Optogenetic modulation of the central nervous system for pain management

Abstract

The present invention generally relates to compositions and methods for modulating cellular activity using multispecific opsins (MCOs). Additionally, the present invention provides methods for treating conditions associated with activity in the dorsal root ganglion (DRG), anterior cingulate cortex (ACC), or spinal cord, including neuropathic pain. Certain embodiments include methods for modulating inhibitory pathways to suppress chronic neuropathic pain. The methods can employ photosensitive optogenetic actuators with inhibitory neuron specificity. Embodiments also include optogenetic pain modulation devices that can modulate the frequency and intensity of optogenetic stimulation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 447,029, filed February 20, 2023, the contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference The entire contents of the following sequence listing, submitted electronically via the Patent Center of the United States Patent and Trademark Office (USPTO) as set forth in the United States Manual of Patent Examining Procedures (MPEP) Section 173011.B.2(a)(C), are incorporated herein by reference in their entirety for all purposes. The sequence listing is identified in the electronically submitted text file as follows: Filename: OB1-003WO; Creation Date: February 20, 2024; Size (bytes): 5KB. [Technical Field]

[0003] The present invention relates to methods of treatment, and more particularly to methods of alleviating pain using optogenetic actuators to activate inhibitory neurons in the dorsal root ganglia (DRG), anterior cingulate cortex (ACC) or spinal cord. [Background technology]

[0004] Pain can be defined as an unpleasant sensory and emotional experience that is related to or similar to actual or potential tissue damage. In the medical field, pain is considered a symptom of an underlying disease. The sensation of pain can provide important information about the state of the body or any current damage, as well as motivate behavior that contributes to the preservation of the body's integrity. However, due to dysfunction or unresolved disease pathology, pain itself can be a debilitating problem.

[0005] Pain can be characterized based on various criteria. For example, it can be described by location, duration, mechanism, and underlying cause. The description of pain by location or cause (often malignant or nonmalignant) is relatively clear. The primary mechanisms by which pain is experienced are nociceptive (i.e., pain from pain nerves reporting information) and neuropathic (i.e., pain due to dysfunction of the pain nerves or associated neurons themselves). Pain can also be classified by its duration. Most pain experiences are acute (e.g., nociceptive pain directly related to injury and dissipating over time with healing or adaptation). However, when pain persists for longer than 3–6 months (i.e., longer than the normal healing process), it becomes chronic pain. Its constant nature and persistence negate the warning benefits of pain and make it a debilitating chronic condition that requires its own treatment. If left untreated long enough, acute pain can produce secondary changes in the central nervous system, resulting in chronic pain syndromes.

[0006] Chronic pain is a common condition affecting an estimated 15-20% of the adult population worldwide. It is estimated that debilitating chronic pain costs $560-635 billion annually and affects approximately 100 million Americans. The economic cost of debilitating chronic pain is further complicated by the unintended effects of common treatments. Opioids are a common and effective treatment for pain, but they are associated with dose-limiting adverse effects, such as respiratory depression and addiction. Given the nature of chronic pain, treatment must be as persistent as the pain itself. According to the CDC, the probability of addiction increases with the duration of treatment. Even a one-day prescription can carry a 2.9% risk of addiction, and a month-long prescription increases the risk to 30%.

[0007] Non-opioid drugs for pain treatment have been developed as an alternative to opioids. For example, antiepileptic drugs are a common class of drugs used to treat neuropathic pain. However, they can induce serious central nervous system-related side effects, including drowsiness, somnolence, loss of consciousness, and cardiac side effects. Non-drug therapies for pain treatment are being pursued for pain treatment. These include spinal cord stimulation, peripheral nerve stimulation, and electrical deep brain stimulation. These methods have similar limitations to opioids in that they are non-specific in their interaction with human biology. For example, discharges activate all neurons within a specific effect area, thus resulting in unintended consequences, including speech impairment, personality changes, and in some cases cognitive impairment.

[0008] Therefore, in developing effective pain management, it is necessary to determine which anatomical structures and cell types are involved in the sensing, transmission, processing, experience, and outcome of pain (making them viable treatment targets), as well as alternative methods for modulating these treatment targets to regulate pain. Efforts are currently underway to develop better methods and drugs that lack serious side effects in order to provide more significant pain relief to a broader patient population.

[0009] Previous studies have established the major nerve types that sense pain. However, because they are localized, targeted delivery of pain treatments to these regions can result in numbness and cannot address more widespread damage / dysfunction or diffuse pain conditions such as visceral pain. Furthermore, central pain conditions, such as dysfunctional changes resulting in chronic pain syndromes, are not fully addressed by this treatment strategy. Therefore, targeting higher-level neurons in the central nervous system (CNS) is warranted. The role of specific CNS structures in pain experience is a much more complex issue, as the study of these anatomical structures is complicated by their sensitivity and essential role in life, as well as the interconnectedness of various substructures that form a functional CNS. Nevertheless, diverse studies have established the role of key structures in pain, such as the dorsal root ganglion (DRG) in the spinal cord, the thalamus (for routing pain information), the insula, and the anterior cingulate cortex (ACC). A variety of case reports and experimental studies in both animals and humans suggest that neurons in the ACC are involved in pain perception (among many other functions of the ACC), specifically the affective-aversive aspects of the pain experience.

[0010] Another complicating factor in pain drug discovery relates to differences in the types of pain. Pain pathologies are diverse, and different pain conditions may require different tailored therapeutic agents. For example, pain associated with postherpetic neuralgia, diabetic painful neuropathy, and fibromyalgia may require three different types of drugs. Therefore, it is important to generate preclinical data that can reliably predict the specific effects of new drugs on human pain pathways and provide information about which type of pain may best be treated by a given drug candidate. In recent years, progress has been made in the field of clinical diagnostic neurology. Thanks to the introduction of various tests performed on patients, it is now possible to begin to establish with some accuracy the types of somatosensory nerve fibers affected by painful conditions and, in some cases, even assess the extent and type of damage. This information can be useful in guiding the selection of treatment options.

[0011] Because traditional pain treatments are generally ineffective, alternative methods for treating pain are needed. Traditional drugs can be addictive and / or produce numerous undesirable side effects. Non-drug therapies, such as electrode implants, lack specificity and are generally ineffective in treating pain. Applicant has discovered a method of optogenetic modulation that overcomes these limitations by enabling cell-specific stimulation at low power. Summary of the Invention [Means for solving the problem]

[0012] The invention described and claimed herein has many attributes and embodiments, including but not limited to those described or illustrated or referenced in this Summary. The invention described and claimed herein is not limited to, and is not limited by, the features or embodiments identified in this Summary, which are included for purposes of example only and not limitation.

[0013] Embodiments of the present invention include recombinant viruses comprising the recombinant nucleic acids disclosed herein. In some embodiments, the virus is a recombinant adeno-associated virus (AAV). In some embodiments, the multi-characteristic opsin (MCO) of SEQ ID NO: 1 is expressed using the virus.

[0014] In some embodiments, the multi-specificity opsin (MCO) expressed in the methods described herein has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1.

[0015] Embodiments also include methods of optically controlling neural activity in a cell, which may include expressing a recombinant nucleic acid provided herein in a cell and controlling the neural activity of the cell with light to modulate expression of a light-sensitive protein.

[0016] Embodiments also include a method for suppressing pain in a subject. The method may include (a) expressing a recombinant nucleic acid encoding a light-sensitive protein in a spinal cord cell, DRG, or ACC cell of the subject, and (b) controlling the expression of the light-sensitive protein to modulate neural activity of the cell with light. The modulation can treat pain by reducing its duration and / or intensity.

[0017] In some embodiments, the light / illumination can be tuned to respond to wavelengths that produce a low power load and propagate well through tissue without damaging cells.

[0018]

[0010] Embodiments also include methods for inhibiting neuropathic pain without affecting nociceptive pain. The methods may include (a) expressing recombinant multispecific opsin (MCO) in neural tissue of a subject, and (b) controlling MCO with light to modulate activity in the neural tissue. Expression of MCO can treat neuropathic pain by reducing its duration and / or intensity.

[0019] In some embodiments, the neuropathic pain is caused by one or more of a traumatic insult, spinal cord injury, limb amputation, contusion, inflammation or surgery, an ischemic event, exposure to an infectious agent, a toxic agent, or disease.

[0020] Embodiments also include methods of treating an ailment in a subject. The method may include (a) expressing a recombinant nucleic acid encoding a light-sensitive protein in cells of neural tissue of the subject, and (b) modulating activity of the neural tissue with light that controls expression of the light-sensitive protein. This modulation can adjust the balance of excitation / inhibition (E / I) in the neural tissue to treat the ailment. The ailment may be, for example, pain and / or a neurological disorder.

[0021] In certain embodiments, the neurological disorder is one or more of fibromyalgia, rheumatoid arthritis, osteoarthritis, chronic arthropathy, spinal nerve compression syndromes associated with neoplasia and / or herniated discs, chronic back pain, chronic joint pain of any etiology associated with inflammation and / or structural joint abnormalities, post-herpetic neuralgia, trigeminal neuralgia, chronic metabolic neuropathy associated with chronic pain, migraine, inflammatory pain, post-surgical pain syndromes including phantom limb pain, post-traumatic stress disorder (PTSD), irritable bowel syndrome, autonomic neuropathy, arachnoiditis, chronic regional pain syndromes, vulvodynia, and chronic pain syndromes associated with activation of central nervous system sensitization pathways, visual disorders, drug addiction, psychiatric disorders, and movement disorders.

[0022]

[0010] Embodiments also include recombinant nucleic acids comprising a nucleic acid fragment encoding a light-sensitive protein and a regulatory nucleic acid fragment capable of directing selective expression of the light-sensitive protein in cells of the CNS. In embodiments, the light-sensitive protein is sensitive to visible light or transdermally delivered light. In some embodiments, the light-sensitive protein can regulate neuronal activity in cells of the spinal cord, DRG, and / or ACC, including activity associated with pain transmission or generation.

[0023] In some embodiments, the light-sensitive protein is a membrane-bound microbial opsin. In some embodiments, the microbial opsin is a light-gated ion channel or pump.

[0024] Embodiments also include a gene encoding a light-activated, ambient light-activatable multi-specificity opsin (MCO). In aspects, the MCO is packaged into a safe viral vector (e.g., AAV) with a fluorescent reporter (e.g., vMCO1-mCherry).

[0025] Embodiments also include AAV vectors carrying red-light sensitive optogenetic actuators with inhibitory neuron specificity to target inhibitory (e.g., GABAergic) neurons of the DRG and / or ACC to treat disease and / or suppress pain responses.

[0026] Another embodiment is a method for alleviating neuropathic pain. The method can include optically modulating activity associated with neural transmission or generation of pain. The method can include expressing a recombinant nucleic acid provided herein in a cell of a subject; and controlling neural activity of the cell with light to modulate expression of a light-sensitive protein, thereby alleviating neuropathic pain. The control can be performed with high spatial and temporal precision using a specifically positioned device that controls light emission over time.

[0027]

[0003] Embodiments include methods of expressing a recombinant nucleic acid in target cells of a subject and controlling the neural activity of the cells with light to modulate the expression of a light-sensitive protein, thereby alleviating neuropathic pain. This method can provide significant analgesia for chronic neuropathic pain without off-target effects such as general central nervous system depression. In embodiments, the target cells (for expression of the recombinant protein) are cells of the spinal cord, DRG, and / or ACC.

[0028] Further embodiments include methods of optogenetic stimulation that reduce inflammatory pain but do not affect the early, alarming nociceptive pain that provides information about the localization and intensity of physical injury.

[0029] In some embodiments, the methods described herein can alter the balance between excitation and inhibition (ie, the E:I ratio) of a neuronal cell.

[0030] The results described herein support modulation of inhibitory pathways within the DRG and ACC as a viable alternative for suppressing chronic neuropathic pain.

[0031] Other features and advantages of aspects of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of aspects of the invention.

[0032] The accompanying drawings illustrate embodiments of the present invention, and show: [Brief explanation of the drawings]

[0033] [Figure 1] 1 is a flowchart of steps in a method of optogenetic stimulation. [Figure 2A] 1 is an image of a wireless optogenetic stimulation device. [Figure 2B] This is an image of the optical fiber of a minimally invasive device that emits low-power red light. [Figure 2C] 1 is an image of mouse brain tissue showing the propagation of red light. [Figure 2D] 1 is a graph showing an intensiometric analysis of light propagation through brain slices. [Figure 2E] FIG. 10 is a graph showing that the presence of the implant does not significantly affect the acute pain response in the formalin assay. [Figure 2F]1A-1C are images of mice showing that the presence of the implant does not significantly affect the acute pain response as measured by paw licking. [Figure 2G] 1A-C are images of mice showing that the presence of the implant does not significantly affect acute pain responses as measured by paw lifting. [Figure 3A] FIG. 1 shows endogenous mCherry fluorescence imaging of GAD67 promoter-driven MCO expression in the anterior cingulate cortex (ACC) 2 weeks after pMCO2 injection (optical fiber outline indicated by arrow). [Figure 3B] 10 is an image of a wireless fiber-coupled red LED implanted in an MCO transfected mouse. [Figure 3C] Confocal immunostained mouse brain sections: DAPI (nuclear stain). [Figure 3D] Images of confocal immunostained mouse brain sections: GAD65 - a marker for GABAergic neurons. [Figure 3E] Confocal immunostained mouse brain sections: MCO reporter-mCherry. [Figure 3F] Confocal immunostained mouse brain sections: GAD65 and mCherry overlay. [Figure 3G] 3F shows an image of a confocal immunostained mouse brain section; the magnified area (indicated by the rectangle in FIG. 3F) shows the colocalization of GAD65. [Figure 3H] Image of a confocal immunostained mouse brain section: the magnified area (indicated by the rectangle in Figure 3F) shows co-localization of mCherry. [Figure 4A] FIG. 1 is a flowchart showing the steps in assessing the effects of optogenetic modulation on an acute pain model (formalin injection into the hind paw). [Figure 4B]Graph comparing baseline formalin-induced pain scores at 10-minute intervals with scores during continuous 5 Hz optogenetic stimulation (5 ms pulses at 630 nm). [Figure 4C] Baseline formalin-induced pain scores 11 days after early transduction are shown. [Figure 4D] The mean cumulative pain scores (measured at 5-minute intervals) during the early (0-11 minutes) non-inflammatory phase and the late (20-41 minutes) inflammatory phase of pain on day 11 after transduction are shown. [Figure 4E] The mean cumulative pain scores 5 weeks after optimal transduction are shown. [Figure 5A] Baseline formalin-induced pain scores over a 10-minute interval are compared to scores following 5 Hz optogenetic stimulation (5 ms pulses at 630 nm) over a 5-minute on-off interval. [Figure 5B] Baseline formalin-induced pain scores are shown along with scores for 2 Hz optogenetic stimulation with a 5 min on-off interval. [Figure 5C] Baseline formalin-induced pain scores are shown along with scores from 5 Hz optogenetic stimulation starting 15 min after the start of the experimental session. Mean cumulative pain scores (measured at 5-min intervals) during the early (0–11 min) non-inflammatory phase and the late (20–41 min) inflammatory phase of pain. [Figure 5D] Baseline formalin-induced pain scores are shown along with scores for intermittent 5 Hz optogenetic stimulation. [Figure 5E] Baseline formalin-induced pain scores are shown along with scores for intermittent 2 Hz optogenetic stimulation. [Figure 5F] Baseline formalin-induced pain scores are shown along with scores for delayed 5 Hz optogenetic stimulation. [Figure 6A] 1 is a flow chart showing the steps of a mechanical hypersensitivity chronic pain experiment. [Figure 6B]16 is an image of a Manual Von Frey apparatus showing the presentation of Von Frey filaments to the hind paw of a baseline mouse. [Figure 6C] 1 is a chart showing that the minimum force required to elicit a hind paw withdrawal response (withdrawal response) to a mechanical stimulus increases with increasing duration of exposure. [Figure 6D] 1 is a chart showing the percent increase or decrease in withdrawal force required to elicit a withdrawal response relative to the untreated baseline threshold of a mouse. [Figure 7A] FIG. 1 is a flow chart showing steps of a chronic pain experiment. [Figure 7B] 1 is an image of the CPP apparatus showing the dark (untreated) chamber (left), the center chamber, and the lit (treatment-related) chamber (right). [Figure 7C] Comparison of chamber preference between naive unimplanted and implanted mice in the CPP apparatus. [Figure 7D] FIG. 1 is a chart showing the increase in percent time spent in the treatment chamber post-conditioning. [Figure 8A] 1 is a graph showing the temperature change of an irradiated spot in a living brain measured by an infrared (IR) camera. [Figure 8B] A series of images demonstrating no loss of viability of ACC-GABAergic neurons expressing MCO-mCherry after chronic optogenetic stimulation. (i) DAPI; (ii) MCO-mCherry; (iii) Caspase-3; and (iv) Bi-iii overlay. [Figure 8C] A series of images demonstrating that AAV-MCOII injection resulted in MCO expression without eliciting an inflammatory response: (i) DAPI; (ii) mCherry immunostaining confirming MCO expression; (iii) CD45 immunostaining (green), absent in the ACC region injected with 3 ml of AAV-MCO (8 x 10 vg / ml). (iv) Overlay of Ci-iii. [Figure 8D] A series of images demonstrating the absence of immune cell response to implants in MCO-mCherry-transfected ACC. (i) DAPI; (ii) MCO-mCherry; (iii) Iba1 (a marker for microglia / macrophages); (iv) Di-iii overlay. Minimal Iba1-positive (green) cells were observed in the vicinity of the implant. [Figure 9A] Graph showing formalin assay scores at 10 minute intervals. [Figure 9B] 1 is a graph showing mean pain scores over time, demonstrating that pain responses are reduced even when treatment is delayed. [Figure 10A] 1 is a graph showing the force required to elicit a hyperalgesic paw withdrawal response over time, illustrating the modulatory effect of optogenetic stimulation. [Figure 10B] Graph showing the percentage of mice that retained an increased sensitivity threshold. [Figure 11A] An image of a three-chamber social novelty apparatus. [Figure 11B] 1 is a graph depicting the results of a social impact study showing that transfection of MCO does not affect social interactions. [Figure 11C] 1 is a graph depicting the results of a social impact study showing that transfection of MCO does not affect social interactions. [Figure 12A] FIG. 1 is a graph showing mean pain scores over time, demonstrating that acute pain responses are reduced by continuous 5 Hz treatment. [Figure 12B] 1A is a bar graph showing the force required to elicit a paw withdrawal response (escape response) in different groups of mice. [Figure 13A] An image of a Y-maze apparatus for studying social behavior. [Figure 13B]1 is a bar graph showing the percentage of alternation in treated and untreated mice. [Figure 14A] 1 is a bar graph showing the results of the formalin assay score at 10 minute intervals with a 5 ms pulse. [Figure 14B] 1 is a bar graph showing the results of the formalin assay score, comparing baseline to treated experimental mice and untreated (ie, discontinued) mice. [Figure 15A] 1 is a series of images showing the results of intrathecal delivery to the spinal cord and associated nerve axial section. [Figure 15B] 1 is a series of images showing the results of paraspinal delivery to the axial section of the spinal cord and associated nerves. [Figure 16A] 1 is a series of images showing the results of delivery to the spinal cord and associated nerves in cross-axial section. [Figure 16B] 1 is a series of images showing the results of delivery to the spinal cord and associated nerves in cross-axial section. DETAILED DESCRIPTION OF THE INVENTION

[0034] definition References herein to "one embodiment / aspect" or "embodiment / aspect" mean that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the present disclosure. Use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places herein does not necessarily all refer to the same embodiment / aspect, nor does it mean that separate or alternative embodiments / aspects are mutually exclusive of other embodiments / aspects. Furthermore, various features are described that are exhibited by some embodiments / aspects but not by others. Similarly, various requirements are described that are requirements of some embodiments / aspects but not others. Embodiment and aspect may be used interchangeably in some cases.

[0035] The terms used in this specification generally have their ordinary meaning in the art, within the context of this disclosure, and within the specific context in which each term is used.Specific terms used to describe this disclosure are discussed below or elsewhere in this specification to provide additional guidance to practitioners regarding the description of this disclosure.It will be understood that the same thing can be said in multiple ways.

[0036] Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein. Furthermore, no special meaning is attached to a term whether it is recited or discussed herein. Synonyms are provided for certain terms. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed herein, is merely illustrative and is not intended to further limit the scope and meaning of the disclosure or any exemplified term. Similarly, the disclosure is not limited to the various embodiments described herein.

[0037] Without intending to further limit the scope of the present disclosure, examples of instruments, devices, methods, and their related results according to embodiments of the present disclosure are provided below. Please note that titles or subtitles may be used in the examples for the convenience of the reader, but this does not limit the scope of the present disclosure in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of conflict, the present specification, including definitions, will prevail.

[0038] As used herein and in the appended claims, the terms "about" or "generally" refer to a margin of ±20% unless otherwise specified. Where applicable, the term "substantially" as used herein and in the appended claims refers to a margin of ±10% unless otherwise specified. It should be noted that not all uses of the above terms are quantifiable enough to allow the referenced range to be applied.

[0039] The term "BLUETOOTH®" refers to a short-range wireless technology standard used to exchange data between fixed and mobile devices over short distances and create personal area networks using UHF radio waves in the 2.402-2.48 GHz ISM band.

[0040] The term "neural cell" refers to a type of cell that sends and receives messages from the body to the brain and from the brain to the body. In the nervous system, neurons, or nerve cells, are electrically excitable cells that emit electrical signals called action potentials throughout the neural network. Neurons communicate with other cells through synapses, specialized connections that pass electrical signals from presynaptic neurons through the synaptic cleft to target cells, usually using trace amounts of chemical neurotransmitters. Neurons are the primary component of nervous tissue in all animals except sponges and placozoans. Neurons are typically classified into three types based on their function: sensory neurons respond to stimuli, such as force, sound, or light, acting on sensory cells and transmit signals to the spinal cord or brain. Motor neurons receive signals from the brain or spinal cord and control everything from muscle contraction to glandular secretions. Interneurons connect neurons within the same region of the brain or spinal cord. When multiple neurons are functionally connected, they form neural circuits.

[0041] The term "GABAergic neuron" refers to neurons located in the hippocampus, thalamus, basal ganglia, hypothalamus, and brainstem. The balance between GABA-mediated inhibitory neurotransmission and glutamate-mediated excitatory neurotransmission is essential for proper cell membrane stability and neuronal function. A neuron is "GABAergic" if it relates to or affects the neurotransmitter GABA.

[0042] The term "pain" refers to any unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a health care provider. Pain can be classified as one of two types: chronic and acute. "Acute pain" is pain of sudden onset and short duration. One type of acute pain is, for example, cutaneous pain felt with injury to the skin or other superficial tissue (e.g., caused by a cut or burn). Cutaneous nociceptors terminate just below the skin and produce well-defined, short-lasting localized pain due to the high concentration of nerve endings. "Chronic pain" is pain other than acute pain. Chronic pain includes neuropathic pain, inflammatory pain, headache, somatic pain, visceral pain, and referred pain.

[0043] The term "neuropathic pain" refers to abnormal sensory inputs that cause discomfort, originating from the peripheral nervous system, the central nervous system, or both. Symptoms of neuropathic pain can include persistent, spontaneous pain, as well as allodynia (i.e., painful responses to normally non-painful stimuli), hyperalgesia (i.e., exaggerated responses to painful stimuli that normally cause only mild discomfort, such as a pinprick), or hyperpathia (i.e., short-term discomfort becomes prolonged, severe pain). Neuropathic pain can be caused by, for example, traumatic insults (e.g., nerve compression injuries, such as nerve crush, nerve stretch, nerve entrapment, or incomplete nerve section), spinal cord injury (e.g., hemisection of the spinal cord), limb amputation, contusion, inflammation (e.g., spinal cord inflammation), or surgical procedures. Neuropathic pain can also be caused by an ischemic event, exposure to an infectious agent, a toxic agent, or a disease such as an inflammatory disorder, a neoplastic tumor, acquired immune deficiency syndrome (AIDS), Lyme disease, leprosy, a metabolic disease, or a peripheral neuropathy. Neuropathic pain also includes chronic pain, such as low back pain, osteoarthritis and joint pain (e.g., knee pain or carpal tunnel syndrome), myofascial pain syndrome, and neuropathic pain.

[0044] Neuropathic pain can be related to pain disorders, a term that refers to diseases, disorders, or conditions associated with or caused by pain. Examples of pain disorders include arthritis, allodynia, classic trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesis, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia dolorosa, causalgia, sciatic pain disorders, degenerative joint disorders, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis, or pain associated with cancer.

[0045] The term "pain disorder" refers to a condition or disorder that is secondary to (i.e., affected by or caused by) a disorder such as chronic pain and / or neuropathic pain.

[0046] The term "somatic pain" refers to pain originating from ligaments, tendons, bones, blood vessels, and even nerves. It is sensed by somatic nociceptors. A lack of pain receptors in these areas results in a duller, less localized pain that lasts longer than cutaneous pain; examples include sprains and fractures. Further examples include: excessive muscle strain, repetitive strain injuries, muscle disorders, muscle pain, infections, and drugs.

[0047] The term "neuralgia" refers to pain that radiates along the course of one or more specific nerves, usually without any obvious pathological changes in the nerve structure. Various causes of neuralgia include chemical irritation, inflammation, trauma (including surgery), pressure from nearby structures (e.g., from a tumor), and infection. However, the cause is often unknown or cannot be identified. Neuralgia includes, for example, trigeminal neuralgia, postherpetic neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica, and atypical facial pain.

[0048] The term "deafferentation" refers to the loss of sensory input from a part of the body and can be caused by blockage of either peripheral sensory fibers or nerves from the central nervous system. Deafferentation pain syndromes include, for example, brain or spinal cord injury, post-stroke pain, phantom limb pain, paraplegia, brachial plexus avulsion injury, and lumbar radiculopathy.

[0049] The term "light-sensitive protein" refers to a protein that responds to light. Membrane light-sensitive proteins are activated by light and cause the exchange of cations or anions across the membrane, resulting in membrane hyperpolarization or depolarization. Therefore, depending on which protein is introduced and expressed, light stimulation can excite or inhibit neural tissue. Light-sensitive proteins can include membrane-bound light-sensitive ion channels or proton pumps that cause cell hyperpolarization or depolarization in response to light stimulation. Light-sensitive proteins include, for example, opsins such as rhodopsin, blue opsin, red opsin, halorhodopsin (NpHR), channelrhodopsin 2, enhanced halorhodopsin (eNpHR), archaerhodopsin-3 (Arch), and Leptosphaeria maculans (Mac), as well as functional fragments or variants thereof. The light-sensitive opsins of the present invention also include light-gated ion channels and ion pumps. Combinations of two or more light-sensitive proteins can also be used in the same manner.

[0050] The term "optogenetics" refers to a biological technique for controlling the activity of neurons or other cell types with light. This is achieved through the expression of light-sensitive ion channels, pumps, or enzymes specifically in target cells. At the individual cell level, light-activated enzymes and transcription factors allow for precise control of biochemical signaling pathways. In systems neuroscience, the ability to control the activity of genetically defined sets of neurons has been used to understand their contribution to decision-making, learning, fear memory, mating, and addiction.

[0051] Optogenetics involves the introduction of light-responsive opsin channels or pump proteins in the plasma membrane of target neurons, allowing for temporally precise manipulation of neuronal membrane potential while maintaining cell-type resolution through the use of specific targeting mechanisms.

[0052] The term "opsin" refers to a group of proteins that become light-sensitive via the chromophore retinal (or its variants) present in the photoreceptor cells of the retina. Five classical opsin groups are involved in vision and mediate the conversion of light photons into electrochemical signals, the first step in the visual transduction cascade.

[0053] Multi-characteristic opsin (MCO) refers to highly photosensitive opsins with unique spectral and temporal properties that generate significant currents in response to ambient light.

[0054] Channelrhodopsin-2 (ChR2) is an opsin that specifically responds to blue light. When ChR2 is introduced into neurons, these neurons can be activated by blue light.

[0055] The term "halorhodopsin" refers to a light-driven ion pump specific for chloride ions found in archaea known as halobacteria. It is a seven-transmembrane retinylidene protein from the microbial rhodopsin family. It is similar in tertiary structure (but not in primary sequence) to vertebrate rhodopsin, the light-sensing pigment in the retina. Halorhodopsin contains all-trans-retinal, an essential photoisomerizable vitamin A derivative. Halorhodopsin utilizes the energy of green / yellow light to translocate chloride ions into cells and control membrane potential. In addition to chloride ions, it transports other halides and nitrate ions into cells. Potassium chloride uptake by cells helps maintain osmotic balance during cell growth. By performing the same task, light-driven anion pumps can significantly reduce metabolic energy use. Halorhodopsin has been the subject of extensive research, and its precise structure is known. Similar in properties to bacteriorhodopsin, these two light-driven ion pumps transport cations and anions in opposite directions.

[0056] The term "GABAergic" refers to agents that modify the effects of GABA in the body or brain. Similarly, GABAergic neurons affect the neurotransmitter gamma-aminobutyric acid (GABA). For example, if a synapse uses GABA as its neurotransmitter, the synapse is GABAergic, and the GABAergic neuron produces GABA. Subclasses of GABAergic neurons include (a) Ca 2+ These include (a) the binding protein parvalbumin (PVALB), (b) the neuropeptide somatostatin (SST), (c) vasoactive intestinal peptide (VIP), and (d) the ionotropic 5-hydroxytryptamine 3a serotonin receptor (HTR3A).

[0057] The term "adeno-associated virus" or "AAV" refers to a small virus that infects humans and some other primate species. These are small (20 nm), replication-deficient, non-enveloped viruses with a linear, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). Several characteristics make AAV attractive candidates for generating viral vectors for gene therapy and for generating isogenic human disease models. AAV-based vectors have emerged as the preferred vector system for neurological gene therapy and have a good safety track record in clinical trials.

[0058] The term "nociception assay" refers to a technique for assessing an animal's (e.g., a mouse's) ability to detect noxious stimuli, such as pain sensations caused by stimulation of nociceptors. These assays measure the presence of pain through behaviors such as withdrawal, licking, immobility, and vocalization. The formalin assay is a common chemical assay of nociception that involves the injection of a dilute solution of formalin into the hind paw of a rodent. The affected hind paw is then monitored and scored for stereotyped behaviors such as flinching, licking, and biting.

[0059] The term "Von Frey assay" refers to a method that uses Von Frey hairs or fibers (i.e., small pieces of nylon rod) to test the sensitivity of rodents to mechanical stimuli. The Von Frey test involves applying a punctate stimulus to a given area of ​​the rodent's body, usually the plantar surface of the hind paw, and recording the stimulus intensity that elicits a withdrawal reflex.

[0060] The term "neurological disorder" broadly refers to disorders of the nervous system. Neurological disorders can affect the brain and nerves located throughout the body and spinal cord. Structural, biochemical, or electrical abnormalities in the brain, spinal cord, or other nerves can cause a variety of symptoms. Neurological disorders include, for example, acute spinal cord injury, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia, Bell's palsy, brain tumors, cerebral aneurysms, epilepsy and seizures, Guillain-Barré syndrome, headache, head trauma, hydrocephalus, meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, Parkinson's disease, stroke, headache, encephalitis, and myasthenia gravis.

[0061] Neuroplasticity, also known as neural plasticity or brain plasticity, is the ability of neural networks in the brain to change through growth and reorganization. These changes range from individual neuronal pathways forming new connections to systematic adjustments such as cortical reorganization. Examples of neuroplasticity include circuit and network changes resulting from learning new skills, environmental influences, training, and psychological stress. Activity-dependent plasticity can have important implications for healthy development, learning, memory, and recovery from brain injury.

[0062] The term "dendrite" refers to the branched plasma extensions of a neuron that transmit electrochemical stimuli received from other neurons to the neuron's soma (cell body) from which the dendrites project. Electrical stimuli are transmitted to dendrites by upstream neurons (usually via their axons) through synapses scattered throughout the dendritic tree. Dendrites appear to be capable of plastic change during adulthood in animals, including invertebrates. Neuronal dendrites contain various compartments known as functional units that are capable of computing incoming stimuli. These functional units are involved in input processing and consist of dendritic subdomains such as spines, branches, or groupings of branches. Therefore, plasticity resulting in changes in dendritic structure affects information transmission and processing in the cell. During development, dendrite morphology is shaped by intrinsic programs within the cell's genome and extrinsic factors, such as signals from other cells. However, in vivo, extrinsic signals become more influential and lead to greater changes in dendritic structure compared to intrinsic signals during development.

[0063] The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein. "Gene product" or "gene expression product" refers to the amino acid sequence (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0064] The term "expression" refers to the two-step process in which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0065] The term "under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence (usually a DNA sequence) is dependent on being operably linked to elements that contribute to or promote the initiation of transcription. "Operably linked" means that the polynucleotide is positioned so that it can function within the cell. In one embodiment, the invention provides a promoter operably linked to a downstream sequence.

[0066] The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if, in its natural state or when manipulated by methods well known to those of skill in the art, it can be transcribed to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0067] The term "promoter" refers to a regulatory sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or transgene. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific. Promoters can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and transcription factors, can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally including the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the ubiquitous chicken β-actin hybrid (CBh) promoter, the small nuclear RNA (U1a or U1b) promoter, the MeCP2 promoter, the MeP418 promoter, the MeP426 promoter, the minimal MeCP2 promoter, the VMD2 promoter, the mRho promoter, or the EFI promoter.

[0068] Additional promoters include, for example, EFla, Ubc, human β-actin, CAG, TRE, Ac5, polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequences of such promoters can be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying the TATA box of the 7SK, U6, and H1 promoters to disable RNA polymerase III transcription and promote RNA polymerase II-dependent mRNA transcription). Synthetic promoters can be used for ubiquitous or tissue-specific expression. In addition, viral promoters (some of which are described above) can be useful in the methods disclosed herein (e.g., CMV, HIV, adenovirus, and AAV promoters). In embodiments, the promoter is used in conjunction with an enhancer to increase transcription efficiency, non-limiting examples of which include the interstitial retinoid-binding protein (IRBP) enhancer, the RSV enhancer, or the CMV enhancer.

[0069] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. An enhancer / promoter may be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to a gene by genetic engineering (i.e., molecular biological techniques) so that transcription of the gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancers / promoters for use in the methods, compositions, and constructs provided herein include a PDE promoter plus an IRBP enhancer, or a CMV enhancer plus an U1a promoter. It is understood in the art that an enhancer can act from a distance and can act regardless of its orientation relative to the location of the endogenous or heterologous promoter. Thus, an enhancer that acts at a distance from a promoter is further understood to be "operably linked" to that promoter regardless of its location within the vector or its orientation relative to the location of the promoter.

[0070] The terms "homology" or "identity" or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. The percent identity can be determined by comparing a position within each sequence that can be aligned for comparison. If a position within the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the present disclosure. Alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined using ClustalW by importing the nucleic acid or amino acid sequence into ClustalW.

[0071] As used herein, an amino acid modification can be an amino acid substitution, an amino acid deletion, or an amino acid insertion. An amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, a "conservative mutation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative mutations include the substitution of hydrophobic residues, such as isoleucine, valine, leucine, or methionine; or the substitution of charged or polar residues, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Other illustrative examples of conservative substitutions include the following: alanine to serine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine, or glutamic acid; phenylalanine to tyrosine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0072] The term "viral vector" refers to a recombinant virus or viral particle containing a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.

[0073] The term "recombinant expression system" or "recombinant vector" refers to a genetic construct or constructs for the expression of specific genetic material formed by recombination.

[0074] The term "gene delivery vehicle" refers to any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers (including natural and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; bacteria; viruses such as baculoviruses, adenoviruses, and retroviruses; bacteriophages, cosmids, plasmids, and fungal vectors; and other recombinant vehicles typically used in the art that have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression. Liposomes that comprise, consist essentially of, or consist of targeting antibodies or fragments thereof can also be used in the methods disclosed herein. In addition to delivery of polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be achieved by protein transfection, which is a non-limiting technique; alternatively, culture conditions that can enhance expression and / or promote activity of the proteins disclosed herein are other non-limiting techniques.

[0075] The polynucleotides disclosed herein can be delivered to cells or tissues using a gene delivery vehicle. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to the introduction of an exogenous polynucleotide (also called a "transgene") into a host cell, regardless of the method used for transfer. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., via viral infection / transfection or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to introduce polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contain an origin of replication compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, numerous vectors are known to be capable of mediating gene transfer into mammalian cells.

[0076] The term "plasmid" typically refers to a DNA molecule that is separate from and can replicate independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population and typically provide a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to natural antibiotics in competitive environmental niches, or the proteins produced may act as toxins under similar circumstances. While plasmid vectors often exist as extrachromosomal circular DNA molecules, they can also be designed to stably integrate into host chromosomes in a random or targeted manner, and it is known in the art that such integration can be achieved using either circular plasmids or plasmids linearized prior to introduction into host cells.

[0077] "Plasmids" used in genetic engineering are also called "plasmid vectors." Many plasmids are commercially available for such uses. The gene to be replicated is inserted into a copy of the plasmid, which also contains a gene that confers resistance to a specific antibiotic to the cell and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites that allows for the easy insertion of DNA fragments at that location. Another major use of plasmids is to produce large amounts of proteins. In this case, researchers can culture bacterial or eukaryotic cells containing a plasmid carrying the gene of interest and induce them to produce large amounts of protein from the inserted gene.

[0078] In embodiments in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide comprising, consisting essentially of, or consisting of the viral genome or portion thereof and the transgene.

[0079] The term "adeno-associated virus" or "AAV" refers to a member of the group of viruses belonging to the Parvoviridae family, Dependoparvovirus genus. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Since it is well known that various serotypes are very closely related structurally, functionally, and even at the genetic level, it is fully expected that the same principles described in these reviews will also be applicable to additional AAV serotypes characterized after the publication date of these reviews. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes; and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes throughout the entire length of the genome; and by the presence of similar self-annealing segments at the termini corresponding to "inverted terminal repeat sequences" (ITRs). Similar infectivity patterns suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes are capable of infecting cells from a variety of tissue types. At least 11 sequentially numbered AAV serotypes are known in the art.Non-limiting exemplary serotypes useful in the methods described herein include any of 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV PHP.B. AAV particles comprise, consist essentially of, or consist of three major viral proteins: VP1, VP2, and VP3. In embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAVrh74.

[0080] The term "AAV vector" refers to a vector comprising, consisting essentially of, or consisting of one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides the functionality of the rep and cap gene products, for example, by transfection of the host cell. In embodiments, the AAV vector contains a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or an enhancer and / or terminator within the flanking ITRs, and is packaged into infectious AAV particles. The encapsidated nucleic acid portion may be referred to as the AAV vector genome. A plasmid containing an AAV vector may contain elements for manufacturing purposes (e.g., antibiotic resistance genes, etc.), but these are not encapsidated and therefore do not form part of the AAV particle.

[0081] The term "viral capsid" or "capsid" refers to the proteinaceous outer shell or coating of a viral particle. The capsid functions to enclose, protect, transport, and release the viral genome into the host cell. A capsid is generally composed of protein oligomeric structural subunits ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3. As described in Sonntag F et al., (June 2010), "A viral assembly factor promotes AAV2 capsid formation in the nucleolus," Proceedings of the National Academy of Sciences of the United States of America. 107 (22): 10220-5, and Rabinowitz JE, Samulski RJ (December 2000), "Building a better vector: the manipulation of AAV virions," each of which is incorporated herein by reference in its entirety, the mixture of VP1, VP2, and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry at a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3).

[0082] "AAV virion" or "AAV viral particle" or "AAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. Thus, since such a vector is contained within an AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0083] The term "subject" or "patient" refers to any single animal, more preferably a mammal (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human animals such as non-human primates), for which treatment is desired. Most preferably, the patient herein is a human.

[0084] The term "active agent" or "active ingredient" refers to a substance, compound, or molecule that is biologically active or otherwise induces a biological or physiological effect in a subject to which it is administered. In other words, "active agent" or "active ingredient" refers to the component(s) of a composition to which the composition's effect is attributable, in whole or in part. An active agent may be a primary active agent, or in other words, a component of a composition to which the composition's effect is attributable, in whole or in part. An active agent may also be a supplemental agent, or in other words, an additional portion of a composition and / or a component of a composition to which other effects are attributable.

[0085] The term "pharmaceutically acceptable carrier" as used herein refers to any solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. The composition may also contain other active compounds that provide complementary, additional, or enhanced therapeutic functions.

[0086] As used herein, the term "pharmaceutically acceptable composition" refers to a composition comprising at least one compound disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0087] As used herein, the term "prevention" refers to any action by which the onset of a disease is inhibited or delayed.

[0088] The term "treating" or "treatment" refers to one or more of: (1) inhibiting the disease (i.e., preventing further development of the pathology and / or symptoms); and (2) ameliorating the disease (i.e., reversing the pathology and / or symptoms), e.g., reducing the severity of the disease.

[0089] The term "administration" refers to the introduction of a certain amount of a predetermined substance into a patient by a specific appropriate method. The compositions disclosed herein can be administered via any common route, such as inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, as long as the desired tissue can be reached.

[0090] The terms "inductive charging" or "wireless charging" refer to a type of wireless power transfer that uses electromagnetic induction to power portable devices. Inductive charging is also used in vehicles, power tools, electric toothbrushes, and medical devices. A portable device can be placed near a charging station or induction pad without the need for precise alignment or electrical contact with a dock or plug. Inductive charging is so named because it transfers energy through inductive coupling. First, an alternating current flows through an induction coil in the charging station or pad. The moving charges generate a magnetic field, and the strength of the magnetic field fluctuates as the amplitude of the current fluctuates. This changing magnetic field creates an alternating current in the induction coil of the portable device, which then passes through a rectifier and is converted to direct current. Finally, the direct current charges the battery or provides operating power.

[0091] All numerical indications, including ranges (e.g., pH, temperature, time, concentration, and molecular weight, etc.), should be understood as approximations, in accordance with common practice in the art. As used herein, the term "about" can mean a variation of ±1%, ±5%, or ±10% of the stated amount, as appropriate depending on the context. It should be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalents of such reagents are known in the art.

[0092] Remington's Pharmaceutical Sciences (13 th Many known useful compounds and the like are described in "Therapeutic Agents for the Treatment of Acetaminophen," Ed., Mack Publishing Company, Easton, PA. As used herein, the term "formulation" means a combination of at least one active ingredient (or active ingredient) with one or more other ingredients (also commonly referred to as excipients), which may be independently active or inactive. The term "formulation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals, and may also include compositions that are useful intermediates for storage or research purposes.

[0093] Other technical terms used herein have their ordinary meaning in the technical field in which they are used, as exemplified in various technical dictionaries. The specific values ​​and configurations discussed in these non-limiting examples may vary and are cited merely to describe at least one embodiment and are not intended to limit its scope.

[0094] Detailed Description It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology as claimed. Additional features and advantages of the subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the description and claims hereof.

[0095] While pain syndromes arise from peripheral input and may be ameliorated by spinal manipulation, other conditions (i.e., central pain syndromes) arise from alterations in supraspinal function. Embodiments include methods of alleviating pain by targeting higher level neurons in the central nervous system (CNS) (e.g., cells of the spinal cord, DRG, and ACC).

[0096] The role of specific CNS structures in pain is complicated because the study of this anatomical structure and its function in pain processing is confounded by its essential role in life and the interconnectedness of various substructures to form a functional CNS. Nevertheless, multiple studies have established the role of key structures in pain, such as the DRG, thalamus (i.e., for routing pain information), insular cortex, and anterior cingulate cortex (ACC). Research suggests that neurons in the DRG and ACC are involved in pain perception (among many other functions of the DRG and ACC), specifically the affective and aversive aspects of the pain experience.

[0097] The ACC receives input from multiple brain regions, including the thalamus, hippocampus, amygdala, and other regions of the cerebral cortex. Electrophysiological analyses reveal that ACC neurons respond to noxious stimuli and are active when pain is anticipated. Furthermore, ACC activation can reduce pain behavior and activity in the dorsal horn. These findings have been confirmed by neuroimaging studies showing that the ACC and other cortical structures are activated by noxious stimuli, including psychosocial pain. Damage to the ACC is associated with attenuated pain responses in animal models. Persistent chronic pain has also been shown to manifest as plastic changes in the ACC. Alterations in the balance between excitation and inhibition (i.e., the E:I ratio) due to long-term potentiation of pyramidal neurons, reduced activity of interneurons, or loss of inhibitory regulation can lead to persistent pain. Because the ACC is involved in multiple other functions, such as social behavior, it has been proposed that modulation (e.g., by electrical stimulation) rather than destruction may be a viable option for pain control.

[0098] Abnormalities in the cortical excitability to inhibition (E / I) balance may also play a role in disorders such as autism, including comorbidities such as reduced pain sensitivity. Previous studies have determined that under pain conditions, the E / I balance in the ACC shifts toward inhibition. Therefore, optogenetic enhancement of inhibitory GABAergic neuronal activity can lower the E / I ratio as a pain treatment strategy. Previous studies have demonstrated that optogenetic manipulation of pain-related central brain structures (e.g., the ACC) alters pain sensation measured using reflex behavioral assays. The proposed mechanism for these results in the ACC is modulation of the cellular E / I balance.

[0099] Previous studies have demonstrated that optogenetic stimulation of inhibitory neurons in the ACC results in a reduction in electrical activity in the ACC and a significant attenuation of pain responses to cutaneous and noxious stimuli. Importantly, modulation of ACC function by electrical stimulation is inherently limited because such stimulation alters the excitability of both excitatory and inhibitory interneurons, potentially resulting in a deterioration of psychological states caused by painful stimuli. An alternative approach, described herein, is the use of optogenetics.

[0100] Challenges with electrode-based brain stimulation for pain relief can be attributed to the inherently nonspecific nature of electrical stimulation. Specific cell types cannot be stimulated without affecting neighboring cells. Furthermore, pharmaceutical drugs are similarly nonspecific. For example, bicuculline has important drawbacks: it is a competitive inhibitor (i.e., its effectiveness depends on the local GABAergic tone in the area) and is unstable. To address these drawbacks, applicants utilize the optogenetic method described herein, which specifically targets GABAergic neurons via promoter-directed expression. The use of optogenetics with precise fiber optic stimulation allows for local modulation at controlled stimulation frequencies. Results demonstrate that continuous optogenetic stimulation has the strongest effect on inflammatory pain during the late phase of the formalin pain response. Furthermore, optogenetic modulation of chronic pain only has a strong effect on behavior in a biased (light-dark) conditional place preference when modulation is applied in real time. Bright light itself may induce pain that minimizes the strength of post-conditioning preference.

[0101] Applicants herein present a novel and effective approach to pain modulation by focusing on the regulatory and transduction pathways connecting the injury / pain site with cortical sensory structures. In embodiments, cell-specific treatment (e.g., via promoter-driven opsin gene expression) targets the spine (e.g., neurons of the spinal cord, ACC, and / or DRG).

[0102] Embodiments of the present invention include compositions and methods for specifically delivering light-sensitive proteins to sensory neurons in the ACC to optically silence pain-related neurotransmission and reduce the sensation of chronic pain. This approach provides for the control of activity in specifically targeted neuronal families in a spatially and temporally specific manner using light.

[0103] 1 is a flowchart summarizing the steps of the optogenetic stimulation method described herein. In the first step (105), neural cells are transfected with a virus to express multispecific opsin (MCO). In some embodiments, the neural cells are cells of the spinal cord, cells of the dorsal root ganglion (DRG), or cells of the anterior cingulate cortex (ACC).

[0104] The method can also include implanting a wireless optogenetic stimulation device (110), such as an optical fiber or similar light source. The implantable device can be made of glass, polymer, PMMA, silicone, or PDMS; it can be coated with a biocompatible material to minimize neuroinflammatory responses without compromising light guidance. While transcutaneous light transmission is also available, optical fiber avoids the limitations of external light sources (e.g., thermal transmission). In some embodiments, the light source is activated via a wireless (radio) signal. In some embodiments, the light source is wirelessly charged (e.g., via inductive charging), thus avoiding the need for an internal or connected power source.

[0105] Optical devices can activate transfected neurons using visible light. Light-induced activation of multispecific opsin (MCO) results in depolarization of only cells expressing MCO. (115) Subjects (e.g., mice) can then be studied by pain scoring for acute (120) and chronic (125) pain.

[0106] The methods described herein utilize optogenetics to enable highly selective neuronal stimulation with millisecond-level temporal precision. This opsin-based approach can be applied to pain modulation, as well as conditions such as visual, drug, psychiatric, or movement disorders. Through the use of opsins, sensitizing cells to specific wavelengths of light creates a means by which cells involved in essential aspects of pain syndromes (e.g., from nociception to allodynia) can be modulated.

[0107] Embodiments also include wireless (e.g., BLUETOOTH®) optogenetic pain modulation devices capable of modulating the frequency and intensity of optogenetic stimulation. The sensitivity of the optogenetic actuator, multispecific opsin (MCO), minimizes the power requirements for pain modulation by activating it with low-intensity light. MCO's sensitivity to wavelengths in the red spectrum of visible light, which have deeper tissue penetration, allows for deeper activation with more shallowly implanted light sources. As described in the Examples below, stimulation of inhibitory neurons in the spinal cord, DRG, or ACC expressing MCO resulted in attenuation of reflex acute pain responses and altered conditioned place preference in a mouse model of chronic pain. Furthermore, measurements show that the suppression of pain responses depends on the optogenetic stimulation schedule of the DRG / ACC. The results described herein support modulation of inhibitory pathways within the DRG / ACC as a viable alternative for suppressing chronic neuropathic pain.

[0108] The methods described herein can be used to treat, for example, fibromyalgia, rheumatoid arthritis, osteoarthritis, chronic arthropathy, spinal nerve compression syndromes associated with neoplasms and / or herniated discs, chronic back pain, chronic joint pain of any etiology associated with inflammation and / or structural joint abnormalities, post-herpetic neuralgia, trigeminal neuralgia, chronic metabolic neuropathy associated with chronic pain, migraine, inflammatory pain, post-surgical pain syndromes including phantom limb pain, post-traumatic stress disorder (PTSD), irritable bowel syndrome, autonomic neuropathies, arachnoiditis, chronic regional pain syndromes, vulvodynia, and chronic pain syndromes associated with activation of central sensitization pathways.

[0109] The method can be used one or more times as a preventative measure (i.e., to avoid disease). Alternatively, a series of methods can be used any time after diagnosis. The method can be the sole treatment or can be used in conjunction with other drugs or therapies useful in treating the condition in question.

[0110] Administration Channelrhodopsin-2 (ChR2) is a nonselective cation channel used to depolarize neurons by light activation. Selective activation and silencing of neurons (ChR2) by millisecond (ms) pulses of light have been demonstrated in cell cultures, brain slices, and animals. Currently, optogenetic approaches for neuronal modulation utilizing ChR2 or halorhodopsin (NpHR, a chloride channel) require moderate light intensities, which raises the possibility of phototoxic damage to cells in the region. Multispecific opsin (MCO) is a novel optogenetic molecule with red light sensitivity. This wavelength of light propagates through tissue without attenuation or cytotoxicity, making it useful for sensitizing deep tissues. MCO combines this wavelength specificity with high sensitivity, allowing for effective stimulation with very low light intensities.

[0111] Embodiments also include vectors having a nucleotide sequence encoding the light-sensitive opsin protein described herein or any variant thereof (e.g., rhodopsin). In some aspects, the opsin is identified as SEQ ID NO: 1. Vectors that can be administered according to the present invention include vectors containing nucleotide sequences encoding DNA, RNA (e.g., mRNA), or siRNA, which, when transcribed from the vector polynucleotide, result in expression of the light-sensitive protein in the cell membrane of the target animal cell. Vectors that can be used include, for example, lentiviral vectors, HSV, adenovirus, and adeno-associated virus (AAV). Lentiviruses include HIV-1, HIV-2, VIS, VIF, and VAIE. Lentiviruses can be pseudotyped with envelope proteins of other viruses, such as VSV, rabies virus, Mo-MLV, baculovirus, and Ebola virus. Such vectors can be prepared using conventional methods in the art.

[0112] In some embodiments, the vector is a recombinant AAV vector. AAV vectors are relatively small DNA viruses that can be stably and site-specifically integrated into the genome of infected cells. They can infect a wide range of cells without inducing any effects on growth, morphology, or cell differentiation, and are not thought to be involved in human pathology. The AAV genome has been cloned, sequenced, and characterized. Its total length is approximately 47.00 bases, and it contains approximately 145 bases of inverted terminal repeat (ITR) regions at each end that function as viral replication origins. The remainder of the genome is divided into two essential regions responsible for encapsidation functions: the left part of the genome contains the rep gene, which is involved in viral replication and viral gene expression, and the right part of the genome contains the Cap gene, which encodes the viral capsid protein. AAV vectors can be prepared using standard methods in the art. The resulting AAV recombinants are then purified using standard techniques.

[0113] In some embodiments, vectors for use in the methods of the invention are packaged into viral particles (e.g., VAA viral particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the invention includes recombinant viral particles (recombinant because they contain a recombinant polynucleotide) comprising any of the vectors described herein. Methods for producing such particles are known in the art.

[0114] In some embodiments, the promoter used to drive the expression of MCO targets inhibitory neurons. Suitable promoters for such inhibitory GABAergic neurons include, for example, GAD65 / 67, VGAT, SLC32A1, GAD1, and GAD2. In some embodiments, enhancer elements are included (e.g., DLX1, DLX2, DLX5, or DLX6). Suitable promoters for satellite glial cells include Bestrophin 1, Glial Fibrillary Acidic Protein, Bcan, Fdps, Mlc1, Gja1, Ednrb, Slc1a3, Plp1, and Fabp7.

[0115] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventor anticipates that such variations will be adopted by those skilled in the art as appropriate, and the inventor also intends that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto to the extent permitted by applicable law. Furthermore, this invention includes any combination of the above-described embodiments in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0116] The therapeutic agent in the pharmaceutical composition can be formulated in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount can vary depending on the condition being treated, the severity and course of the condition, the method of administration, whether the agent is administered for prophylactic or therapeutic purposes, the bioavailability of the particular agent, the ability of the therapeutic small molecule to elicit a desired response in the individual, previous treatment history, the patient's age, weight, and sex, the patient's medical history and response to the agent, the type of therapeutic small molecule used, the discretion of the attending physician, etc. A therapeutically effective amount is also an amount in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

[0117] In aspects of this embodiment, the methods of the invention reduce the occurrence of pain by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In still other aspects of this embodiment, the methods disclosed herein provide for reducing pain occurrence by, for example, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, or about 10% to about 80%. The reduction is about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0118] Aspects of the present specification disclose, in part, treating an individual susceptible to pain or suffering from pain (acute or chronic). As used herein, the term "treating" refers to reducing or eliminating pain; or reducing or depleting the occurrence of pain. For example, the term "treating" can mean reducing pain by, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Those skilled in the art will know the appropriate symptoms or indicators associated with a particular type of disease and will know how to determine whether an individual is a candidate for the treatments disclosed herein.

[0119] In aspects of this embodiment, the methods disclosed herein reduce pain by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In other aspects of this embodiment, the methods disclosed herein reduce pain by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In still other aspects of this embodiment, the methods herein reduce pain by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0120] method Generation of GAD67-MCO2-mCherry and packaging into viral vectors The pAAV-GAD67-MCO2-mCherry plasmid was designed by Nanoscope Technologies LLC. The plasmid was packaged into AAV5 (hereafter referred to as pMCO2) using the triple transduction method.

[0121] Mouse model C57BL / 6J (wild-type) male and female mice were obtained from Jackson Laboratory. Experimental mice were maintained on a 12:12 light cycle in strict compliance with IACUC guidelines for the use of animals in research. Mice were housed and experimented on humanely.

[0122] Fiber optic stimulation implants and transduction of ACC with AAV-MCO2 in wt mice Aseptic technique was used for all surgical procedures, and surgical instruments were sterilized by autoclaving. Wild-type (wt) mice were anesthetized with 2-3.5% isoflurane, and the fur in the target area was chemically removed. A midline incision was made, and the skin in the ACC region was removed. A burr hole was created over the ACC (0.7 mm anterior to bregma, 0.4 mm lateral to the midline, and 1.8 mm deep from the skull surface), and a 1.5 mm long implant was placed and secured to the skull with cyanoacrylate and dental cement. At this point, AAV-MCO2 was injected into the ACC (dose-dependent) using a syringe. Mice were maintained with the implant for 2 weeks to allow for appropriate expression before use in experiments. A novel Bluetooth®-controlled, dorsal-mounted optogenetic stimulation device activated the ACC via this permanently implanted cannula.

[0123] Transduction of DRG with AAV-borne MCO2 in wt mice Aseptic technique was used for all surgical procedures, and surgical instruments were sterilized by autoclaving. Wild-type (wt) mice were anesthetized with 2-3.5% isoflurane, and the fur of the target area was chemically removed. MCO2 loaded onto AAV was delivered intrathecally.

[0124] Spinal transfection with laser-delivered pMCO2 in wt mice Aseptic technique was used for all surgical procedures, and surgical instruments were sterilized by autoclaving. Wild-type (wt) mice were anesthetized with 2-3.5% isoflurane, and the fur in the target area was chemically removed. A syringe was aseptically filled, and anesthetized animals received a single intrathecal injection of gold nanorods (GNRs) mixed with the MCO plasmid. 10 μL of the appropriate transfection solution was drawn into a 20 μL Hamilton syringe. Convection-enhanced delivery infusion was performed through the spine at a flow rate of approximately 1 μl / min for approximately 10 minutes. After the procedure was completed, the solution was left in place to infiltrate the spine for 2 hours. The spine was then illuminated with 1080 nm light.

[0125] Formalin assay Mice were injected with 20 microliters of 1% formalin and placed in a holding chamber. At 5-minute intervals, mice were observed for 1 minute each for paw lifting and paw licking. The total time in seconds during each observation during which this behavior was observed was recorded. This continued for 45 minutes. Pain scoring was performed according to the following formula: ((2 * Licking time) + (1 * Limb raising time)) / 60 This was repeated in implanted wt mice with and without stimulation (2 Hz or 5 Hz at approximately 630 nm on various schedules) to determine statistically measurable differences in activity. The experimental design is outlined in Figure 4A.

[0126] Chronic pain induced by sciatic nerve ligation Mice were anesthetized with isoflurane or an intraperitoneal injection of 4 ml / kg of a mixture of ketamine (17 mg / ml) and xylazine (2.5 mg / ml) in sterile phosphate-buffered saline (PBS). Fur was chemically removed from one hind limb. The surgical area was cleaned with 70% ethanol. A muscle-sparing incision was made along the sciatic vein between the semitendinosus and biceps muscles to expose the sciatic nerve. The two muscles were gently spread apart to expose 1.5 cm of the sciatic nerve. The main branch of the sciatic nerve was elevated using a sterile glass hook. The sciatic nerve was hydrated with sterile PBS. The main branch of the sciatic nerve was tightly ligated using 4-0 silk suture. The muscle was then sutured using 4-0 suture. The overlying skin was closed using staples. Mice were allowed to recover on a heating pad. By the day after ligation, and for several months thereafter, mice develop guarding behavior of the ipsilateral hind paw in response to mechanical stimuli (lifting the affected limb), a common model of neuropathy-induced pain. This mechanical allodynia is accompanied by thermal hyperalgesia, which lasts approximately 3 weeks.

[0127] Conditioned Placement Preference Assay Preconditioning phase: All mice used for the study underwent sciatic nerve ligation. During this phase (days 1-3), the guillotine door was removed to allow free access to the entire apparatus. Experimental mice were placed in the central chamber. Free movement of each experimental animal through the chambers was recorded for 15 minutes. The time spent in the two side chambers on day 3 was used as the baseline preference. Mice that entered either side chamber less than four times were excluded from the study.

[0128] Conditioning phase: During conditioning (days 4-9), mice were confined to either the treatment (light-on) or the light-off (non-treatment) chamber for 45 minutes. Ten hours later, each mouse was confined to the chamber opposite the one from that morning's association session for 45 minutes. While in the treatment-associated chamber, mice received optogenetic stimulation at a rate of 5 Hz. While in the non-treatment paired chamber, a Bluetooth™ stimulation device was placed on the mouse but not activated.

[0129] Test phase: During testing, mice were placed in the center aisle and allowed free access to the entire apparatus. Their activity was recorded for 15 min during exploration. These post-conditioning tests were conducted on days 3, 6, and 10 after conditioning. During live testing, mice were placed in the middle aisle as usual, but optogenetic stimulation was turned on when the mice entered the lit chamber and turned off when the mice entered the middle aisle or the unlit chamber.

[0130] The percentage of time spent in either chamber ("chamber time") across the 15-minute test session was calculated for each time point. The overall experimental overview and CPP apparatus are shown in Figure 7B.

[0131] Von Frey assay For each experimental mouse, a baseline mechanical threshold for withdrawal responses was established using an ascending or descending presentation method. After an acclimation period of at least 30 min, the test phase began, and optogenetic stimulation, if applicable, was initiated. Starting at 0.6 g, the hindpaw was stimulated with a Von Frey filament (pressing the tip against the plantar surface until it bent). This presentation was repeated five times, and the presence or absence of a response was recorded. A 2-min interval was allowed between presentations. If at least three out of five results were positive, a lower filament was used; if fewer than three out of five results were positive for withdrawal, the next higher filament was used. This continued until a minimum threshold of at least three out of five positive results was established. After sciatic nerve ligation (see above), mouse hindpaws were tested in a similar manner. For timed treatments, the procedure was adapted to a standard "up-down" method, where the next higher or lower filament was presented (respectively) immediately after testing (with an approximately 1-min interval), depending on whether the withdrawal response was negative or positive. This continued until at least three presentations were positive for a particular filament. This was necessary because the usual 2 min time frame was not sufficient to complete the test. The overall experimental process and Von Frey assay setup are shown in Figure 6A and Figure 6B, respectively.

[0132] Immunostaining of tissues for immunogenicity and viability assays Mice were sacrificed, their brains removed, and immersed in PFA for 8 hours. They were then transferred to 30% sucrose (w / v) and stored until cryoprotected. The brains were then sectioned, and the collected sections were stained with either anti-IBA1, anti-CD45, anti-Gad65 (1:100), or anti-caspase (1:250) primary antibodies, as well as anti-MCherry (1:250 or 1:500). After overnight incubation with the primary antibodies, secondary antibodies (1:500) were added. Finally, each slide was stained with DAPI, and a coverslip was placed over the sample. Each sample was then imaged using a confocal microscope.

[0133] statistical analysis Data collected from experimental rodents were analyzed using GraphPad Prism 9.3 (Windows, GraphPad Software, San Diego, CA, USA). Statistical analysis was performed by parametric T-test. [Example]

[0134] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the representative embodiments contemplated herein. These examples are intended to be merely a subset of all possible contexts in which the components of the formulations may be combined. Thus, these examples should not be construed as limiting any of the embodiments described herein, including the types and amounts of the components of the formulations and / or their methods and uses.

[0135] Example 1 Wireless optogenetic stimulation device enables pain modulation in freely moving animals using minimally invasive implants Optogenetic stimulation devices enable freely mobile optogenetic modulation of pain using minimally invasive implants that do not significantly affect pain responses. Figures 2A-2B show a minimally invasive wireless optogenetic stimulation device. This device enables freely mobile optogenetic modulation of pain without significantly affecting pain responses. Figure 2A is an image of a Bluetooth®-enabled optogenetic stimulation device for wireless control of optogenetic modulation of pain. Figure 2B is an image of an optical fiber emitting low-power red (i.e., 635 nm) light. Although the optical fiber emits low-power red light, it is minimally invasive because the red light propagates within brain tissue. Figure 2C is a sagittal section image of a mouse brain, demonstrating minimal invasiveness of the optical fiber (indicated by the arrow) in the dorsal aspect of the ACC. Figure 2D shows the normalized transmittance spectrum of visible light through a 1 mm-thick brain slice. This is a graph depicting the propagation of red light through brain tissue. Minimal absorption is observed above 630 nm. Figure 2E demonstrates that the presence of the fiber implant did not significantly affect acute pain responses in the formalin assay, as measured by quantifiable (ns: not significant) behaviors such as paw licking (Figure 2F) and paw lifting (Figure 2G).

[0136] Example 2 MCO2-mCherry colocalization in ACC GABAergic neurons To examine MCO2 expression in the anterior cingulate cortex (ACC), sections from pMCO2-transduced brains were immunostained using cell-specific antibodies along with GFP antibodies. Analysis of the sectioned brain tissue demonstrated that ACC transduction colocalized with GAD65-labeled cells. Two weeks after pMCO2 injection, endogenous mCherry fluorescence imaging of GAD67 promoter-driven MCO2 expression in the ACC was performed. The outline of the optical fiber is indicated by the arrow. Figure 3A shows GAD65- and mCherry-positive labeled cells in the ACC. Figure 3B shows an image of a wireless fiber-coupled red LED implanted in an MCO-transfected mouse.

[0137] Figures 3C-3E are confocal immunostaining images of mouse brain sections. Figure 3C shows DAPI (nuclear staining). Figure 3D shows GAD65, a marker for GABAergic neurons, and Figure 3E shows the multispecific opsin (MCO) reporter, mCherry. Figure 3F shows an overlay of GAD65 and mCherry. Analysis of sectioned brain tissue showed that ACC transduction colocalized with GAD65-labeled cells. Magnified areas are indicated by rectangles in Figures 3G and 3H. Images show high-magnification coexpression images of ACC and colocalization of GAD65 and mCherry.

[0138] Example 3 Acute inflammatory pain responses are attenuated by optogenetic stimulation with MCO transduction of GABAergic neurons in the ACC The following example involves testing mice for their response to pain using optogenetic stimulation. Figure 4A is a flowchart of the experimental design for assessing the effects of optogenetic modulation on an acute pain model (i.e., formalin injection into the hind paw). Baseline formalin-induced pain scores at 10-min intervals were compared to scores during continuous 5 Hz optogenetic stimulation (5 ms pulses at 630 nm). Figure 4B shows results 11 days after early transduction; Figure 4C shows results 5 weeks after optimal transduction. The chart depicts the mean cumulative pain scores (measured at 5-min intervals) during the early (0-11 min) non-inflammatory phase of pain and the late (20-41 min) inflammatory phase. Figure 4D shows results 11 days after early transduction; Figure 4E shows results 5 weeks after optimal transduction. Mean ± standard error (SEM). N = 7 for baseline and N = 4 for the MCO-transduced group (mice with incorrectly placed fibers were excluded). *p < 0.05.

[0139] Example 4 Optogenetic stimulation of GABAergic neurons in the ACC reduced acute pain behavior Untreated mice showed higher overall pain scores compared to intermittently treated mice. During the formalin assay, experimental mice received 5 Hz optogenetic stimulation on a 5-min on-off schedule (Figures 5A-5D). The effect of this treatment schedule on the late phase of pain was reduced compared to the late phase pain response 5 weeks after transduction with constant treatment (87.4292% reduction to 45.5202%).

[0140] Results of the formalin test are shown in Figure 5A-5F. Baseline formalin-induced pain scores at 10-min intervals were compared with the following: (Figure 5A) 5 Hz optogenetic stimulation (5 ms pulses at 630 nm) with a 5-min on-off interval; (Figure 5B) 2 Hz optogenetic stimulation with a 5-min on-off interval; (Figure 5C) 5 Hz optogenetic stimulation starting 15 min after the start of the experimental session. Mean cumulative pain scores (measured at 5-min intervals) during the early (0-11 min) non-inflammatory phase of pain and the late (20-41 min) inflammatory phase were compared with the following: (Figure 5D) intermittent 5 Hz optogenetic stimulation; (Figure 5E) intermittent 2 Hz optogenetic stimulation; (Figure 5F) delayed 5 Hz optogenetic stimulation. Mean ± SEM. N = 7 for baseline and N = 4 for MCO-transduced groups (mice with incorrectly placed fibers were excluded). *p < 0.05.

[0141] Example 5 Mechanical hyperalgesia and allodynia are attenuated in chronic pain models by MCO-based optogenetic stimulation of GABAergic neurons in the ACC Mechanical hyperalgesia and allodynia are attenuated by MCO-based optogenetic stimulation of the ACC compared to baseline thresholds. Figure 6A shows a flowchart of the experimental design for the mechanical hypersensitivity chronic pain experiment. Figure 6B shows an image of a manual Von Frey apparatus presenting a Von Frey filament to the hind paw of a baseline mouse. The inset shows the Von Frey filament contacting the mouse's paw. Figure 6C shows that the minimum force required to elicit a hind paw withdrawal response to a mechanical stimulus increases with increasing duration of exposure. Figure 6D shows the percentage increase or decrease in withdrawal force relative to the untreated baseline threshold of the mice. N=4. Mean ± SEM. **=p<0.05, **=p<0.01.

[0142] Example 6 Conditioned place preference responses to implanted and MCO-based optogenetic stimulation Figure 7A is a flowchart of the experimental design for the chronic pain experiment. Figure 7B is an image of the CPP apparatus showing the dark (untreated) chamber (left), the center chamber, and the lit (treatment-associated) chamber (right). Figure 7C is a graphical comparison of chamber preference between naive unimplanted and implanted mice in the CPP apparatus. There was no statistically significant change in preference between unimplanted and ACC-implanted mice. N=5. Figure 7D shows an increase in the percentage of time spent in the treatment-associated chamber after conditioning. Despite rodents' instinctive preference for the dark area, preference increased toward the treatment-associated chamber after conditioning (while maintaining preference for the dark chamber). However, during live reinforcement, preference for the treatment-associated chamber significantly increased. Mean ± SEM. N=4, **p<0.01.

[0143] Example 7 Long-term safety of optogenetic implantation and stimulation Figure 8A shows the temperature change of the irradiated spot in a living brain measured with an IR camera. Inset: Thermal image of a mouse under red LED light stimulation. Figure 8B is a series of images showing no loss of viability of ACC-GABAergic neurons expressing MCO2-mCherry after chronic optogenetic stimulation with i) DAPI, (ii) MCO2-mCherry, and (iii) caspase-3. This is also shown in the overlay (iv) of images (i), (ii), and (iii). After eight sessions of 1 h each (3 ms, 5 Hz, 0.4 mW), no apoptotic (caspase-positive, green) cells were observed. Figure 8C shows the results of (i) DAPI, (ii) immunostaining for mCherry confirming MCO2 expression, and (iii) administration of 3 ml of AAV-MCO2 (8 × 10 12 The absence of CD45 immunostaining (green) in the ACC area injected with AAV-MCOII (vg / ml) indicates that injection of AAV-MCOII resulted in MCO expression without causing an inflammatory response. This is also shown in the merged image (iv) of (i), (ii), and (iii). Figure 8D shows the absence of immune cell response to the implant in the MCO-mCherry-transfected ACC. (i) DAPI, (ii) MCO-mCherry, (iii) Iba1 (a microglia / macrophage marker). This is also shown in the merged image (iv) of images (i), (ii), and (iii). Minimal Iba1-positive (green) cells were observed near the implant.

[0144] Changes in mechanical allodynia, as reflected in the Von Frey assay, also indicate a role for the ACC in pain. Notably, while mechanical thresholds are immediately elevated, significant reductions in mechanical allodynia are only evident 20 minutes after stimulation. Therefore, we infer that a strong direct inhibition of sensation does not occur; rather, there is some evolving regulation of how that input is interpreted and processed in the CNS. The comparable sensitivity of mechanical thresholds in the treated and unligated limbs is encouraging, in that the goal is not to numb pain to the point of potential danger, but rather to reduce chronic pain to a manageable level that allows for the return of normal, beneficial function.

[0145] The use of optogenetic stimulation allows for effective pain modulation in a cell-specific and highly customizable manner, which is suitable for addressing the diversity of pain experiences. In our current study, we found that central methods enable us to address intractable pain syndromes of multiple etiologies and symptomologies with a single implant. This is reflected in the attenuation of reflex pain responses in mice undergoing optogenetic-based anterior cingulate cortex stimulation. The results indicate that the stimulation schedule (i.e., the frequency of stimulation sessions and the intensity of those sessions) correlates with the level of acute pain reduction, as an intermittent treatment schedule showed higher mean pain scores than constant stimulation. Furthermore, optogenetic stimulation-mediated reduction of chronic neuropathic pain motivated increased time spent in a lit chamber, which is normally aversive. This supports the value of optogenetic methods as an alternative to traditional pharmacological methods.

[0146] Example 8 Effects of delayed optogenetic stimulation on pain modulation Delayed optogenetic treatment (stimulation of MCO-transduced inhibitory neurons in the ACC) still demonstrated a statistically significant reduction in pain scores during the inflammatory phase of the formalin assay (Figure 9).

[0147] Figures 9A and 9B show that even delayed (starting 15 min after the start of the experimental session) treatment with 5 Hz (630 nm, 5 ms pulses) optogenetic stimulation attenuates the inflammatory pain response. Figure 9A shows the results of formalin assay scores at 10-min intervals comparing untreated baseline and treated experimental mice. Figure 9B shows the mean cumulative pain scores at 5-min intervals during the early (0-11 min) and late (20-41 min) phases of the experimental session in delayed-treated mice. Mean ± SEM. N = 7 for baseline, N = 4 for treatment. *p < 0.05.

[0148] Example 9 Persistence of pain modulation after cessation of optogenetic stimulation The analgesia effect persisted in a chronic pain (sciatic nerve ligation) model even after cessation of CNS optogenetic stimulation. Typical mechanical hyperalgesia in the affected limb remained compensated long after optogenetic stimulation was terminated. Mechanical thresholds remained elevated from pre-stimulation baselines after a single 20-minute optogenetic stimulation (Figures 10A and 10B).

[0149] Figures 10A and 10B show the durability of the modulatory effects of MCO-based optogenetic stimulation of the CNS. Figure 10A shows the force (g) required to induce hyperalgesic paw withdrawal (escape response) in experimental mice after cessation of optogenetic stimulation (20 min, indicated by the red bar). Figure 10B shows the percentage of the experimental population that maintained an elevated mechanical sensitivity threshold after optogenetic stimulation. N=6, mean ± SEM.

[0150] Example 10 Opsin expression and optogenetic stimulation of the ACC do not alter social behavior CNS implantation and MCO expression did not significantly affect typical social novelty behavior in experimental mice. Both wild-type (non-implanted) and implanted mice (with MCO transfected into the CNS) showed similar affinities for novel social interactions (Figure 11).

[0151] Figures 11A-11C show that CNS MCO transfection-implantation does not affect social interactions. Figure 11A shows an image of a three-chamber social novelty apparatus. Preference for novel stimuli results in a statistically significant difference in the time spent with a previously introduced stranger versus a completely new stranger in both (Figure 11B) wild-type controls and (Figure 11C) mice with CNS MCO expression. N=4. Mean ± SEM. **p<0.01, *p<0.05.

[0152] Example 11 Pain modulation by optogenetic stimulation of DRG sensitized by virally delivered MCO As shown in Figure 12A, significant pain suppression was observed with optical stimulation of the spine (after intrathecal delivery of a plasmid encoding MCO). A typical formalin test shows early (Phase 1) and late (Phase 2) pain responses, reflecting direct pain and inflammatory pain, respectively. Furthermore, we modeled chronic neuropathic pain using a peripheral nerve injury model, and optogenetic stimulation compensated for the hyperalgesia seen in these models, as reflected by a statistically significant increase in the minimum force required to elicit a paw withdrawal response in the affected limb (Figure 12B).

[0153] Figures 12A and 12B show that acute pain responses are attenuated by continuous 5 Hz treatment (5 ms pulses at 630 nm). Figure 12A shows that quantifiable behavioral pain responses during a formalin assay at 5-minute intervals with continuous spinal optogenetic stimulation reflect this change. Figure 12B shows that the minimum force required to elicit a hind paw withdrawal response (escape response) to mechanical stimulation was increased by spinal optogenetic stimulation. Mean ± SEM. N=13 for untreated group and N=4 for spinal treatment group. *p<0.05.

[0154] Example 12 Opsin expression in the DRG does not alter social behavior Spinal expression of MCO similarly had no significant effect on spontaneous alternation behavior in the Y-maze. Comparison of wild-type mice with MCO-expressing mice (intrathecally injected with MCO-carrying AAV) showed no significant difference in their typical exploratory activity through the Y-maze (Fig. 13).

[0155] Figures 13A and 13B show the results of spontaneous alternation behavior in a Y-maze. Figure 13A is an image of an open Y-maze with labeled arms. Figure 13B shows a comparison of spontaneous alternation rates between wild-type mice and MCO-expressing mice. N=4. Mean ± SEM.

[0156] Example 13 Suppression of pain responses persists after early termination of optogenetic stimulation Optogenetic stimulation of the anterior cingulate cortex resulted in a significant attenuation of the mean pain response, even after stimulation ended. Comparison of wild-type mice with MCO-expressing mice (injected with MCO-carrying AAV into the ACC) showed a striking difference in pain responses after cessation of optogenetic stimulation.

[0157] Figures 14A and 14B show that acute pain responses remain attenuated after termination of optogenetic stimulation. A) Formalin assay scores at 10-minute intervals with 5 ms pulses (630 nm) comparing baseline and treated experimental mice that received 20 minutes of 5 Hz treatment (treatment was discontinued). C) Mean cumulative pain scores at 5-minute intervals during the early (0-11 minutes) and late (20-41 minutes) phases of the experimental session in mice that had treatment discontinued. Mean ± SEM. N=7 for baseline, N=6 for treatment. *p<0.05

[0158] Example 14 Treating sciatica and lower back pain using optogenetic stimulation Targeting central nervous structures that play an essential role in pain perception avoids many of the drawbacks and challenges inherent in conventional approaches related to electrode implantation and non-specificity. Optogenetic modulation addresses these drawbacks by enabling cell-specific stimulation at low power. Illumination occurs at low power loads and can be tuned to respond to wavelengths that better propagate through tissue without damaging cells.

[0159] Sciatica refers to pain that radiates along the path of the sciatic nerve. It begins along the sciatic nerve and radiates down the buttocks and back of the thigh. It is usually caused by a herniated (or bulging) disc in the spine that presses on the sciatic nerve. The pain can range from a mild ache to a sharp, burning pain. Sometimes it feels like a shock or electric shock.

[0160] In this case, the patient (i.e., a 50-year-old man) presented to a medical institution complaining of persistent pain, tingling, and muscle weakness radiating from his lower back down to his extremities. The patient was otherwise in good health and wished to avoid taking pain medications. Medical professionals recommended a treatment regimen involving optogenetic modulation of the CNS to relieve sciatica.

[0161] Multispecific opsin (MCO2) is packaged into adenoviral, adeno-associated viral, or lentiviral vectors and injected into the patient's neural cells (i.e., cells in the spinal cord, DRG, and / or ACC). Two to four weeks after injection, healthcare providers confirm expression in the target cells.

[0162] The patient is also given an implanted optical fiber that serves as a light source for delivering light to the target cells. In this example, the light source is activated via a wireless signal. Furthermore, the light source is powered wirelessly (e.g., via inductive charging), thus avoiding the need for a built-in or connected power source.

[0163] The light-emitting diode (LED) is controlled to generate pulses with pulse widths between 1 and 100 milliseconds, with a duty cycle between 1 and 100 percent. The LED is positioned to illuminate the target area, and the tip is flat or tapered to control the shape of the diverging light beam. The intensity of the light emitted from the light source or a waveguide coupled to the light source is 1 mW / mm. 2 ~100mW / mm 2 The range is.

[0164] The light source generates pulses of light when activated by the patient (or healthcare provider) or by a pre-set program. The light activates the transfected neurons using visible light. Light-induced activation of multispecific opsin (MCO) depolarizes only cells that express MCO2.

[0165] The patient is then evaluated for sciatica. The patient reports that nearly all (approximately 90%) of the pain has subsided. The patient is advised to continue a healthy lifestyle and activate the fiber optics for pain as needed. * * *

[0166] The dosage and frequency of administration of the pharmaceutical compositions disclosed herein are determined by the type of active ingredient, as well as various factors such as the disease to be treated, the administration route, the age, sex, and weight of the patient, and the severity of the disease. Furthermore, the pharmaceutical composition can be administered alone or in combination with or simultaneously with other pharmaceutical preparations that exhibit prophylactic or therapeutic effects.

[0167] In various embodiments, the formulations can include, but are not limited to, combinations of bioactive agents (e.g., viruses, proteins, antibodies, peptides, etc., as described herein) in the formulation. For example, the formulations described herein can include a single bioactive agent for the treatment of one or more conditions, including, but not limited to, a disease. The formulations described herein can also, in one embodiment, include, but are not limited to, two or more different bioactive agents for single or multiple conditions. The use of multiple bioactive agents in a formulation can be, for example, for the same or different indications. Similarly, in another embodiment, multiple bioactive agents can be used in a formulation to, for example, treat both a disease state and one or more side effects caused by a primary treatment. In further embodiments, multiple bioactive agents may be included in the formulations described herein to achieve different medical goals, such as, but not limited to, the simultaneous treatment and monitoring of the progression of a disease state. In further embodiments, multiple simultaneous therapies, such as those exemplified herein, as well as other combinations known in the art, are particularly useful for patient compliance, as a single formulation may be sufficient for some or all of the proposed treatments and / or diagnoses. Those of skill in the art are familiar with bioactive agents that can be combined for a wide range of combination therapies. Similarly, in various embodiments, the formulations can be used in combination with small molecule drugs, and can be used in combination with one or more biologically active agents and one or more small molecule pharmaceuticals. Thus, in various embodiments, formulations are provided that include one, two, three, four, five, six, or more different biologically active agents, as well as formulations that combine one or more biologically active agents with one or more small molecule pharmaceuticals.

[0168] Packaging and equipment for administration may be determined by various considerations, such as the amount of substance to be administered, storage conditions, administration by skilled medical personnel or patient self-administration, dosing regimen, geopolitical environment (e.g., exposure to extreme temperature conditions in developing countries), and other practical considerations.

[0169] Injection devices include pen-type injectors, auto-injectors, safety syringes, injection pumps, infusion pumps, glass pre-filled syringes, plastic pre-filled syringes, and needleless syringes, and syringes can be pre-filled with liquid or can be dual-chambered, for example, for use with lyophilized substances.One example of a syringe for such use is Lyo-Ject®, a dual-chamber pre-filled lyosyringe available from Vetter GmbH (Lavenburg, Germany).Another example is LyoTip, a pre-filled syringe designed to conveniently deliver lyophilized formulations, available from LyoTip, Inc. (Camarillo, California, USA).Injection administration can be, but is not limited to, intravenous, intramuscular, intraperitoneal, or subcutaneous administration as needed.Non-injection administration can be, but is not limited to, nasal, oral, ocular, cutaneous, or pulmonary administration as appropriate.

[0170] In certain embodiments, the kit may include one or more single-chamber or multi-chamber syringes (e.g., liquid syringes and lyosyringes) for administering one or more formulations described herein. In various embodiments, the kit may include formulation components for parenteral, subcutaneous, intramuscular, or IV administration, sealed in a vial under partial vacuum in a form ready for loading into a syringe and administration to a subject. In this regard, the composition may be placed therein under partial vacuum. In all of these and other embodiments, the kit may include one or more vials according to any of the foregoing, each vial containing a single unit dose for administration to a subject.

[0171] The formulations described herein can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures.

[0172] Also provided herein are combinatorial methods for developing suitable viral formulations using combinations of amino acids. These methods are useful for developing stable liquid or lyophilized formulations, particularly pharmaceutical viral formulations.

[0173] Compositions according to embodiments described herein have desirable properties such as solubility, viscosity, syringability, and stability. Lyophilizates according to embodiments described herein also have desirable properties such as reconstitution, stability, and reconstitution.

[0174] In certain embodiments, the method reduces pain (eg, neuropathic pain or chronic pain) by 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% or more.

[0175] In certain embodiments, the duration of the treatment methods described herein is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In further embodiments, the period of time for which administration is discontinued is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0176] In aspects of this embodiment, the methods of treatment described herein reduce signs / symptoms in an individual suffering from pain by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In other aspects of this embodiment, the methods of treatment disclosed herein reduce signs / symptoms such as pain, for example, by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In still other aspects of this embodiment, the methods of treatment disclosed herein reduce signs / symptoms, such as pain, by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0177] In other embodiments, the methods of treatment disclosed herein reduce the level of pain (e.g., neuropathic pain, nociceptive pain, or chronic pain) in an individual by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In other aspects of this embodiment, the methods of treatment disclosed herein reduce pain by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In still other aspects of this embodiment, the methods of treatment disclosed herein reduce pain (compared to an untreated subject) by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0178] Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein, the term "about" means that the feature, item, quantity, parameter, characteristic, or term so identified encompasses a range of ±10 percent above and below the value of the stated feature, item, quantity, parameter, characteristic, or term. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be construed, at least in light of the number of reported significant digits and with ordinary rounding techniques applied. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise stated herein, each separate value within a range is incorporated herein as if it were individually set forth herein.

[0179] The grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification includes the group as modified and is therefore deemed to satisfy the description of all Markush groups used in the appended claims.

[0180] Finally, while aspects of the present specification are emphasized by reference to specific embodiments, those skilled in the art will recognize that these embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methodology, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or variations of the disclosed subject matter, or alternative configurations, can be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

[0181] TIFF2026507627000002.tif168170

Claims

1. 1. A method of inhibiting pain in a subject suffering from pain, said method comprising the steps of: a) expressing a recombinant nucleic acid encoding a light-sensitive protein in a neuronal cell of the subject; and b) controlling the expression of the light-sensitive protein and modulating the neural activity of the cells with light wherein said modulating reduces the duration and / or intensity of pain.

2. 2. The method of claim 1, wherein the neuronal cells are spinal cord cells, dorsal root ganglion (DRG) cells, and / or anterior cingulate cortex (ACC) cells.

3. The method of claim 1, wherein the light-sensitive protein is multispecific opsin (MCO).

4. The method of claim 2, wherein the multispecific opsin (MCO) has at least 80% identity to SEQ ID NO:

1.

5. 10. The method of claim 1, wherein the pain is one or more of neuropathic pain, inflammatory pain, headache, somatic pain, visceral pain, and referred pain.

6. 6. The method of claim 5, wherein the neuropathic pain is chronic neuropathic pain.

7. 6. The method of claim 5, wherein the neuropathic pain is caused by one or more of a traumatic insult, spinal cord injury, limb amputation, contusion, inflammatory or surgical procedure, ischemic event, exposure to an infectious agent, a toxic agent, or disease.

8. 10. The method of claim 1, which does not affect nociceptive pain.

9. 2. The method of claim 1, wherein a viral vector is used in the step of expressing a recombinant nucleic acid encoding a light-sensitive protein in the subject's neural cells.

10. 10. The method of claim 9, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AA) vector.

11. 10. The method of claim 9, wherein the viral vector targets inhibitory neurons in the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC) of the subject.

12. 10. The method of claim 1, further comprising the step of implanting an optical fiber into the subject to control expression of the light-sensitive protein.

13. The method of claim 12 , wherein the optical fiber is activated from a wireless signal.

14. The method of claim 12, wherein the optical fiber is either electrically charged and embedded or wirelessly powered.

15. The method of claim 12 , wherein the light is a low-power red light.

16. The method of claim 12 , wherein the light has a wavelength of about 635 nm.

17. The method of claim 1 , wherein the step of controlling cellular neural activity with light comprises exposing the subject to light through an optical fiber.

18. 1. A method of inhibiting neuropathic pain in a subject without affecting nociceptive pain, said method comprising the steps of: a) expressing a recombinant multispecific opsin (MCO) in neural tissue of a subject; and b) controlling the MCO with light to regulate neural tissue activity wherein expression of MCO reduces the duration and / or intensity of neuropathic pain.

19. 19. The method of claim 18, wherein the neuropathic pain is chronic neuropathic pain.

20. 19. The method of claim 18, wherein the multispecific opsin (MCO) has at least 80% identity to SEQ ID NO:

1.

21. 19. The method of claim 18, wherein the neural tissue comprises cells of the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC).

22. 20. The method of claim 19, wherein the neuropathic pain is caused by one or more of a traumatic insult, spinal cord injury, limb amputation, contusion, inflammatory or surgical procedure, ischemic event, exposure to an infectious agent, a toxic agent, or disease.

23. The method of claim 18, wherein a viral vector is used in the step of expressing the multispecific opsin (MCO).

24. 24. The method of claim 23, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

25. 24. The method of claim 23, wherein the viral vector targets inhibitory neurons of the spinal cord, dorsal root ganglion (DRG), and / or anterior cingulate cortex (ACC).

26. 20. The method of claim 18, wherein the light is visible light.

27. 20. The method of claim 18, wherein the light is low power red light having a wavelength of about 635 nm.

28. 20. The method of claim 18, wherein the subject is exposed to light through an optical fiber.

29. 20. The method of claim 18, further comprising implanting an optical fiber in the subject to control expression of MCO.

30. 30. The method of claim 29, wherein the optical fiber is activated from a wireless signal.

31. 30. The method of claim 29, wherein the optical fiber is implanted with a charge or is wirelessly powered.

32. 30. The method of claim 28, wherein the light is low power red light having a wavelength of about 635 nm.

33. 1. A method of treating a disease in a subject in need thereof, said method comprising the steps of: a) expressing a recombinant nucleic acid encoding a light-sensitive protein in cells of the subject's neural tissue; and b) modulating the activity of neural tissue with light that controls the expression of said light-sensitive protein; wherein the modulation modulates the balance of excitation and inhibition (E / I) in nervous tissue.

34. 34. The method of claim 33, wherein the condition is a neurological disorder.

35. 35. The method of claim 34, wherein the neurological disorder is one or more of fibromyalgia, rheumatoid arthritis, osteoarthritis, chronic arthropathy, spinal nerve compression syndromes associated with neoplasms and / or herniated discs, chronic back pain, chronic joint pain of any etiology associated with inflammation and / or structural joint abnormalities, post-herpetic neuralgia, trigeminal neuralgia, chronic metabolic neuropathy associated with chronic pain, migraine, inflammatory pain, post-surgical pain syndromes including phantom limb pain, post-traumatic stress disorder (PTSD), irritable bowel syndrome, autonomic neuropathies, arachnoiditis, chronic regional pain syndromes, vulvodynia, and chronic pain syndromes associated with activation of central sensitized pathways, visual disorders, drug addiction, psychiatric disorders, and movement disorders.

36. 34. The method of claim 33, wherein the disease is neuropathic pain.

37. 37. The method of claim 36, wherein the neuropathic pain is chronic neuropathic pain.

38. 38. The method of claim 37, wherein the neuropathic pain is inflammatory pain, headache, somatic pain, visceral pain, or referred pain.

39. 34. The method of claim 33, wherein the light-sensitive protein is multispecific opsin (MCO).

40. 34. The method of claim 33, wherein the light-sensitive protein is a multispecific opsin having at least 80% identity to SEQ ID NO:

1.

41. 34. The method of claim 33, wherein a viral vector is used in the step of expressing a recombinant nucleic acid encoding a light-sensitive protein in cells of the subject's neural tissue.

42. 42. The method of claim 41, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

43. 42. The method of claim 41, wherein the viral vector targets inhibitory neurons in the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC) of the subject.

44. 42. The method of claim 41, wherein the viral vector targets GABAergic neurons in the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC) of the subject.

45. 34. The method of claim 33, wherein the light is visible light.

46. 34. The method of claim 33, wherein the light is low power red light having a wavelength of about 635 nm.

47. 34. The method of claim 33, wherein an optical fiber is used as the light source.

48. 34. The method of claim 33, wherein the durability of the treatment is maintained by adjusting light parameters (intensity, duty cycle, duration, etc.).

49. 34. The method of claim 33, wherein pain modulation has minimal or no effect on social cognitive function.

50. 1. A method of reducing pain in a subject, the method comprising the steps of: a) transfecting neural cells of a subject with a virus to express multispecific opsin (MCO); b) implanting a wireless light source in or near the subject's neurons, spinal cord, ACC, or DRG; and c) activating MCO in specific cells of the spinal cord, ACC, or DRG with light emitted from the light source, thereby reducing pain. The method comprising:

51. The light is emitted in pulses having a width of 1-100 milliseconds and a duty cycle of 1-100%, and has an output of 1 mW / mm ２ ~100mW / mm ２ 51. The method of claim 50, wherein the light intensity is adjusted to

52. 51. The method of claim 50, wherein the pain is neuropathic pain.

53. 53. The method of claim 52, wherein the neuropathic pain is chronic neuropathic pain.

54. 53. The method of claim 52, wherein the neuropathic pain is inflammatory pain, headache, somatic pain, visceral pain, or referred pain.

55. 51. The method of claim 50, wherein the multi-specificity opsin (MCO) has at least 80% identity to SEQ ID NO:

1.

56. 51. The method of claim 50, wherein a viral vector is used in the step of transfecting the neuronal cells.

57. 57. The method of claim 56, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

58. 57. The method of claim 56, wherein the viral vector targets inhibitory neurons in the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC) of the subject.

59. 57. The method of claim 56, wherein the viral vector targets GABAergic neurons in the spinal cord, dorsal root ganglion (DRG) and / or anterior cingulate cortex (ACC) of the subject.

60. 51. The method of claim 50, wherein the light is visible light.

61. 51. The method of claim 50, wherein the light is low power red light having a wavelength of about 635 nm.

62. 51. The method of claim 50, wherein the light source is an optical fiber.

63. 51. The method of claim 50, wherein the reduction in pain modulation has minimal or no effect on social cognitive function.

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