCATGCTGCGGATG GACCGGAGAGGTGGCACCTGGAAGCTTCTGGGCTCAGCCATCTGTGCCCACAGCAAAGAA CTCATCACGGCCTGGTACATCGGTTTCCTGACACTCATCCTTTCTTCATTTCTTGTCTAC CTGGTTGAGAAAGACGTCCCAGAGGTGGATGCACAAGGAGAGGAGATGAAAGAGGAGTTT GAGACCTATGCAGATGCCCTGTGGTGGGGCCTGATCACACTGTACACCATTGGCTATGGA GACAAGACACCCAAAACGTGGGAAGGCCGTCTGATTGCCGCCACCTTTTCCTTAATTGGC GTCTCCTTTTTTGCCCTTCCAGCGGGCATCCTGGGGTCCGGGCTGGCCCTCAAGGTGCAG GAGCAACACCGTCAGAAGCACTTTGAGAAAAGGAGGAAGCCAGCTGCTGAGCTCATTCAG GCTGCCTGGAGGTATTATGCTACCAACCCCAACAGGATTGACCTGGTGGCGACATGGAGA TTTTATGAATCAGTCGTCTCTTTTCCTTTCTTCAGGAAAGAACAGCTGGAGGCAGCATCC AGCCAAAAGCTGGGTCTCTTGGATCGGGTTCGCCTTTCTAATCCTCGTGGTAGCAATACT AAAGGAAAGCTATTTACCCCTCTGAATGTAGATGCCATAGAAGAAAGTCCTTCTAAAGAA CCAAAGCCTGTTGGCTTAAACAATAAAGAGCGTTTCCGCACGGCCTTCCGCATGAAAGCC TACGCTTTCTGGCAGAGTTCTGAAGATGCCGGGACAGGTGACCCCATGGCGGAAGACAGG GGCTATGGGAATGACTTCCCCATCGAAGACATGATCCCCACCCTGAAGGCCGCCATCCGA GCCGTCAGAATTCTACAATTCCGTCTCTATAAAAAAAAATTCAAGGAGACTTTGAGGCCT TACGATGTGAAGGATGTGATTGAGCAGTATTCTGCCGGGCATCTCGACATGCTTTCCAGG ATAAAGTACCTTCAGACGAGAATAGATATGATTTTCACCCCTGGACCTCCCTCCACGCCA AAACACAAGAAGTCTCAGAAAGGGTCAGCATTCACCTTCCCATCCCAGCAATCTCCCAGG AATGAACCATATGTAGCCAGACCATCCACATCAGAAATCGAAGACCAAAGCATGATGGGG AAGTTTGTAAAAGTTGAAAGACAGGTTCAGGACATGGGGAAGAAGCTGGACTTCCTCGTG GATATGCACATGCAACACATGGAACGGTTGCAGGTGCAGGTCACGGAGTATTACCCAACC AAGGGCACCTCCTCGCCAGCTGAAGCAGAGAAGAAGGAGGACAACAGGTATTCCGATTTG AAAACCATCATCTGCAACTATTCTGAGACAGGCCCCCCGGAACCACCCTACAGCTTCCAC CAGGTGACCATTGACAAAGTCAGCCCCTATGGGTTTTTTGCACATGACCCTGTGAACCTG CCCCGAGGGGGACCCAGTTCTGGAAAGGTTCAGGCAACTCCTCCTTCCTCAGCAACAACG TATGTGGAGAGGCCCACGGTCCTGCCTATCTTGACTCTTCTCGACTCCCGAGTGAGCTGC CACTCCCAGGCTGACCTGCAGGGCCCCTACTCGGACCGAATCTCCCCCCGGCAGAGACGT AGCATCACGCGAGACAGTGACACACCTCTGTCCCTGATGTCGGTCAACCACGAGGAGCTG GAGAGGTCTCCAAGTGGCTTCAGCATCTCCCAGGACAGAGATGATTATGTGTTCGGCCCC AATGGGGGGTCGAGCTGGATGAGGGAGAAGCGGTACCTCGCCGAGGGTGAGACGGACACA GACACGGACCCCTTCACGCCCAGCGGCTCCATGCCTCTGTCGTCCACAGGGGATGGGATT T C T GAT T C AGT AT G GAG C C C T T C C AAT AAG C C C AT T T AA

[0267] SEQ ID No. 9 - wild type Kv7.3

[0268] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLATIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0269] SEQ ID No. 10 - Kv7.3 A315T

[0270] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315T MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLTTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0271] SEQ ID No. 11- KV7.3 A315S

[0272] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315S MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLSTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0273] SEQ ID No. 12 - Kv7.3 A315V

[0274] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315V MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLVTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0275] SEQ ID No. 13 - Kv7.3 A315C

[0276] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315C MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLCTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0277] SEQ ID No. 14 - Kv7.3 A315N

[0278] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315N MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLNTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0279] SEQ ID No. 15 - Kv7.3 A315Q

[0280] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315Q MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLQTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

[0281] SEQ ID No. 16 - Kv7.3 A315Y

[0282] >sp | 043525 | KCNQ3 HUMAN Potassium voltage-gated channel subfamily KQT member 3 0S=Homo sapiens OX=9606 GN=KCNQ3 PE=1 SV=2 A315Y MGLKARRAAGAAGGGGDGGGGGGGAANPAGGDAAAAGDEERKVGLAPGDVEQVTLALGAG ADKDGTLLLEGGGRDEGQRRTPQGIGLLAKTPLSRPVKRNNAKYRRIQTLIYDALERPRG WALLYHALVFLIVLGCLILAVLTTFKEYETVSGDWLLLLETFAIFIFGAEFALRIWAAGC CCRYKGWRGRLKFARKPLCMLDIFVLIASVPWAVGNQGNVLATSLRSLRFLQILRMLRM DRRGGTWKLLGSAICAHSKELITAWYIGFLTLILSSFLVYLVEKDVPEVDAQGEEMKEEF ETYADALWWGLITLYTIGYGDKTPKTWEGRLIAATFSLIGVSFFALPAGILGSGLALKVQ EQHRQKHFEKRRKPAAELIQAAWRYYATNPNRIDLVATWRFYESWSFPFFRKEQLEAAS SQKLGLLDRVRLSNPRGSNTKGKLFTPLNVDAIEESPSKEPKPVGLNNKERFRTAFRMKA YAFWQSSEDAGTGDPMAEDRGYGNDFPIEDMIPTLKAAIRAVRILQFRLYKKKFKETLRP YDVKDVIEQYSAGHLDMLSRIKYLQTRIDMIFTPGPPSTPKHKKSQKGSAFTFPSQQSPR NEPYVARPSTSEIEDQSMMGKFVKVERQVQDMGKKLDFLVDMHMQHMERLQVQVTEYYPT KGTSSPAEAEKKEDNRYSDLKTIICNYSETGPPEPPYSFHQVTIDKVSPYGFFAHDPVNL PRGGPSSGKVQATPPSSATTYVERPTVLPILTLLDSRVSCHSQADLQGPYSDRISPRQRR SITRDSDTPLSLMSVNHEELERSPSGFSISQDRDDYVFGPNGGSSWMREKRYLAEGETDT DTDPFTPSGSMPLSSTGDGISDSVWTPSNKPI

Claims

Claims1. A system for use in the treatment of endometriosis, the system comprising:a gene therapy vector comprising:a. a capsid that selectively transduces target nociceptive neurons, and b. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No. 9 to SEQ ID No. 15,wherein, when the gene therapy vector transduces the target nociceptive neurons, expression of the Kv7.3 ion channel subunit is upregulated and functional Kv7.3-containing ion channels are formed; anda neuromodulatory drug that targets the Kv7.3-containing ion channels.

2. The system for use according to claim 1 wherein the neuromodulatory drug is selected from Retigabine / Ezogabine and derivatives thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine.

3. A method of treating endometriosis comprising the steps:a. administering to a subject in need thereof, a therapeutically effective amount of a gene therapy vector comprising:a. a capsid that selectively transduces target nociceptive neurons, and b. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No. 9 to SEQ ID No. 15,such that the gene therapy vector transduces the target nociceptive neurons to produce transduced nociceptive neurons;b. upregulating expression of the Kv7.3 ion channel subunit in the transduced nociceptive neurons, such that the transduced nociceptive neurons form functional Kv7.3-containing ion channels; andc. administering to the subject a neuromodulatory drug known to target Kv7.3- containing ion channels, wherein the neuromodulatory drug is administered at a dose lower than the dose that would be required to elicit an equivalent response in nociceptive neurons that are not transduced by the gene therapy vector.

4. The method of claim 3 wherein the neuromodulatory drug is selected from Retigabine / Ezogabine and derivatives thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine.

5. A gene therapy vector comprising:a. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ ID No.9 to SEQ ID No.15, and b. a capsid that selectively transduces target nociceptive neurons,wherein, when the gene therapy vector transduces the target nociceptive neurons, expression of the Kv7.3 ion channel subunit is upregulated and functional Kv7.3- containing ion channels are formed.

6. The system for use, method or the gene therapy vector according to any preceding claim, wherein the Kv7.3-containing ion channel assembles as a Kv7.3 homomer, a Kv7.2 / 7.3 heteromer, or a Kv7.3 / 7.5 heteromer.

7. The system for use, method or the gene therapy vector according to any preceding claim wherein the upregulation of the Kv7.3 ion channel subunit expression reduces the excitability of the target nociceptive neuron.

8. The system for use, method or the gene therapy vector according to any preceding claim wherein the upregulation of the Kv7.3 ion channel subunit expression by the target nociceptive neuron reduces activity of cells innervated by axon terminals of the target nociceptive neuron in the presence of the neuromodulatory drug.

9. A gene therapy vector comprising:a. a nucleotide sequence encoding a Kv7.3 ion channel subunit comprising the amino acid sequence of any of SEQ. ID No.9 to SEQ ID No.15, andb. a capsid that selectively transduces target nociceptive neuronsfor use in the treatment of endometriosis.

10. Use of the gene therapy vector according to claim 5 in the preparation of a medicament for use in the treatment of endometriosis.

11. A therapeutic system comprising a gene therapy vector according to claim 5, and a neuromodulatory drug that targets Kv7.3-containing ion channels.

12. The therapeutic system according to claim 11 wherein the neuromodulatory drug is selected from the group consisting of: retigabine or a derivative thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine.

13. A neuromodulatory drug selected from the group consisting of: retigabine or a derivative thereof, BHV-7000 and Xen496, Xen 1101, flupirtine, diclofenac, BMS-204352, meclofenamic acid, ETX-123, and linopirdine, for use in the treatment of endometriosis.

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