Long-lasting analgesia via targeted in vivo epigenetic repression

Abstract

To provide epigenetic based approaches and methods for use in the treatment or control of chronic pain in subjects.SOLUTION: The present invention provides a method for treating subjects having pain, using constructs comprising a zinc finger (ZF) fused with a repressor domain and / or a dCas9 fused with a repressor domain. This epigenetic approach enables transient gene therapy advantageous in the chronic pain framework, since permanent pain insensitivity is not desired.SELECTED DRAWING: None

Description

Cross - References to Related Applications

[0001] This application claims priority under 35 U.S.C.§119 to Provisional Application Serial No. 62 / 831,706, filed on April 9, 2019, and Provisional Application Serial No. 62 / 877,810, filed on July 23, 2019. For all purposes, the entire disclosures of those are incorporated herein by reference. Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under CA222826, GM123313, and HG009285 awarded by the National Institutes of Health. The government has certain rights in this invention.

Technical Field

[0003] The present disclosure provides epigenetic - based approaches and methods for treating and controlling pain in a subject in need thereof using a genome - editing construct comprising a zinc finger fused to a repressor domain and / or dCas9 fused to a repressor domain. Incorporation of Sequence Listing by Reference

[0004] A sequence listing entitled “Sequence - Listing_ST25.txt” (created on April 9, 2020, 121,918 bytes, machine - formatted on an IBM - PC, MS - Windows OS) is filed herewith. For all purposes, the entire sequence listing is incorporated herein by reference.

Background Art

[0005] Between 19% and 50% of the world's population is affected by chronic pain, and more than 100 million people in the United States alone are suffering from it. Also, due to aging and chronic diseases, the number of people complaining of chronic pain is expected to increase by 2035. Chronic pain is more common than cancer, diabetes, and cardiovascular diseases, but the development of its therapeutic agents has not advanced as significantly as in other therapeutic areas. Furthermore, current standard treatments for chronic pain often rely on narcotic substances. These may have side effects and a significant risk of addiction. Despite decades of research, a widely effective, sustainable, and non-addictive treatment for chronic pain has not yet been achieved. Summary of the Invention

[0006] The present disclosure provides a recombinant gene silencing factor complex comprising a Cas9 (dCas9) protein fused to a transcriptional repressor, associated with at least one guide RNA (gRNA), and inactivated nuclease, wherein the gRNA specifically hybridizes to a target nucleic acid sequence encoding a gene product selected from the group consisting of TRPV1 / 2 / 3 / 4, P2XR3, TRPM8, TRPA1, P23X2, P2RY, BDKRB1 / 2, Hlr3A, ACCNs, TRPV4, TRPC / P, ACCN1 / 2, SCN1 / 3 / 8A / 9A, SCN10A, SCN11A, KCNQ, BDNF, OPRD1 / K1 / M1, CNR1, GABRs, TNF, PLA2, IL1 / 6 / 12 / 18, COX-2, NTRK1, NGF, GDNF, TNF, LIF, CCL1, CNR2, TLR2 / 4, P2RX47, CCL2, CX3CR1, BDNF, NR1 / 2, GR1A1-4, GRC1-5, NK1R, CACNA1A-S, and CACNA2D1, and the expression of the gene product is inhibited. In one embodiment, the target nucleic acid sequence is located at position 2q24.3 of chromosome 2. In another or further embodiment, the gRNA comprises a sequence encoded by a sequence shown in any of 11 to 107. In another or further embodiment, the gRNA specifically hybridizes to a nucleic acid sequence encoding the SCN9A product (Nav1.7). In another or further embodiment, the transcriptional repressor is selected from the group consisting of mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Kruppel-associated box ( JPEG2025084803000001.jpg1279, KRAB). In another embodiment, the transcriptional repressor domain is a KRAB domain.

[0007] The present disclosure also provides a polynucleotide encoding one or more components of the recombinant gene repressor complex described above and herein. In one embodiment, the polynucleotide has codons optimized for expression in human cells.

[0008] The present disclosure also provides a vector or vector system comprising a polynucleotide of the present disclosure that encodes one or more components and generates the recombinant gene silencing complex of the present disclosure. In one embodiment, the polynucleotide is operably linked to a promoter. In another embodiment, the promoter is selected from the group consisting of the human cytomegalovirus (CMV) promoter, the CAG promoter, the Rous sarcoma virus (RSV) LTR promoter / transcription enhancer, the SV40 promoter, the EF1-α promoter, the CMV immediate / early gene transcription enhancer / CBA promoter, the Nav1.7 promoter, the Nav1.8 promoter, the Nav1.9 promoter, the TRPV1 promoter, the synapsin promoter, the calcium / calmodulin-dependent protein kinase II promoter, the tubulin αI promoter, the neuron-specific enolase promoter, and the glial fibrillary acidic protein (GFAP) promoter. In yet another embodiment, the vector comprises a polIII promoter upstream of at least one guide RNA coding sequence. In a further embodiment, the polIII promoter is selected from U6 and H1 promoters. In another embodiment, the vector further comprises a regulatory control sequence. In a further embodiment, the regulatory control sequence is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In another embodiment, the vector is a recombinant adeno-associated virus vector (rAAV vector). In another embodiment, the rAAV is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vectors (scAAV). In yet another or further embodiment, the polynucleotide comprises one or more inverted repeats (ITRs).In yet another or further embodiment, the polynucleotide comprises a polyA sequence. In another embodiment, the vector and the polynucleotide are engineered to be expressed intracellularly. In yet another embodiment, the vector is a lentiviral vector, a gammaretroviral vector, or a herpes simplex virus vector. In yet another embodiment, the vector comprises a split dCas9 vector system. In another embodiment, the vector comprises a nucleic acid encoding a dCas9 having a sequence as set forth in SEQ ID NO: 2 or a sequence that is at least 90% identical thereto, capable of complexing with a gRNA, and lacking nuclease activity. In yet another embodiment, the vector comprises a nucleic acid encoding a KRAB sequence of SEQ ID NO: 7 or a sequence that is at least 90% identical thereto, and capable of suppressing transcription. In yet another embodiment, the split vector system comprises a vector sequence selected from SEQ ID NO: 3, 4, and 10, or a sequence that is at least 90% to 99% identical thereto.

[0009] The present disclosure provides a construct of a zinc-finger repressor comprising an engineered zinc-finger DNA-binding domain bound to a transcriptional repressor, wherein the zinc-finger DNA-binding domain comprises 1 to 6 zinc-finger sequences, and the zinc-finger sequences bind to a target nucleic acid sequence in a gene encoding a gene product selected from the group consisting of TRPV1 / 2 / 3 / 4, P2XR3, TRPM8, TRPA1, P23X2, P2RY, BDKRB1 / 2, Hlr3A, ACCNs, TRPV4, TRPC / P, ACCN1 / 2, SCN1 / 3 / 8A / 9A, SCN10A, SCN11A, KCNQ, BDNF, OPRD1 / K1 / M1, CNR1, GABRs, TNF, PLA2, IL1 / 6 / 12 / 18, COX-2, NTRK1, NGF, GDNF, TNF, LIF, CCL1, CNR2, TLR2 / 4, P2RX47, CCL2, CX3CR1, BDNF, NR1 / 2, GR1A1-4, GRC1-5, NK1R, CACNA1A-S, and CACNA2D1, and the expression of the gene product is inhibited. In one embodiment, the target nucleic acid sequence is the sequence shown in Table 2. In yet another embodiment, the transcriptional repressor is selected from the group consisting of mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( JPEG2025084803000002.jpg1279, KRAB).

[0010] The present disclosure also provides a polynucleotide encoding the construct of the zinc-finger repressor described above and herein. In one embodiment, the polynucleotide has codons optimized for expression in human cells.

[0011] The present disclosure also provides a vector comprising a polynucleotide encoding a zinc-finger repressor construct of the present disclosure. In one embodiment, the polynucleotide is operably linked to a promoter. In a further embodiment, the promoter is selected from the group consisting of the human cytomegalovirus (CMV) promoter, CAG promoter, Rous sarcoma virus (RSV) LTR promoter / transcription enhancer, SV40 promoter, EF1-α promoter, CMV immediate / early gene transcription enhancer / CBA promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, TRPV1 promoter, synapsin promoter, calcium / calmodulin-dependent protein kinase II promoter, tubulin αI promoter, neuron-specific enolase promoter, and glial fibrillary acidic protein (GFAP) promoter. In another embodiment, the vector further comprises a regulatory control sequence. In a further embodiment, the regulatory control sequence is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In another embodiment, the vector is a recombinant adeno-associated virus vector (rAAV vector). In yet another embodiment, the rAAV is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vector (scAAV). In another embodiment, the vector or polynucleotide comprises one or more inverted repeats (ITRs). In another embodiment, the vector or polynucleotide comprises a polyA sequence. In yet another embodiment, the nucleic acid is engineered to express one or more components in a cell.In another embodiment, the vector is a lentiviral vector, a gammaretroviral vector, or a herpes simplex virus vector. In another embodiment, the vector comprises a nucleic acid encoding the KRAB sequence of SEQ ID NO:7 or a sequence that is at least 90% to 99% identical thereto.

[0012] The present disclosure also provides an epigenetically based method for treating or controlling chronic pain in a subject. The method comprises administering an effective amount of the complex or construct described above and herein.

[0013] The present disclosure also provides an epigenetically based method for treating or controlling pain in a subject in need thereof. The method includes administering to the subject an effective amount of a zinc finger-repressor construct and / or a complex of a dCas9-repressor domain. Here, the dCas9 is a catalytically inactivated Cas9 that does not cleave DNA but maintains the ability to bind to the genome via a guide-RNA (gRNA). In one embodiment, the pain is selected from neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-related neuropathy, nerve injury, rheumatoid arthritis pain, arthritic pain, burns, back pain, eye pain, visceral pain, cancer pain, bone cancer pain, migraine, pain due to carpal tunnel syndrome, fibromyalgia, neuroinflammatory pain, sciatic pain, pain of pelvic hyperalgesia, pelvic pain, pain of postherpetic neuralgia, postoperative pain, pain after stroke, and menstrual pain. In another embodiment, the pain is associated with a disease or disorder selected from the group consisting of neuropathic peripheral neuropathy, diabetic neuropathy, postprandial neuralgia, trigeminal neuralgia, back injury, cancer neuropathy, HIV neuropathy, limb loss, carpal tunnel syndrome, stroke, alcohol dependence, hyperthyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, and epilepsy. In another embodiment, the method is used to treat a subject having chronic pain. In yet another embodiment, the zinc finger-repressor construct is an mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( It comprises a repressor domain selected from the group consisting of JPEG2025084803000003.jpg1279, KRAB). In a further embodiment, the repressor domain comprises KRAB. In another embodiment, the zinc finger-repressor construct binds to the target of Table 2. In another embodiment, the complex of the dCas9-repressor domain is a mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Kruppel-associated box( It comprises a repressor domain selected from the group consisting of (JPEG2025084803000004.jpg1279, KRAB). In a further embodiment, the repressor domain comprises KRAB. In another embodiment, the construct of the dCas9-repressor domain comprises a guide RNA spacer sequence having a sequence selected from SEQ ID NOs: 11 to 106 and 107. In yet another embodiment, the construct of the zinc finger-repressor and / or the construct of the dCas9-repressor domain provides non-permanent gene repression of voltage-gated sodium channels. In a further embodiment, the voltage-gated sodium channel is selected from NaV1.7, NaV1.8, and NaV1.9. In yet another embodiment, the voltage-gated sodium channel is NaV1.7. In another embodiment, the construct of the zinc finger-repressor and / or the construct of the dCas9-repressor domain are packaged and delivered by a recombinant virus. In a further embodiment, the recombinant virus is an adenovirus, a gammaretrovirus, an adeno-associated virus (AAV), a herpes simplex virus (HSV), or a lentivirus. In a further embodiment, the recombinant virus is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 mutant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 mutant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vector (scAAV). In another embodiment, the construct of the zinc finger-repressor and / or the construct of the dCas9-repressor domain are administered intravenously, intraperitoneally, intrathecally, intraganglionically, intraneurally, intracranially, or intramuscularly.

Brief Description of the Drawings

[0014] Figures 1A-D show that in situ NaV1.7 inhibition leads to pain improvement in the carrageenan model of inflammatory pain. (A) Schematic overview of the overall strategy for in situ NaV1.7 inhibition using ZFP-KRAB and KRAB-dCas9. NaV1.7 is a DRG channel involved in the transmission of nociceptive stimuli to electrical impulses at the peripheral terminals of DRG neurons. In situ NaV1.7 inhibition via AAV-ZFP-KRAB and AAV-KRAB-dCas9 is achieved by intrathecal injection and disrupts pain signals before they reach the brain. (B) Schematic of the carrageenan-induced inflammatory pain model. On day 0, mice were intrathecally injected with either AAV9-zinc-finger-4-KRAB, AAV9-mCherry, AAV9-KRAB-dCas9-dual gRNA, or AAV9-KRAB-dCas9 without gRNA. Twenty-one days later, thermal pain sensitivity was measured for all mice using the Hargreaves test. To establish the baseline level of this sensitivity, mouse tactile thresholds were tested using von Frey filaments before carrageenan injection. Next, carrageenan was injected into the left hind paw (ipsilateral) of the mouse, and saline was injected into the right hind paw (contralateral) as an internal control in the mouse. Then, the paw withdrawal latency to heat was tested 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours after carrageenan administration. (C) In vivo NaV1.7 inhibition efficiency: Twenty-four hours after carrageenan administration, mouse DRG (L4-L6) was collected, and the NaV1.7 inhibition effect was measured by qPCR (n = 5; error bars are SEM; Student's t-test; ***p = 0.0008, **p = 0.0033). (D) The total paw withdrawal latency was calculated as the area under the curve (AUC) of both the carrageenan- and saline-injected paws. In mice treated with zinc-finger-4-KRAB and KRAB-dCas9-dual gRNA, the paw withdrawal latency of the carrageenan-injected paw was significantly increased (n = 10; error bars are SEM; Student's t-test, ****p < 0.0001).

[0015] Figures 2A-C show the evaluation criteria for in situ NaV1.7 inhibition using zinc-finger-KRAB with the established small molecule drug gabapentin. (A) Schematic of the experimental approach. (B-C) Time course of thermal hyperalgesia after injection of carrageenan (solid line) or saline (dotted line) into the hind paw of mice injected with gabapentin (100 mg / kg), mCherry, and zinc-finger-4-KRAB. The mean paw withdrawal latency (PWL) is shown. The area under the curve (AUC) of the time course of thermal hyperalgesia is plotted in the right panel. A significant increase in PWL is seen in the carrageenan-injected paw of mice injected with gabapentin and zinc-finger-4-KRAB (n = 5 for mCherry and gabapentin, n = 6 for zinc-finger-KRAB; error bars are SEM; Student's t-test, *p = 0.0208, **p = 0.0021).

[0016] Figures 3A-E show the in vivo efficacy of zinc-finger-KRAB and KRAB-dCas9 in two neuropathic pain models. (A) Schematic of the paclitaxel-induced neuropathic pain model. AAV9-mCherry, AAV9-zinc-finger-4-KRAB, AAV9-KRAB-dCas9 without gRNA, AAV9-KRAB-dCas9-dual gRNA, or saline was injected i.t. into mice. After a baseline von Frey threshold test on day 14, 8 mg / kg of paclitaxel was injected i.p. into the mice on days 14, 16, 18, and 20 after the i.t. injection. On day 21 after the i.t. injection, the mice were tested for tactile allodynia with von Frey filaments and cold allodynia with acetone application. (B) In situ NaV1.7 suppression via zinc-finger-4-KRAB and KRAB-dCas9-dual gRNA reduces paclitaxel-induced tactile allodynia. (n = 8; error bars are SEM; Student's t-test; ***p = 0.0007, ***p = 0.0004) (C) In situ NaV1.7 suppression via zinc-finger-4-KRAB and KRAB-dCas9-dual gRNA reduces paclitaxel-induced cold allodynia. (n = 8; error bars are SEM; Student's t-test; ****p < 0.0001, **p = 0.008). (D) Schematic of the BzATP pain model. On day 0, AAV9-mCherry, AAV9-KRAB-dCas9 without gRNA, or AAV9-KRAB-dCas9-dual gRNA was injected into mice. After 21 days, the baseline von Frey tactile threshold of the mice was set, and 30 nmol of BzATP was injected i.t. The mice were tested for tactile allodynia 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours after BzATP administration. (E) In the BzATP model of neuropathic pain, in situ NaV1.7 suppression via KRAB-dCas9-dual gRNA reduces tactile allodynia (n = 5 for the case without KRAB-dCas9-gRNA, n = 6 for the other groups, two-way ANOVA with Bonferroni post-test; ****p < 0.0001, *p = 0.0469).

[0017] Figures 4A-E show the long-term efficacy of zinc-finger-KRAB and KRAB-dCas9 in two independent pain models. (A) Schedule of the carrageenan-induced inflammatory pain model. (B) Time course of thermal hyperalgesia after injection of carrageenan (solid line) or saline (dotted line) into the hind paw of mice 42 days after i.t. injection of AAV9-mCherry and AAV9-zinc-finger-4-KRAB. Mean paw withdrawal latency is shown. The AUC of the time course of thermal hyperalgesia is plotted in the right panel. A significant increase in PWL is seen in the carrageenan-injected paw of mice injected with AAV9-zinc-finger-4-KRAB (n = 8; error bars are SEM; Student's t test; ****p < 0.0001). (C) Schematic of the paclitaxel-induced neuropathic pain model. (D) In situ NaV1.7 inhibition via zinc finger-4-KRAB and KRAB-dCas9-dual gRNA reduces paclitaxel-induced tactile allodynia 49 days after the final injection of paclitaxel (n = 7 for zinc-finger-4-KRAB, n = 8 for other groups, error bars are SEM, Student's t test, ****p < 0.0001). (E) In situ NaV1.7 inhibition via zinc-finger-4-KRAB and KRAB-dCas9-dual gRNA reduces paclitaxel-induced cold allodynia. (n = 7 for zinc-finger-4-KRAB, n = 8 for other groups, error bars are SEM, Student's t test, ****p < 0.0001, ***p = 0.0001).

[0018] Figures 5A-B show the in vitro optimization of an epigenetic genome engineering tool enabling NaV1.7 suppression. (A) A panel of four zinc finger proteins and ten gRNAs was designed to target NaV1.7 in a mouse neuroblastoma cell line (Neuro2a) and screened for suppression effects by qPCR. A non-targeting gRNA (no gRNA) was used as a control for the KRAB-dCas9 construct targeting NaV1.7, and mCherry was used as a control for the ZFP-KRAB construct targeting NaV1.7 (n = 3; error bars are SEM; one-way ANOVA; ****p < 0.0001). (B) In vitro NaV1.7 western blotting in Neuro2a cells transfected with mCherry, zinc-finger-2-KRAB, zinc-finger-4-KRAB, KRAB-dCas9-no gRNA, KRAB-dCas9-dual gRNA (1+2), and KRAB-dCas9-gRNA-8+10.

[0019] Figures 6A-D show that in situ NaV1.7 suppression leads to pain improvement in a carrageenan model of inflammatory pain. (A) Confirmation of AAV9-mCherry transduction in mouse DRG via RNA FISH (red = mCherry, pink = NaV1.7, green = NeuN; scale bar = 50 μm). (B) Time course of thermal hyperalgesia after injection of carrageenan (solid line) or saline (dotted line) into the hind paw of mice 21 days after i.t. injection of AAV9-KRAB-dCas9-no gRNA and AAV9-KRAB-dCas9-dual gRNA. Mean paw withdrawal latency is shown (n = 10; error bars are SEM). (C) Time course of thermal hyperalgesia after injection of carrageenan (solid line) or saline (dotted line) into the hind paw of mice 21 days after i.t. injection of AAV9-mCherry and AAV9-zinc-finger-4-KRAB. Mean paw withdrawal latency is shown (n = 10; error bars are SEM). (D) Plot of the thickness of the ipsilateral paw at baseline and 4 h after carrageenan injection (n = 10).

[0020] Figures 7A-D show the evaluation of zinc-finger-KRAB in an inflammatory pain model. (A) In vivo NaV1.7 inhibition efficiency from treated mouse DRG. Twenty-four hours after carrageenan administration, mouse DRGs (L4-L6) were collected and the NaV1.7 inhibitory effect was measured by qPCR (n = 5 in the mCherry and gabapentin groups; n = 6 in the zinc-finger-4-KRAB group; error bars are SEM; one-way ANOVA with Dunnett's post hoc test; ***p = 0.0007, *p = 0.0121). (B) The thickness of the ipsilateral foot at baseline and 4 hours after carrageenan injection was plotted. (C) Significance of the paw withdrawal latency in mice injected with AAV9-zinc-finger-4-KRAB and gabapentin (100 mg / kg) compared to the paw injected with AAV9-mCherry carrageenan (negative control) (two-way ANOVA using Bonferroni's post hoc test). (D) Single replication of the experiment in (a): Time course of thermal hyperalgesia after injection of carrageenan (solid line) or saline (dotted line) into the hind paw of mice 21 days after injection of AAV9-mCherry and AAV9-zinc-finger-4-KRAB. The mean paw withdrawal latency is shown. The AUC of the time course of thermal hyperalgesia is plotted in the right panel. A significant increase in PWL was seen in the carrageenan-injected paw of mice injected with AAV9-zinc-finger-4-KRAB (n = 8; error bars are SEM; Student's t-test; ***p < 0.0001).

[0021] Figures 8A-C show the plasmid constructs used in the methods and compositions of the present disclosure. (A) shows the plasmid construct of AAV, dNCas9 (SEQ ID NO: 3), (B) shows the plasmid construct of AAV, dNCas9-KRAB (SEQ ID NO: 10), and (C) shows the plasmid construct of AAV, dCCas9 (SEQ ID NO: 4).

[0022] Figure 9 shows the zinc-finger plasmid construct (SEQ ID NO: 5) used in the methods and compositions of the present disclosure.

[0023] Figures 10A-B show that in situ NaV1.7 inhibition restores chemotherapy-induced neuropathic pain. (A) Schematic of the treatment of a chronic neuropathic pain model induced by paclitaxel. To establish the baseline level of sensitivity, mouse tactile thresholds were tested using von Frey filaments. Then, on days 1, 3, 5, and 7, 8 mg / kg of paclitaxel was injected into the mice. On day 9, after confirming tactile allodynia with von Frey filaments, either AAV9-zinc-finger-4-KRAB (1E+11 or 1E+12 vg / mouse) or AAV9-mCherry (1E+11 or 1E+12 vg / mouse) was injected intrathecally into the mice. After 14 and 21 days, mouse tactile allodynia was tested using von Frey filaments. (B) In situ NaV1.7 inhibition via zinc-finger-4-KRAB reduces paclitaxel-induced tactile allodynia (n = 8; error bars are SEM; Student's t-test; week 2 ****p<0.0001, *p = 0.0029; week 3 ****p<0.0001, ***p = 0.0012).

[0024] Figure 11 shows genes involved in pain perception and potential modes of therapeutic intervention. Detailed description

[0025] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a zinc finger" includes a plurality of such zinc fingers, and reference to "an adeno-associated virus" includes one or more adeno-associated viruses and their equivalents known to those skilled in the art.

[0026] Also, the use of "or" means "and / or" unless specified otherwise. Similarly, "comprises", "comprising", "includes", and "including" are interchangeable and not intended to be limiting.

[0027] It should be further understood that when the term "comprising" is used in the description of various embodiments, one of ordinary skill in the art can understand that in some specific cases, the embodiments may be described alternatively with the terms "consisting essentially of" or "consisting of".

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, exemplary methods and materials are disclosed herein.

[0029] All publications mentioned herein are hereby incorporated by reference in their entirety for the purpose of explaining and disclosing the methodologies that may be used in connection with the description herein. Further, with respect to any terms presented in one or more publications that are similar or identical to terms explicitly defined herein, the definitions provided explicitly in this disclosure shall govern in all respects.

[0030] As used herein, "chronic pain" means pain characterized by the duration and / or frequency of recurrence, excluding acute pain of only limited duration without recurrence. In some cases, "chronic pain" persists for six months or more, or longer, than the course of the natural healing process associated with a particular injury, condition or disease. "Chronic pain" includes, but is not limited to, neuropathic pain, inflammatory pain, cancer pain, thermal pain, and organic pain, or combinations of two or more of the foregoing.

[0031] The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that, in its native state or when manipulated by methods well known to one of ordinary skill in the art, is said to "encode" a polypeptide. It can be transcribed and / or translated to produce the mRNA of the polypeptide and / or its fragment. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0032] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological or cellular substance, and are intended to have minimal homology while still maintaining the desired structure or functionality.

[0033] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. When the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells. The level of gene expression can be measured by measuring the amount of mRNA or protein in a cell or tissue sample. Further, the expression levels of multiple genes can be measured to establish an expression profile for a particular sample.

[0034] As used herein, the term "functionality" can be used to modify a molecule, biological or cellular substance such that it is intended to achieve a particular effect.

[0035] A "fusion molecule" is a molecule in which two or more subunit molecules are linked, typically by covalent bonds. The subunit molecules may be of the same chemical molecular species or of different chemical molecular species. Examples of the first type of fusion molecule include, but are not limited to, fusion polypeptides (e.g., a fusion between a ZFP DNA-binding domain and a transcriptional repression domain, or a fusion between Cas9 or dCas9 and a transcriptional repression domain) and fusion nucleic acids (e.g., a nucleic acid encoding the above-described fusion polypeptide).

[0036] "Gene silencing" and "inhibition of gene expression" refer to any process that results in a decrease in the production of a gene product. The gene product may be either RNA (including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) or protein. Thus, gene silencing includes processes that reduce gene transcription and / or mRNA translation. Examples of gene silencing processes that reduce transcription include those that inhibit the formation of the transcription initiation complex, those that decrease the rate of transcription initiation, those that decrease the rate of transcription elongation, those that decrease the progress of transcription, and those that antagonize transcriptional activation (e.g., by blocking the binding of transcriptional activators), but are not limited thereto. Gene silencing can include, for example, prevention of activation and inhibition of expression below existing levels. Examples of gene silencing processes that reduce translation include those that decrease the initiation of translation, those that decrease the elongation of translation, and those that decrease the stability of mRNA. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription. In general, gene silencing includes a detectable decrease in the production of a gene product. In some cases, a decrease in gene product production of about 2-fold, in other cases, about 2-fold to about 5-fold or any integer in between, in still other cases, about 5 to about 10-fold or any integer in between, in yet other cases, about 10 to about 20-fold or any integer in between, sometimes, about 20 to about 50-fold or any integer in between, in other cases, about 50 to about 100-fold or any integer in between, and in still other examples, a decrease in gene product production of more than 100-fold is included. In still other cases, gene silencing results in complete inhibition of gene expression and the gene product cannot be detected.

[0037] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized through hydrogen bonding between the bases of nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or other sequence-specific ways. The complex may include a double-stranded structure formed by two strands, three or more strands forming a multiple-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can consist of steps of a broader process such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide. Examples of stringent conditions for hybridization include an incubation temperature of about 25°C to about 37°C; a concentration of hybridization buffer of about 6x SSC to about 10x SSC, a concentration of formamide of about 0% to about 25%, and a washing solution of about 4x SSC to about 8x SSC. Examples of moderate conditions for hybridization include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9x SSC to about 2x SSC, a concentration of formamide of about 30% to about 50%, and a washing solution of about 5x SSC to about 2x SSC. Examples of highly stringent conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1x SSC to about 0.1x SSC, a concentration of formamide of about 55% to about 75%, and a washing solution of about 1x SSC, 0.1x SSC, or deionized water. Generally, the hybridization incubation time is from 5 minutes to 24 hours and includes one, two, or more washing steps, and the washing incubation time is about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. As understood, equivalents of SSC using other buffer systems can be used.

[0038] "Identity" or "sameness" or "similarity" means sequence similarity between two peptides or between two nucleic acid molecules. Identity can be measured by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching or identical positions shared by the sequences. "Unrelated" or "non-identical" sequences share less than 40% or less than 25% identity with one of the sequences of the present disclosure. Methods and algorithms available for measuring "identity" or "sameness" between two polypeptides, or between two nucleic acid molecules, are well known in the art and are available online via the World Wide Web.

[0039] As used herein, the term "isolated" refers to a molecule, biological material or cell material that is substantially free of other substances.

[0040] "Nav1.7" (also known as SCN9A, ETHA, FEB3B, GEFSP7, HSAN2D, NE-NA, NENA, PN1, SFNP, and sodium voltage-gated channel alpha subunit 9) is a sodium ion channel encoded by the SCN9A gene in humans. It is normally expressed at high levels in two types of nerve cells, nociceptive (pain-sensing) nerve cells in the dorsal root ganglion (DRG) and trigeminal ganglion / sympathetic ganglion nerve cells. These are part of the autonomic (involuntary) nervous system. The Nav1.7 channel generates currents that activate and inactivate rapidly and are sensitive to levels of tetrodotoxin. The sequence and chromosomal location of the SCN9A gene are known.

[0041] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In some instances, the disclosure provides nucleic acid sequences in the form of a sequence listing. Where the sequence listing provides a DNA sequence, the disclosure is further intended to encompass the RNA (i.e., "T" is replaced by "U" in any sequences provided herein).

[0042] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence, such as a gene. The promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. The term includes mini- and core-promoters of 50 to 500 base pairs and includes both polII and polIII promoters. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. This may include genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Examples of promoters include, but are not limited to, the CMV promoter and the U6 promoter. Suitable polIII promoters include, but are not limited to, U6 (mouse and human) and H1 (mouse and human). PolIII promoters are suitable for processing small RNA strands (e.g., shRNA, etc.). The present disclosure contemplates the use of various promoters (polII) for driving transcription of CRISPRi, CRISPRi-suppressors, etc. For example, non-specific promoters such as CMV, CAG, SV40, RSV, EF1α, etc. can be used, but are not limited thereto. Alternatively, cell-specific promoters such as Nav1.7-, Nav1.8-, and Nav1.9-specific promoters, the TRPV1 promoter, etc. can be used, but are not limited thereto. In yet another embodiment, neuron-specific promoters such as the synapsin I promoter, the calcium / calmodulin-dependent protein kinase II promoter, the tubulin αI promoter, and the neuron-specific enolase promoter can be used, but are not limited thereto.

[0043] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and, in their broadest sense, refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids and is not limited to the maximum number of amino acids that can include the sequence of the protein or peptide. As used herein, the term “amino acid” means any natural and / or non-natural, or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0044] As used herein, the term “recombinant expression system” means a gene construct for the expression of a certain type of genetic material formed by recombination. Examples of recombinant expression systems include the AAV vectors of the present disclosure that include a number of recombinant domains (see, e.g., FIGS. 8A-C and 9) for the expression of the components of the present disclosure.

[0045] As used herein, the term “subject” is intended to mean any animal. In some embodiments, the subject may be a mammal, and in further embodiments, the subject may be a cow, horse, cat, mouse, pig, dog, human, or rat.

[0046] As used herein, the term “vector” is intended to mean a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and is integrated into the genome of the target cell or remains epigenetic. The vector may be derived from a wild-type virus or be based on a wild-type virus. Aspects of the present disclosure relate to adeno-associated virus vectors (see, e.g., FIGS. 8A-C and 9).

[0047] Pain arising from somatic or nerve injury / pathology typically results from the activation of primary afferent neuronal populations characterized by an activation threshold related to tissue injury and sensitivity to products released by local tissue injury and inflammation. These afferent nerves terminate in the dorsal horn of the spinal cord. Here, the input is encoded and transmitted to the brain by long ascending pathways. In the brain, it is processed into the pain experience. The cell bodies of the primary afferent nerves are located in their dorsal root ganglia (DRGs). These neuronal cell bodies synthesize voltage-dependent sodium channels that help initiate and propagate action potentials. Local anesthetics can provide a high density of anesthesia, but in practice, as shown in previous studies, nonspecific sodium channel blockers such as lidocaine administered systemically at concentrations below the anesthetic dose have been able to produce selective effects on animal models and human hypersensitivity.

[0048] As is currently known, there are nine subtypes of voltage-dependent sodium channels and numerous splice variants. Of these isoforms, three, Nav1.7, Nav1.8, and Nav1.9, have been found to be expressed primarily in primary afferent nociceptors. The association of these isoforms with human pain is suggested by the following observations. Namely, mutations causing loss of function in Nav1.7 (SCN9A) have been observed to result in congenital insensitivity to pain (CIP), a rare genetic disorder. Conversely, gain-of-function mutations result in abnormal hyperalgesic states. Based on these observations, the Nav1.7 channel is considered an attractive target for treating pathological pain states and developing chronic pain therapies. However, efforts to develop selective small molecule inhibitors have been hampered by sequence similarity among Nav subtypes. A number of small molecule drugs targeting Nav1.7 have failed, accordingly, due to off-target specificity via the systemic route or side effects caused by lack of their bioavailability. Also, antibodies have faced a similar situation, as there is a trade-off between selectivity and efficacy by binding to the specific (open / closed) conformation of the channel, and binding is not always translated into successful channel inhibition. Furthermore, it is not clear that such antibodies can gain access to the appropriate Nav1.7 channels and produce a reliable block of their function. Thus, despite preclinical studies showing that a decrease in NaV1.7 activity leads to a reduction in inflammatory and neuropathic pain, there are still no molecules targeting gene products that have reached the final stage of clinical trials.

[0049] The present disclosure uses two genome engineering tools, namely, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR-Cas9) and zinc finger proteins (ZFPs), to genetically regulate the expression of Nav1.7 and provide the alternative approach described above, such that highly specific, persistent, and reversible pain therapies can be engineered.

[0050] The CRISPR-Cas9 system has emerged as a powerful tool for genome engineering through its ability to precisely target DNA mutations that cause disease and has shown therapeutic effects in multiple animal models of human disease. However, permanent genome editing may undesirably lead to permanent changes in pain perception. For example, while pain can be an unpleasant sensation and an emotional experience, it serves an important role in warning of tissue damage. Thus, its permanent loss can have detrimental consequences. For these reasons, in this specification, catalytically inactivated "dead" Cas9 (dCas9, also known as CRISPRi) has been used. This maintains the ability to bind to the genome via guide-RNA (gRNA) without cleaving DNA and further fuses the inactivated Cas9 to a repressor domain (Krüppel-associated box, KRAB) to enable transient gene repression of Nav1.7.

[0051] As shown herein, by adding the KRAB epigenetic repressor motif to dCas9, gene repression can be enhanced with high levels of specificity both in vitro and in vivo. This transcriptional regulatory system takes advantage of the high specificity of CRISPR-Cas9 and at the same time does not perform permanent modifications of the genome, thus increasing the safety profile.

[0052] As a second approach for in situ epigenomic Nav1.7 repression, this specification describes Cys fused to a KRAB repressor 2 His 2Zinc-finger-KRAB proteins (ZFP-KRAB) containing a DNA-binding domain consisting of zinc fingers have been utilized. ZFPs constitute the largest individual family of transcriptional regulators encoded by the genomes of higher organisms, and in a common synthetic version engineered on the human protein scaffold, they represent a potentially low-immunogenic in vivo targeting approach. The present disclosure further provides specific anatomical targeting of gene regulation by delivering epigenetic tools (described herein) of both adeno-associated virus (AAV) constructs (e.g., AAV1-9, rh.8, rh.10, rh.39, and rh.43) into the intrathecal space of the spinal cord. Notably, many AAVs have been shown to produce robust transduction of dorsal root ganglia. This approach has several advantages as it can use minimal viral payload and reduce the potential for systemic immunogenicity.

[0053] The terms “CRISPR system,” “Cas system,” or “CRISPR / Cas system” refer to a set of molecules that together induce modification of nucleic acids at a target sequence by an RNA-guided nuclease or other effector molecule and result in an effect, including the RNA-guided nuclease or other effector molecule and a gRNA molecule. In one embodiment, the CRISPR system includes a gRNA and a Cas protein (e.g., Cas9 protein). Such a system containing Cas9 or a modified Cas9 molecule is referred to herein as a “Cas9 system” or a “CRISPR / Cas9 system.” In one example, the gRNA molecule and the Cas molecule may be combined to form a ribonucleoprotein (RNP) complex.

[0054] The term “Cas9” or “Cas9 molecule” refers to an enzyme from the bacterial type II CRISPR / Cas system responsible for DNA cleavage. Cas9 also includes wild-type proteins, as well as functional and non-functional variants thereof. In embodiments, the Cas9 is Cas9 of S. pyogenes or C. jejuni. In another embodiment, the Cas9 is a modified or “dead” Cas9 (dCas9). In yet another embodiment, the Cas9 is a dead Cas9 that has been further cleaved to limit its size. In the present disclosure, the use of Cas9 nuclease-null orthologs from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Treponema denticola, Neisseria meningitidis, Campylobacter jejuni, etc. is contemplated.

[0055] In some embodiments, the Cas protein is modified (e.g., genetically engineered) to lack nuclease activity. For example, the dead Cas9 (dCas9) protein binds to a target locus but does not cleave nucleic acids at that locus. In some embodiments, the dCas9 protein comprises the sequence of SEQ ID NO: 2, the nucleic acid sequence of which is provided in SEQ ID NO: 1. In other embodiments, the dCas9 comprises a sequence that is at least 70%, 80%, 85%, 87%, 90%, 92%, 95%, 98%, or 99% identical to SEQ ID NO: 2 and is capable of binding to a target sequence but lacks nuclease activity.

[0056] In some embodiments, a catalytically dead Cas9 protein (e.g., dead Cas9, “dCas9”) is fused (e.g., covalently) to a transcriptional regulatory factor domain and regulates (e.g., inhibits) the expression of a target gene (e.g., Nav1.7). In some embodiments, dCas9 comprises a sequence that is 70% to 100% identical to SEQ ID NO: 2. Without wishing to be bound by a particular theory, in some embodiments, dCas9 (or other catalytically dead Cas proteins) mediates transcriptional repression by sterically inhibiting the binding of the transcriptional machinery (e.g., RNA polymerase complex) to the target sequence.

[0057] In some embodiments, the present disclosure provides a split Cas9 system in which the N- and C-domains are separated into different vectors and co-expressed with the N- and C-domains on an intein molecule. For example, two or more parts or segments of Cas9 are provided to a cell, such as by being expressed from corresponding nucleic acids introduced into the cell. The two or more parts combine intracellularly to form a Cas9 having the ability to co-localize with a guide RNA at a target nucleic acid. It should be understood that Cas9 may have one or more modifications (e.g., dCas9) from the full-length Cas9 known to those skilled in the art, but still retains, or has, the ability to co-localize with a guide RNA at a target nucleic acid. Thus, when the two or more parts or segments are joined together, they produce, or result in, a Cas9 having the ability to co-localize with a guide RNA at a target nucleic acid. In one embodiment, a first nucleic acid encodes a first part of a Cas9 protein having a first split intein, a second nucleic acid encodes a second part of a Cas9 protein having a second split intein complementary to the first split intein, and the first part of the Cas9 protein and the second part of the Cas9 protein bind to form a Cas9 protein. In one embodiment, C-intein-dCCas9 comprises the sequence of SEQ ID NO: 8 or a sequence that is 70-99% identical to SEQ ID NO: 8, and dNCas9-N-intein comprises the sequence of SEQ ID NO: 9 or a sequence that is 70-99% identical to SEQ ID NO: 9.

[0058] In some embodiments, the Cas protein (e.g., dCas9) is fused to a transcriptional regulatory factor domain. As used herein, a "transcriptional regulatory factor domain" is a protein domain that catalyzes a structural or chemical change in chromatin molecules that results in altered transcriptional activity (e.g., transcriptional activation or transcriptional repression). In some embodiments, the transcriptional regulatory factor domain is a transcriptional repressor domain. In some embodiments, the repressor domain includes a Kruppel-associated box domain (KRAB domain). Non-limiting examples of the KRAB domain include the KOX1 KRAB domain, the KOX8 KRAB domain, the ZNF43 KRAB domain, and the ZNF184 KRAB domain. In some embodiments, the KRAB domain is the KOX1 KRAB domain. Further non-limiting examples of the repressor domain include the chromo-shadow (CS) domain (e.g., the CS domain of HP1α) and the WRPW domain (e.g., the WRPW domain of Hes1). In certain embodiments, the KRAB domain comprises the nucleic acid sequence of SEQ ID NO: 6 and the polypeptide sequence of SEQ ID NO: 7, or a sequence that is at least 70%, 80%, 85%, 87%, 90%, 92%, 95%, 98%, or 99% identical thereto.

[0059] In some embodiments, the dCas9 comprises one or more transcriptional repressors. For example, in some embodiments, the general structure of an exemplary dCas9 fusion protein having a transcriptional repressor domain is [NH 2 -[NLS]-[dCas9 or Cas9]-[(transcriptional repressor) n -[COOH], [NH 2 -[NLS]-[(transcriptional repressor) n -[dCas9 or Cas9]-[COOH ], [NH 2 -[dCas9 or Cas9]-[(transcriptional repressor) n -[COOH], or [NH 2 -[(transcriptional repressor) n-[dCas9 or Cas9]-[COOH] structure, where NLS is a nuclear localization signal, NH 2NH₂ is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the fusion protein comprises one or more repeats of the transcriptional repressor, for example, n = 1 to 10 (for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n = 1 to 20. In some embodiments, the linker is inserted between the dCas9 and the transcriptional repressor domain. In some embodiments, the linker is inserted between the nuclear localization signal (NLS) and the transcriptional repressor and / or the dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the transcriptional repressor and / or the dCas9 domain. In some embodiments, the NLS is located between the transcriptional repressor domain and the dCas9 domain. Additional features such as sequence tags may also be present. In some embodiments, the transcriptional repressor is selected from the group consisting of the KRAB (Krüppel-associated box) domain of Kox1, the SID (mSin3 interaction domain), the CS (chromoshadow) domain of HP1α, the WRPW domain of Hes1, MBD2, MBD3, the DNMT family (DNMT1, DNMT3A, DNMT3B, DNMT2A), Rb, Mecp2, Fog1, ROM2, AtHD2A, and LSD1. These and other repressor domains are known in the art and, in some embodiments, correspond to those described in published U.S. patent application Ser. No. 14 / 105,017, published as US2014 / 0186958 A1 (the entire content of which is incorporated herein by reference), Urrutia, zinc-finger repressor proteins containing KRAB, Genome. Biol. 2003; 4(10):231; Gilbert et al., modular RNA-guided transcriptional regulation via CRISPR in eukaryotes, Cell. 2013; 154, 442-451; Konermann et al., optical control of mammalian endogenous transcription and epigenetic states, 2013;500, 472-476.In some embodiments, the transcriptional repressor domain comprises one or more repeats of the KRAB domain (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats). In some embodiments, the KRAB domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7. In some embodiments, the transcriptional repressor domain comprises one or more repeats of the SID protein. In some embodiments, the repressor domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the SID protein. In some embodiments, the repressor domain comprises 4 repeats of SID.

[0060] In some embodiments, the transcriptional regulator is present in one or both constructs of the split Cas9 system. For example, the KRAB domain can be present in either or both of SEQ ID NO: 3 and / or 4. For example, if the KRAB sequence is present in SEQ ID NO: 3, the resulting construct is provided in FIG. 8B and SEQ ID NO: 10.

[0061] The terms “guide RNA”, “guide RNA molecule”, “gRNA molecule” or “gRNA” are used interchangeably and refer to a set of nucleic acid molecules that direct an RNA-guided nuclease or other effector molecule (typically in complex with the gRNA molecule) to a specific sequence. Techniques for designing gRNAs for target specificity and donor therapeutic polynucleotides are well known in the art. See, for example, Doench, J. et al., Nature biotechnology 2014;32(12):1262-7, Mohr, S. et al., (2016) FEBS Journal 283:3232-38, and Graham, D. et al., Genome Biol.2015;16:260. A gRNA comprises, consists essentially of, or consists of a fusion polynucleotide comprising a CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA), or a polynucleotide comprising a CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA). In some embodiments, the gRNA is synthetic (Kelley, M. et al., (2016) J.Biotechnology 233((2016)74-83). In some embodiments, targeting is achieved by hybridizing a portion of the gRNA to DNA (e.g., the gRNA targeting domain) and binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule. In some embodiments, the gRNA molecule consists of a single contiguous polynucleotide molecule, herein referred to as a “single guide RNA” or “sgRNA” for example. In other embodiments, the gRNA molecule typically consists of multiple, usually two, polynucleotide molecules that can be associated via hybridization, herein referred to as “dual guide RNA” or “dgRNA” for example. The gRNA molecule generally comprises a targeting domain and a tracr. In embodiments, the targeting domain and tracr are located on a single polynucleotide. In other embodiments, the targeting domain and tracr are located on separate polynucleotides.

[0062] The term "target domain", when used in connection with a gRNA, is part of a gRNA molecule that recognizes (e.g., is complementary to) a target sequence (e.g., a target sequence within a nucleic acid of a cell (e.g., within a gene)).

[0063] The term "target sequence" refers to a sequence of nucleic acids that is complementary to, e.g., fully complementary to, the gRNA targeting domain. In embodiments, the target sequence is located on genomic DNA. In one embodiment, the target sequence is adjacent (either on the same strand or on the complementary strand of the DNA) to a protospacer adjacent motif (PAM) sequence that is recognized by a protein having nuclease or other effector activity, e.g., Cas9. In embodiments, the target sequence is a target sequence within a gene or locus that affects the expression of the Nav1.7 gene (e.g., one that affects the expression of voltage-gated sodium channel 1.7).

[0064] The present disclosure provides the gRNA target sequences in Table 1. As will be appreciated, the gRNA target sequences in Table 1 can vary by substitution of about 1 to 5 base pairs (e.g., 1, 2, 3, 4, or 5 base pairs), so long as the target sequence can hybridize to the target sequence in the SCN9A gene in the genome.

[0065] Table 1: JPEG2025084803000005.jpg211133JPEG2025084803000006.jpg211133JPEG2025084803000007.jpg169132

[0066] In one embodiment, the present disclosure provides a recombinant gene repressor complex comprising a nuclease-inactivated Cas9 protein fused to a transcriptional repressor, wherein the nuclease-inactivated Cas9 is associated with a guide RNA, wherein the guide RNA has a sequence selected from SEQ ID NOs: 11-107 and any of SEQ ID NOs: 11-107 having 1-5 pairs (e.g., 1, 2, 3, 4, or 5 pairs) of base pair substitutions, wherein the gRNA can bind to a target sequence in the SCN9A gene that suppresses gene expression. In one embodiment, the nuclease-inactivated Cas9 is in the form of split Cas9. The inactivated Cas9 can be derived from and / or induced by any Cas9 protein. In another embodiment, the nuclease-inactivated Cas9 is engineered from C. jejuni. In another embodiment, the nuclease-inactivated Cas9 comprises SEQ ID NO: 2 or a sequence at least 70%, at least 90%-99% identical thereto. In another or further embodiment, the transcriptional repressor is KRAB. In a further embodiment, the KRAB comprises a sequence as set forth in SEQ ID NO: 7, or a sequence 70%-99% identical to SEQ ID NO: 7 and capable of suppressing gene transcription.

[0067] Referring to the constructs of FIGS. 8A-C, at least one of the gRNA sequences of Table 1 is cloned into the AgeI site immediately downstream of the U6 promoter.

[0068] As further described below, the recombinant gene repressor complex can be delivered using various viral vectors including lentiviral vectors and adenoviral vectors.

[0069] The present disclosure also provides a recombinant gene repressor complex comprising a zinc finger DNA binding protein.

[0070] As used herein, "polyA" refers to a polymer of adenosine. The polyA sequence can be obtained, for example, from SV40 poly(A), bovine growth hormone poly(A) (bGHpA), rabbit β-globin poly(A), etc.

[0071] "Regulatory elements" can be used in methods and compositions for improving expression from viral vectors. For example, the Woodchuck hepatitis virus regulatory element (WPRE) can be used to enhance the expression of dCas9 constructs from viral vectors. The sequence of WPRE is known (see, for example, "WHP Posttranscriptional Response Element" in Wikipedia; [https: / / ]en.wikipedia.org / wiki / WHP_Posttranscriptional_Response _Element).

[0072] As those skilled in the art will appreciate by reference to the constructs shown in FIGS. 8A-C and FIG. 9, since these structures are schematic, different "polyA" sequences as provided herein can be cloned into the vectors of the disclosure, different promoters can be substituted for "CMV" in FIGS. 8A-C and FIG. 9 in the vector (e.g., the CMV promoter can be substituted with another polII promoter such as the RSV promoter), and different polIII promoters can be substituted for the U6 promoter in FIGS. 8A-C.

[0073] "Zinc finger DNA-binding protein" (or binding domain) is a protein or a domain within a larger protein that binds DNA sequence-specifically via one or more zinc fingers, and is a region of the amino acid sequence within a binding domain whose structure is stabilized by coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein, ZF, or ZFP. Individual DNA-binding domains are typically referred to as "fingers". ZFPs have at least one finger, typically two, three, four, five, six, or more fingers. Each finger binds to DNA of 2-4 base pairs, typically 3 or 4 base pairs. ZFPs bind to a nucleic acid sequence called the target site or target segment. Each finger typically contains about 30 amino acids, a zinc-chelating, DNA-binding subdomain. An exemplary motif characterizing one class (C 2 H 2 lass) of these proteins is -Cys-(X) 2-4 -Cys-(X) 12 -His-(X) 3-5-His (where X is any amino acid) (SEQ ID NO: 143). Other classes of zinc finger proteins are known and are useful in the practice of the methods and in the manufacture and use of the compositions disclosed herein (see, for example, Rhodes et al., (1993) Scientific American 268:56-65 and U.S. Patent Application Publication No. 2003 / 0108880). Studies have demonstrated that a single zinc finger of this class consists of an α helix containing two invariant histidine residues that coordinate with zinc along with two cysteine residues of a single β turn (see, for example, Berg & Shi, Science 271:1081-1085 (1996)). A single target site for a ZFP typically has from about 4 to about 10 base pairs. Typically, a two-finger type ZFP recognizes a target site of 4 to 7 base pairs, a three-finger type ZFP recognizes a target site of 6 to 10 base pairs, a four-finger type ZFP recognizes a target site of 12 to 14 base pairs, and a six-finger type ZFP recognizes a target site of 18 to 20 base pairs. These can include two adjacent target sites of 9 to 10 base pairs or three adjacent target sites of 6 to 7 base pairs.

[0074] In these embodiments that include an engineered zinc finger binding domain, the zinc finger domain is engineered to bind to a particular target site. The binding domain contains a plurality of zinc fingers (e.g., two, three, four, five, six, or more zinc fingers). Generally, an individual zinc finger binds to a subsite of three to four nucleotides. The subsites may be adjacent to the target site (and in some cases overlapping); alternatively, the subsites may be separated from adjacent subsites by a gap of one, two, three, or more nucleotides. Binding to subsites separated by a gap of one or more nucleotides is facilitated by the use of a non-standard longer linker sequence between adjacent zinc fingers.

[0075] The present disclosure provides zinc finger targets as shown in Table 2. Table 2 shows both mouse and human target sites in the SCN9A gene sequence.

[0076] Table 2: JPEG2025084803000008.jpg97139

[0077] Using the above target sequences, one of ordinary skill in the art can design zinc-finger proteins that bind to these sequences. For example, using methods known in the art, zinc-finger repressor constructs that bind to SEQ ID NO: 108, 109, 110, or 111 were designed. The zinc-finger repressor construct contains six zinc fingers as shown in Table 3 below, where the target amino acids of the zinc fingers contain 6 to 12 amino acids.

[0078] Table 3 (The bold / underlined part is the KRAB repressor sequence, and the double-underlined region is the zinc finger target region.) JPEG2025084803000009.jpg170139

[0079] Human Nav1.7 expression is regulated via binding of the ZFP to target sites having the nucleic acid sequences shown in Table 2 (e.g., SEQ ID NO: 108 - 122 or subsequences thereof). Mouse Nav1.7 expression is regulated via binding of the ZFP to target sites having the sequences of SEQ ID NO: 108, 109, 110, or 111 (e.g., such as mouse SCN10A). Species variants of Nav1.7 (e.g., mouse SCN10A) can be regulated at sites corresponding to SEQ ID NO: 108, 109, 110, or 111 in that species (i.e., the site having the greatest sequence identity). Nucleotides containing the subsites mainly contacted by individual zinc fingers are shown above, and nucleotides between subsites are shown below.

[0080] Exemplary ZFP-KRAB sequences capable of suppressing Nav1.7 expression in the mouse homolog (SCN10A) are shown in SEQ ID NOs: 123-126. Here, each sequence contains six finger domains and the KRAB sequence from amino acids 220-282.

[0081] The zinc finger or CRISPR / Cas proteins described herein can be delivered using a vector comprising a sequence encoding one or more zinc finger or CRISPR / Cas proteins. Any vector system can be used, including but not limited to plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxviral vectors, herpesviral vectors, and adeno-associated viral vectors. See U.S. Pat. Nos. 6,534,261, 6,607,882, 6,824,978, 6,933,113, 6,979,539, 7,013,219, and 7,163,824, which are hereby incorporated by reference in their entirety. Furthermore, it will be apparent that any of these vectors may also contain a sequence encoding one or more zinc finger proteins. Thus, when one or more ZFPs or CRISPR / Cas proteins are introduced into a cell, the sequence encoding the ZFP or CRISPR / Cas protein can be carried on the same vector or a different vector. When multiple vectors are used, each vector may contain a sequence encoding one or more ZFP or CRISPR / Cas systems.

[0082] Using general virus- and non-virus-based gene transfer methods, nucleic acids encoding engineered ZFPs or CRISPR / Cas systems can be introduced into cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding ZFPs or CRISPR / Cas systems to cells in vitro. In certain embodiments, nucleic acids encoding ZFPs or CRISPR / Cas systems are administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses that have either episomal or genomic integration after delivery to the cell. Gene therapy procedures are known; see Anderson (1992) Science 256:808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani and Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu et al., (1994) Gene Therapy 1:13-26.

[0083] Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virus particles, and enhanced uptake of DNA. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. In one embodiment, one or more nucleic acids are delivered as mRNA. Also, in some embodiments, capped mRNA can be used to increase translation efficiency and / or mRNA stability.

[0084] Other exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Patent No. 6,008,336). Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner International Patent Publications WO 91 / 17424 and WO 91 / 16024. It can be delivered to cells (ex vivo administration) or target tissues (in vivo administration).

[0085] The preparation of nucleic acid complexes containing target liposomes such as immunolipid complexes is well known to those skilled in the art (see, for example, Crystal (1995) Science 270:404-410 (1995), Blaese et al., (1995) Cancer Gene Ther. 2:291-297, Behr et al., (1994) Bioconjugate Chem. 5:382-389, Remy et al., (1994) Bioconjugate Chem. 5:647-654, Gao et al., (1995) Gene Therapy 2:710-722, Ahmad et al., (1992) Cancer Res. 52:4817-4820, U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0086] Additional delivery methods include methods of encapsulating the nucleic acid to be delivered into an EnGenelC delivery vehicle (EDV). These EDVs are specifically delivered to the target tissue using a bispecific antibody in which one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody brings the EDV to the surface of the target cell, and then the EDV is introduced into the cell by endocytosis. Once inside the cell, its contents are released (see MacDiarmid et al., (2009) Nature Biotechnology 27(7):643).

[0087] The use of RNA- or DNA virus-based systems for delivery of nucleic acids encoding engineered ZFP or CRISPR / Cas systems takes advantage of highly evolved processes for targeting the virus to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to the patient (in vivo) or cells can be treated in vitro and the modified cells administered to the patient (ex vivo). Virus-based systems for delivering general ZFP or CRISPR / Cas systems include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia viral, and herpes simplex viral vectors for gene transfer.

[0088] Integration into the host genome is possible by retroviral, lentiviral, and adeno-associated viral gene transfer methods and often results in long-term expression of the inserted transgene. High transduction efficiencies have also been observed in many different cell types and target tissues.

[0089] The tropism of retroviruses can be altered by introducing foreign envelope proteins and expanding the potential target population of the target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically can produce high viral titers. The choice of retroviral gene delivery system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with the ability to package foreign sequences up to 6 - 10 kb. The minimal cis-acting LTR is sufficient for vector replication and packaging, which is then used to integrate the therapeutic gene into the target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., (1992) J. Virol. 66:2731 - 2739, Johann et al., (1992) J. Virol. 66:1635 - 1640, Sommerfelt et al., (1990) Virol. 176:58 - 59, Wilson et al., (1989) J. Virol. 63:2374 - 2378, Miller et al., (1991) J. Virol. 65:2220 - 2224 (1991), and PCT / US94 / 05700).

[0090] For applications where transient expression is desired, an adenovirus-based system can be used. Adenovirus-based vectors enable very high transduction efficiencies in many cell types and do not require cell division. High titers and high levels of expression have been obtained with such vectors. The vectors can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used, for example, to transduce cells with a target nucleic acid, for example, for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, for example, West et al., (1987) Virology 160:38-47, U.S. Patent No. 4,797,368, International Patent Publication No. WO 93 / 24641, Kotin (1994) Human Gene Therapy 5:793-801, and Muzyczka (1994) J. Clin. Invest. 94:1351). The construction of recombinant AAV vectors has been described in numerous publications including U.S. Patent No. 5,173,414, Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260, Tratschin et al., (1984) Mol. Cell. Biol. 4:2072-2081, Hermonat and Muzyczka (1984) PNAS 81:6466-6470, and Samulski et al., (1989) J. Virol. 63:3822-3828.

[0091] Currently, at least six viral vector approaches are available for gene transfer in clinical trials, and these utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.

[0092] pLASN and MFG-S are examples of retroviral vectors used in clinical trials (Dunbar et al., (1995) Blood 85:3048-305, Kohn et al., (1995) Nat. Med. 1:1017-102, and Malech et al., (1997) PNAS 94(22):12133-12138). PA317 / pLASN was the first therapeutic vector used in gene therapy trials (Blaese et al., (1995) Science 270:475-480). In the MFG-S packaging vector, transduction efficiency of over 50% has been observed (Ellem et al., (1997) Immunol Immunother 44(1):10-20, and Dranoff et al., (1997) Hum. Gene Ther. 1:111-112).

[0093] Recombinant adeno-associated virus vectors (rAAV) are promising alternative gene delivery systems based on defective and non-pathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that retain only the AAV 145 bp inverted terminal repeats (ITRs) adjacent to the transgene expression cassette. Efficient gene transfer and stable transgene delivery by integration into the genome of transduced cells are important features of this vector system (Wagner et al., (1998) Lancet 351(9117):1702-1703, Kearns et al., (1996) Gene Ther. 9:748-55). Other AAV serotypes including AAV 1, AAV3, AAV4, AAVS, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, and pseudotyped AAV (such as AAV2 / 8, AAV2 / 5, and AAV2 / 6) can also be used according to the present invention.

[0094] Replication-deficient recombinant adenovirus vectors (Ad) can be produced at high titers and can be easily infected into many different cell types. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes. The replication-deficient vector is then propagated in human 293 cells that supply the deleted gene functions in trans. Ad vectors can transduce multiple types of tissues, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle in vivo. Conventional Ad vectors have a large carrying capacity. Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for anti-tumor immunity by intramuscular injection (Sterman et al., (1998) Hum. Gene Ther. 7:1083-1089). Examples of the use of adenovirus vectors for gene transfer in other clinical trials include Rosenecker et al., (1996) Infection 24(1):5-10, Sterman et al., (1998) Hum. Gene Ther. 9(7):1083-1089, Welsh et al., (1995) Hum. Gene Ther. 2:205-218, Alvarez et al., (1997) Hum. Gene Ther. 5:597-613, Topf et al., (1998) Gene Ther. 5:507-513, and Sterman et al., (1998) Hum. Gene Ther. 7:1083-1089.

[0095] Packaging cells are used to form virus particles that can infect host cells. Such cells include 293 cells that package adenoviruses, and Ψ2 cells or PA317 cells that package retroviruses. Virus vectors used in gene therapy are usually produced by producer cell lines that package nucleic acid vectors into virus particles. The vector usually contains the minimal viral sequences necessary for packaging and subsequent integration into the host (if applicable), and other viral sequences are replaced by an expression cassette encoding the protein to be expressed. The defective viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the inverted terminal repeat (ITR) sequences from the AAV genome necessary for packaging and integration into the host genome. The viral DNA is packaged into a cell line containing a helper plasmid that encodes other AAV genes (i.e., rep and cap) but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector from the helper plasmid and the expression of the AAV genes. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Contamination by adenovirus can be reduced, for example, by heat treatment to which adenovirus is more sensitive than AAV.

[0096] In many gene therapy applications, it is desirable for the gene therapy vector to be delivered with a high degree of specificity to a particular tissue type. Thus, viral vectors can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have an affinity for a receptor known to be present on the cell type of interest. For example, Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia virus can be modified to express human hereditary insulin fused to gp70, and that the recombinant virus infects certain human breast cancer cells that express the human epidermal growth factor receptor. This principle can be extended to other pairs of viral target cells where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., Fab or Fv) with specific binding affinity for substantially any selected cell receptor. The above description applies mainly to viral vectors, but the same principle can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that are advantageous for uptake by specific target cells.

[0097] Gene therapy vectors can typically be delivered in vivo by administration to individual patients by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, or intracranial injection) or local application as described below. Alternatively, the vector can be delivered ex vivo to cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or cells such as universal donor hematopoietic stem cells, and then usually the cells incorporating the vector are selected and then re-transplanted into the patient.

[0098] Vectors containing therapeutic ZFP nucleic acids or CRISPR (e.g., retroviruses, adenoviruses, liposomes, etc.) can also be administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA can also be administered. Administration is usually by any of the routes commonly used to introduce molecules that ultimately contact blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Methods suitable for administering such nucleic acids are available and well known to those skilled in the art, and while multiple routes can be used to administer a particular composition, a particular route can often provide a more rapid and effective response than another route.

[0099] The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, as described below, a wide variety of suitable formulations of the available pharmaceutical compositions exist (see, e.g., Remington's Pharmaceutical Sciences (17th edition)).

[0100] Using the Cas9 complex and zinc finger fusion constructs of the present disclosure, pain perception was suppressed in various animal models. Since pain perception is etiologically diverse and multifaceted, several rodent pain models were utilized to study pain signaling and pain behavior. The studies described herein evaluated the effect of CRISPR-mediated knockdown of Nav1.7 using three mechanistically distinct models. (i) Heat sensitivity in the control (normal) and ipsilateral inflamed hind paw, (ii) polyneuropathy resulting in tactile allodynia in both hind paws induced by chemotherapy, and (iii) spinal cord-induced tactile allodynia in both hind paws induced by spinal activation of purinergic receptors. Pain resulting from tissue damage and inflammation arises from the release of factors that sensitize the peripheral endings of nociceptive afferent neurons. This phenotype can be studied by topically applying carrageenan to the paw, which results in inflammation, swelling, increased expression of Nav1.7, and a sharp increase in thermal and mechanical sensitivity (hyperalgesia). Chemotherapy for treating cancer often results in polyneuropathy characterized by sensitivity to light touch (e.g., tactile allodynia) and cold sensation. Paclitaxel is a commonly used chemotherapeutic agent that increases the expression of Nav1.7 in nociceptive afferent nerves and induces intense allodynia in animal models. Finally, ATP (adenosine triphosphate) acting on various purinergic receptors expressed on nociceptive nerve terminals, secondary neurons, and non-neuronal cells is widely involved in inflammatory, visceral, and neuropathic pain. Thus, intrathecal delivery of a stable ATP analog (BzATP: 2',3'-O-(4-benzoylbenzoyl)-ATP) produces persistent allodynia in mice.

[0101] In this specification, various KRAB-CRISPR-dCas9 and ZFP-KRAB constructs were provided and studied for the suppression of Nav1.7 (Neuro2a) expression in a mouse neuroblastoma cell line. These constructs can be packaged into a retroviral system for delivery. For example, the tested constructs with in vitro suppression were packaged into AAV9 and injected intrathecally into adult C57BL / 6J mice. After 21 days, foot inflammation was induced via injection of carrageenan. Thermal hyperalgesia was then evaluated. In the exemplary studies presented herein, in vivo suppression of Nav1.7 using the constructs of the present disclosure resulted in a reduction of thermal hyperalgesia. The constructs were further tested in two models of neuropathic pain (chemotherapy-induced (paclitaxel) neuropathic pain and BzATP-induced neuropathic pain). As shown in the results of the model of paclitaxel-induced neuropathic pain, suppression of Nav1.7 using the constructs of the present disclosure reduced tactile allodynia and cold allodynia. Also, mice injected with KRAB-CRISPR-dCas9 showed a reduction of tactile allodynia after administration of the ATP analog BzATP. Since many pain states occur after chronic inflammation and nerve injury and usually cause a persistent state that requires continuous re-dosing, the genetic approach of the present disclosure provides continuous and controllable modulation of this abnormal process and persistent pain. The in situ epigenetic approach described herein functions as a potential therapeutic approach for chronic pain that replaces narcotic substances and persists for a long time.

[0102] In the experiments presented herein, the efficacy of Nav1.7 suppression in dorsal root ganglia was evaluated using two separate genome engineering constructs of the present disclosure (KRAB-dCas9 and zinc-finger-KRAB proteins). The genome editing constructs of the present disclosure were found to be effective in suppressing acute and persistent nociceptive processing generated in animal models of peripheral inflammation and polyneuropathy. The genome editing constructs of the present disclosure were discovered by testing multiple guide RNA (gRNA) clones rationally designed using an in silico tool that predicts effective gRNAs based on chromatin location and sequence features on the split-dCas9 platform. Similarly, multiple ZFP-KRAB Nav1.7 DNA targeting constructs were also discovered. The genome editing constructs of the present disclosure were transfected into a mouse neuroblastoma cell line expressing Nav1.7 (Neuro2a). Suppression of Nav1.7 was confirmed. For subsequent in vivo studies, the construct showing the highest suppression level was selected.

[0103] Other techniques such as RNAi have been utilized to target Nav1.7, but tests have shown that the non-specific effects of RNAi are much stronger and more prevalent than generally understood compared to CRISPRi. Also, as exogenous systems, CRISPR and ZFP (unlike RNAi) do not compete with endogenous mechanisms such as microRNA or RISC complex function. Thus, RNAi can affect the normal homeostasis mechanisms of RNA synthesis and degradation. Furthermore, the CRISPR and ZFP methods target genomic DNA instead of RNA. That is, to achieve an effect, the RNAi method usually has poor pharmacokinetic prospects and requires higher dosages because of the high RNA turnover rate.

[0104] Studies have shown that partial inhibition of Nav1.7 is sufficient to improve pain. This knockdown helps to produce a significant recovery of hyperalgesia induced by hind paw inflammation. Using antisense oligonucleotides, organic pain can be improved at a Nav1.7 inhibition level of 30 - 80%. Using microRNA 30b, 50% inhibition of Nav1.7 reduces neuropathic pain, while more recently, microRNA182 has improved pain and prevented overexpression of NaV1.7 in rats that were spared nerve injury. Similarly, knockdown via shRNA of Nav1.7 prevented overexpression and alleviated pain in burns. In other studies, the level of Nav1.7 inhibition required to reduce pain was not quantified. Furthermore, an shRNA lentiviral vector can reduce pain in bone cancer by inhibiting Nav1.7 by 40 - 60%.

[0105] The role of Nav1.7 is relevant to various preclinical models, including those associated with strong inflammation such as the carrageenan and CFA models in rodents. Thus, the effect of Nav1.7 knockdown in paclitaxel-induced polyneuropathy was studied using the genome editing constructs of the present disclosure. Previous studies have shown that the treatment induces NaV1.7. Both epigenetic suppressors improve allodynia to the same extent as gabapentin in the internal comparison. Finally, the role of Nav1.7 knockdown using the genome editing constructs of the present disclosure in BzATP-induced hyperalgesia was evaluated. Spinal purinergic receptors have been shown to play an important role in nociceptive processing initiated by various stimulus conditions, including inflammatory / incisional pain and various neuropathies. As shown in this study, suppression of centripetal Nav1.7 expression in nociceptors leads to suppression of centrally induced enhancement of tactile sensitivity. The mechanism underlying these results may reflect the observation that a decrease in Nav1.7 in centripetal nerves may help minimize the activation of microglia and astrocytes. These results suggest that at least part of the pain signaling via Nav1.7 is downstream of ATP signaling. Gabapentin was selected as a positive control because of evidence that it reduces carrageenan-induced thermal hyperalgesia in rodents and is known to suppress Nav1.7. The results using the genome editing constructs of the present disclosure are consistent with previous studies showing an inhibitory effect of gabapentin on Nav1.7 expression levels, ultimately resulting in a decrease in neuronal excitability.

[0106] The methods disclosed herein demonstrate the efficacy of spinal cord reduction in Nav1.7 in three models of hyperalgesia using the genome editing constructs disclosed herein. The studies presented herein clearly establish significant target engagement and a clear therapeutic effect without obvious adverse events following intrathecal knockdown of NaV1.7 when using the genome editing constructs of the present disclosure. The role played by NaV1.7 is in nociceptive afferent nerves, and their cell bodies are present in the DRG neurons of each segment. Thus, the DRG represents the target for this gene delivery motif. The intrathecal delivery route efficiently places AAV in DRG neurons that minimize the potential for off-target biodistribution and reduce the amount of virus required to obtain transduction. In certain embodiments, the genome editing constructs of the present disclosure are administered intrathecally. Importantly, because the cerebrospinal fluid has relatively few B and T cells, potential immune responses are minimized. In this regard, ZFPs are engineered on the human protein scaffold, so they essentially constitute a targeted approach with lower potential immunogenicity. Indeed, studies in non-human primates (NHPs) have found that intrathecal delivery of a non-self protein (AAV9-GFP) produced an immune response not seen with delivery of a self protein.

[0107] The results described herein demonstrate that the genome editing constructs of the present disclosure have favorable target engagement properties and efficacy over short and long time periods. Thus, methods of using the genome editing constructs of the present disclosure for controlling persistent pain are clearly shown. The genome editing constructs of the present disclosure (e.g., KRAB-dCas9 and ZFP-KRAB) show promise as potential clinical therapies for treating chronic inflammatory and neuropathic pain. These systems enable transient gene therapy, which is advantageous for the chronic pain framework, as permanent pain insensitivity is not desired. This transient yet multi-week treatment represents a significant advantage compared to existing drugs that need to be taken daily or every few hours and can have undesirable toxic effects. In summary, the results of these studies indicate a promising new avenue for treating chronic pain, an important and increasingly urgent problem in our society. It should be noted that this treatment regimen addresses an important pain phenotype, namely a persistent but recoverable pain state. Chronic pain, defined as a pain state lasting more than three months, is not necessarily irreversible. Thanks to medical advancements, the number of cancer survivors has been steadily increasing over the past few decades. This increase has led to a subsequent increase in the number of cancer-related side effects, and chemotherapy-induced polyneuropathy is one of the most common adverse events. In this case, a treatment approach lasting several months is preferred over an irreversible approach. Furthermore, the use of multiple spinal epidural anesthetics over time is a common motif of clinical intervention, as in the case of epidural steroids, and epidural delivery may be repeated over a year at several month intervals.

[0108] Accordingly, the present disclosure provides an epigenetic approach for treating a subject having pain using a construct comprising a zinc finger (ZF) fused to a repressor domain and / or dCas9 fused to a repressor domain. Here, dCas9 is a catalytically inactivated Cas9 that does not cleave DNA but maintains the ability to bind to the genome via a guide-RNA (gRNA). The epigenetic approach described herein enables transient gene therapy, which is advantageous for chronic pain frameworks since permanent pain insensitivity is not desired. This transient yet multi-week treatment offers significant advantages compared to existing drugs that need to be taken daily or every few hours and can have undesirable toxic effects. In summary, the epigenetic approach described herein using the ZF-repressor domain construct and / or the dCas9-repressor domain construct provides a promising new avenue for treating chronic pain, an important and increasingly urgent problem in society.

[0109] In a further embodiment, the ZF-repressor domain construct and / or the dCas9-repressor domain construct are packaged and expressed by an adeno-associated virus (AAV). The use of such an AAV system is ideal for patients to whom the virus is administered prior to surgery or for use in patients with chronic pain who can experience pain relief for about one month at a time. In certain embodiments, the AAV is AAV9. As a result of the results presented herein, the epigenetic approach using the ZF-repressor domain construct and / or the dCas9-repressor domain construct of the present disclosure is effective in an inflammatory chronic pain model and can be used in other pain modalities including, but not limited to, neuropathic pain, postoperative pain, migraine, and cancer-induced pain. In a further embodiment, the ZF-repressor domain construct and / or the dCas9-repressor domain construct described herein can be designed to regulate other genes including, but not limited to, Nav1.3, Nav1.9, TRPV1 / 2 / 3 / 4, Nav1.8, P2X4, P2X7, Atp1b3, Mapk8, Avpr1a, Calca, Htr1b, Oprm1, Mc1r, Kcnk9, KCNQ, and TLR2 / 3.

[0110] The present disclosure further provides pharmaceutical compositions and formulations comprising the ZF-repressor domain constructs and / or dCas9-repressor domain constructs described herein for particular modes of administration. In one embodiment, the ZF-repressor domain constructs and / or dCas9-repressor domain constructs described herein are the active ingredients in a composition comprising a pharmaceutically acceptable carrier. Such compositions are referred to herein as pharmaceutical compositions. "Pharmaceutically acceptable carrier" means any pharmaceutically acceptable means for mixing and / or delivering a target delivery composition. As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and, for example, must be compatible with administration to a human subject. Such compositions can be specially formulated for administration via one or more of a number of routes, such as the routes of administration described herein. Additional active ingredients can also be incorporated into the compositions. When the agents, formulations or pharmaceutical compositions described herein are administered to a subject, they are preferably administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount that results in an improvement or treatment of the condition.

[0111] Administration of the pharmaceutical composition to the subject is effected by means of contact of the ZF-repressor domain construct and / or the dCas9-repressor domain construct contained therein with the target cells. The specific route varies depending on specific variables such as the target cells and can be determined by those skilled in the art. Methods suitable for administering the ZF-repressor domain construct and / or the dCas9-repressor domain construct described herein to a patient include any in vivo administration route suitable for delivering the ZF-repressor domain construct and / or the dCas9-repressor domain construct described herein to the patient. Preferred administration routes vary depending on the type of viral gene therapy formulation used, the target cell population, and the disease or condition of the subject and will be apparent to those skilled in the art. Typical methods of in vivo administration include, but are not limited to, intravenous administration, intraperitoneal administration, intrathecal administration, intramuscular administration, intraperitoneal administration, intracranial administration, intraarterial administration (e.g., into the carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intra-articular administration, intracerebral administration, inhalation (e.g., aerosol), intracerebral, nasal, oral, pulmonary administration, catheter impregnation, and direct injection into tissue. In embodiments where the target cells are within or in the vicinity of a tumor, the preferred administration route is by direct injection into the tumor or the surrounding tissue. For example, preferred methods of administration in the case of breast cancer tumors include catheter impregnation and direct injection into the tumor.

[0112] Intravenous, intraperitoneal, intrathecal, intragastric, intraneural, intracranial, and intramuscular administrations can be carried out using standard methods in the art. Aerosol (inhalation) delivery can also be carried out using standard methods in the art (see, e.g., Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference in its entirety). Oral delivery can be effected by complexing the zinc finger-repressor domain construct described herein with a carrier that is resistant to degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include plastic capsules or tablets as known in the art.

[0113] One method of local administration is direct injection. The direct injection technique is particularly useful for administering the ZF - repressor domain constructs and / or dCas9 - repressor domain constructs described herein to cells or tissues accessible by surgery, particularly those on or near the surface of the body. Administering the composition locally within the region of the target cells refers to injecting the composition from a few centimeters, preferably a few millimeters, from the target cells or tissues. For example, in this specification, advantageous results were obtained by the intrathecal administration route.

[0114] The appropriate dosages and treatment regimens for the treatment methods described herein vary depending on the particular disease being treated, the ZF - repressor domain constructs and / or dCas9 - repressor domain constructs described herein being delivered, and the specific condition of the subject. A skilled practitioner will determine the dosage and frequency on a case - by - case basis. In one embodiment, administration is over a period until the desired effect (e.g., symptoms are alleviated) is achieved. In certain embodiments, administration is once, twice, three times, four times, five times, six times, or seven times a week. In particular embodiments, administration is over one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks. In another embodiment, administration is over a period of two months, three months, four months, five months, six months, or a longer period. In yet another embodiment, treatment is resumed after a remission period.

[0115] The ZF-repressor domain constructs and / or dCas9-repressor domain constructs described herein can be administered in combination with one or more other active agents that inhibit nociceptive pain signaling. For example, the ZF-repressor domain constructs and / or dCas9-repressor domain constructs can be administered with one or more other agents that target one or more genes, by delivering one or more gRNAs or by designing ZFs that bind to one or more genes. Other gene targets include, but are not limited to, P2x3, P2x4, P2x7, Nav1.3, capsaicin receptors (TRPV1 / 2 / 3 / 4), TRPA1, SHANK3, voltage-dependent calcium channels (Cav2.2, Cav3.1, Cav3.2). Figure 11 shows other targets that can be inhibited in combination with the ZF-repressor domain constructs and / or dCas9-repressor domain constructs of the present disclosure. Alternatively or additionally, various analgesics can be used in combination with the ZF-repressor domain constructs and / or dCas9-repressor domain constructs of the present disclosure. Such analgesics and other pain-relieving drugs are known in the art.

[0116] For use in the therapeutic applications described herein, kits and manufacturer's instructions are also described herein. Such kits can include a carrier, package, or container compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing one of the distinct elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be made of various materials such as glass or plastic.

[0117] For example, the container can optionally contain one or more ZF - repressor domain constructs and / or dCas9 - repressor domain constructs described herein, either as a composition or in combination with another agent disclosed herein. The container may optionally have a sterile access port (e.g., the container may be a bag or vial of intravenous solution having a stopper pierceable by a hypodermic needle). Such a kit can contain the compounds disclosed herein, optionally together with an identifying description, label, or instructions for its use in the methods described herein.

[0118] The kit typically includes one or more other containers, each containing one or more various materials (e.g., reagents, and / or devices, optionally in concentrated form) that are desirable from a commercial and user perspective for the use of the compounds described herein. Examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials and / or tube labels describing the contents and / or instructions for use, and package inserts with instructions for use. Usually, a set of instructions is also included.

[0119] The label can be on the container or associated with the container. The label can be on the container when the letters, numbers, or other characters forming the label are attached, molded, or etched onto the container itself. The label can be associated with the container, for example, as a package insert, when present in the receptacle or carrier holding the container. The label can be used to indicate that its contents are used for a particular therapeutic use. The label can also indicate how to use the contents, such as the methods described herein. These other therapeutic agents can be used, for example, in the amounts indicated in the Physician's Desk Reference (PDR) or in amounts determined by one of ordinary skill in the art.

[0120] In view of the above and the following examples, the present disclosure provides several aspects illustrated below.

[0121] Embodiment 1: A recombinant gene silencing complex comprising a Cas9 (dCas9) protein fused to a transcriptional repressor, associated with at least one guide RNA (gRNA), and in which a nuclease is inactivated. Here, the gRNA specifically hybridizes to a target nucleic acid sequence encoding a gene product selected from the group consisting of TRPV1 / 2 / 3 / 4, P2XR3, TRPM8, TRPA1, P23X2, P2RY, BDKRB1 / 2, Hlr3A, ACCNs, TRPV4, TRPC / P, ACCN1 / 2, SCN1 / 3 / 8A / 9A, SCN10A, SCN11A, KCNQ, BDNF, OPRD1 / K1 / M1, CNR1, GABRs, TNF, PLA2, IL1 / 6 / 12 / 18, COX-2, NTRK1, NGF, GDNF, TNF, LIF, CCL1, CNR2, TLR2 / 4, P2RX47, CCL2, CX3CR1, BDNF, NR1 / 2, GR1A1-4, GRC1-5, NK1R, CACNA1A-S, and CACNA2D1, and the expression of the gene product is inhibited.

[0122] Embodiment 2: The recombinant gene silencing complex according to Embodiment 1, wherein the target nucleic acid sequence is located at position 2q24.3 of chromosome 2.

[0123] Embodiment 3: The recombinant gene silencing complex according to Embodiment 1 or 2, wherein the gRNA comprises a sequence encoded by a sequence shown in any one of 11 to 107.

[0124] Embodiment 4: The recombinant gene silencing complex according to any one of Embodiments 1 to 3, wherein the gRNA specifically hybridizes to a nucleic acid sequence encoding the SCN9A product (Nav1.7).

[0125] Aspect 5: The transcriptional repressor is selected from the group consisting of mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( JPEG2025084803000010.jpg1279, KRAB), and is the recombinant gene repressor complex according to any one of Aspects 1 to 4.

[0126] Aspect 6: The transcriptional repressor domain is a KRAB domain, and is the recombinant gene repressor complex according to Aspect 5.

[0127] Aspect 7: A polynucleotide encoding one or more components of the recombinant gene repressor complex according to any one of Aspects 1 to 6.

[0128] Aspect 8: The polynucleotide is a codon optimized for expression in human cells, and is the polynucleotide according to Aspect 7.

[0129] Aspect 9: A vector containing the polynucleotide according to Aspect 7 or 8.

[0130] Aspect 10: The polynucleotide is operably linked to a promoter, and is the vector according to Aspect 9.

[0131] Aspect 11: The vector according to Aspect 10, wherein the promoter is selected from the group consisting of a human cytomegalovirus (CMV) promoter, a CAG promoter, a Rous sarcoma virus (RSV) LTR promoter / transcription enhancer, an SV40 promoter, an EF1-α promoter, a CMV immediate / early gene transcription enhancer / CBA promoter, a Nav1.7 promoter, a Nav1.8 promoter, a Nav1.9 promoter, a TRPV1 promoter, a synapsin promoter, a calcium / calmodulin-dependent protein kinase II promoter, a tubulin αI promoter, a neuron-specific enolase promoter, and a glial fibrillary acidic protein (GFAP) promoter.

[0132] Aspect 12: The vector according to Aspect 9, wherein the vector contains a polIII promoter upstream of at least one guide RNA coding sequence.

[0133] Aspect 13: The vector according to Aspect 12, wherein the polIII promoter is selected from U6 and H1 promoters.

[0134] Aspect 14: The vector according to any one of Aspects 9 to 13, further comprising a regulatory control sequence.

[0135] Aspect 15: The vector according to Aspect 14, wherein the regulatory control sequence is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0136] Aspect 16: The vector according to any one of Aspects 9 to 15, wherein the vector is a recombinant adeno-associated virus vector (rAAV vector).

[0137] Aspect 17: The vector according to aspect 16, wherein the rAAV is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vector (scAAV).

[0138] Aspect 18: The vector according to aspect 16, wherein the polynucleotide comprises one or more inverted repeats (ITRs).

[0139] Aspect 19: The vector according to any one of aspects 9 to 18, wherein the polynucleotide or vector comprises a polyA sequence.

[0140] Aspect 20: The vector according to aspect 9, wherein the polynucleotide is engineered to express one or more components in a cell.

[0141] Aspect 21: The vector according to aspect 9, wherein the vector is a lentiviral vector, a gammaretroviral vector, or a herpes simplex virus vector.

[0142] Aspect 22: The vector according to aspect 9, wherein the vector comprises a split dCas9 vector system.

[0143] Aspect 23: The vector according to aspect 9 or 22, wherein the vector comprises a nucleic acid encoding dCas9 having a sequence as shown in SEQ ID NO: 2.

[0144] Aspect 24: The vector according to aspect 9 or 23, wherein the vector comprises a nucleic acid encoding the KRAB sequence of SEQ ID NO: 7.

[0145] Aspect 25: The vector according to any one of Aspects 22 to 24, wherein the split vector system includes a vector sequence selected from SEQ ID NOs: 3, 4, and 10.

[0146] Aspect 26: A construct of a zinc-finger repressor comprising an engineered zinc-finger DNA-binding domain bound to a transcriptional repressor, wherein the zinc-finger DNA-binding domain comprises 1 to 6 zinc-finger sequences, and the zinc-finger sequence binds to a target nucleic acid sequence in a gene encoding a gene product selected from the group consisting of TRPV1 / 2 / 3 / 4, P2XR3, TRPM8, TRPA1, P23X2, P2RY, BDKRB1 / 2, Hlr3A, ACCNs, TRPV4, TRPC / P, ACCN1 / 2, SCN1 / 3 / 8A / 9A, SCN10A, SCN11A, KCNQ, BDNF, OPRD1 / K1 / M1, CNR1, GABRs, TNF, PLA2, IL1 / 6 / 12 / 18, COX-2, NTRK1, NGF, GDNF, TNF, LIF, CCL1, CNR2, TLR2 / 4, P2RX47, CCL2, CX3CR1, BDNF, NR1 / 2, GR1A1-4, GRC1-5, NK1R, CACNA1A-S, and CACNA2D1, and the expression of the gene product is inhibited.

[0147] Aspect 27: The construct of the zinc-finger repressor according to Aspect 26, wherein the target nucleic acid sequence is the sequence shown in Table 2.

[0148] Aspect 28: The transcriptional repressor is an mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl-CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( A construct of the zinc-finger repressor according to embodiment 26 or 27, selected from the group consisting of JPEG2025084803000011.jpg1279, KRAB).

[0149] Embodiment 29 A polynucleotide encoding a construct of the zinc-finger repressor according to embodiment 26, 27 or 28.

[0150] Embodiment 30 The polynucleotide according to embodiment 29, wherein the polynucleotide is a codon optimized for expression in human cells.

[0151] Embodiment 31 A vector comprising the polynucleotide according to embodiment 29 or 30.

[0152] Embodiment 32 The vector according to embodiment 31, wherein the polynucleotide is operably linked to a promoter.

[0153] Embodiment 33 The vector according to embodiment 32, wherein the promoter is selected from the group consisting of human cytomegalovirus (CMV) promoter, CAG promoter, Rous sarcoma virus (RSV) LTR promoter / transcription enhancer, SV40 promoter, EF1-α promoter, CMV immediate / early gene transcription enhancer / CBA promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, TRPV1 promoter, synapsin promoter, calcium / calmodulin-dependent protein kinase II promoter, tubulin αI promoter, neuron-specific enolase promoter, and glial fibrillary acidic protein (GFAP) promoter.

[0154] Embodiment 34 The vector according to embodiment 31, 32 or 33, further comprising a regulatory control sequence.

[0155] Embodiment 35 The vector according to embodiment 34, wherein the regulatory control sequence is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0156] Aspect 36: The vector according to any one of Aspects 31 to 35, wherein the vector is a recombinant adeno-associated virus vector (rAAV vector).

[0157] Aspect 37: The vector according to Aspect 36, wherein the rAAV is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vector (scAAV).

[0158] Aspect 38: The vector according to any one of Aspects 31 to 37, wherein the polynucleotide contains one or more inverted repeats (ITRs).

[0159] Aspect 39: The vector according to any one of Aspects 31 to 38, wherein the polynucleotide contains a polyA sequence.

[0160] Aspect 40: The vector according to Aspect 31, wherein the nucleic acid is engineered to express one or more components in a cell.

[0161] Aspect 41: The vector according to Aspect 31, wherein the vector is a lentiviral vector, a gammaretroviral vector, or a herpes simplex virus vector.

[0162] Aspect 42: The vector according to Aspect 31, wherein the vector contains a nucleic acid encoding the KRAB sequence of SEQ ID NO: 7.

[0163] Aspect 43: An epigenetically based method for treating or controlling chronic pain in a subject, the method comprising administering an effective amount of the complex according to any one of Aspects 1 to 6, the vector according to any one of Aspects 9 to 25, the construct according to any one of Aspects 26 to 28, or the vector according to any one of Aspects 31 to 42.

[0164] Aspect 44: An epigenetically based method for treating or controlling pain in a subject in need of treatment, the method comprising administering to the subject an effective amount of a construct of a zinc finger-suppressor and / or a complex of a dCas9-suppressor domain, wherein the dCas9 is a catalytically inactivated Cas9 that does not cleave DNA but maintains the ability to bind to the genome via a guide-RNA (gRNA).

[0165] Aspect 45: The method according to Aspect 44, wherein the pain is selected from neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-related neuropathy, nerve injury, rheumatoid arthritis pain, arthritic pain, burns, back pain, eye pain, visceral pain, cancer pain, bone cancer pain, migraine, pain due to carpal tunnel syndrome, fibromyalgia, neuropathic inflammatory pain, sciatic pain, pain of pelvic hyperalgesia, pelvic pain, pain of postherpetic neuralgia, postoperative pain, post-stroke pain, and menstrual pain.

[0166] Aspect 46: The method according to Aspect 44, wherein the pain is related to a disease or disorder selected from the group consisting of neuropathic peripheral neuropathy, diabetic neuropathy, postprandial neuralgia, trigeminal neuralgia, back injury, cancer neuropathy, HIV neuropathy, limb loss, carpal tunnel syndrome, stroke, alcohol dependence, hyperthyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, and epilepsy.

[0167] Aspect 47: The epigenetic method according to Aspect 44, wherein the method is used to treat a subject having chronic pain.

[0168] Aspect 48. The construct of the zinc finger-suppressor factor includes a suppressor domain selected from the group consisting of mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), Friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( JPEG2025084803000012.jpg1279, KRAB), and is an epigenetic method according to any one of Aspects 44 to 47.

[0169] Aspect 49. The epigenetic method according to Aspect 48, wherein the suppressor domain includes KRAB.

[0170] Aspect 50. The construct of the zinc finger-suppressor factor binds to the target in Table 2, and is an epigenetic method according to any one of Aspects 44 to 49.

[0171] Aspect 51. The complex of the dCas9-suppressor domain includes a suppressor domain selected from the group consisting of mSin3 interaction domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), Friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis thaliana HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-associated box ( JPEG2025084803000013.jpg1279, KRAB), and is an epigenetic method according to any one of Aspects 44 to 47.

[0172] Aspect 52: The epigenetic method according to aspect 51, wherein the inhibitory factor domain contains KRAB.

[0173] Aspect 53: The epigenetic method according to any one of aspects 44-47 or 51-52, wherein the construct of the dCas9-inhibitory factor domain contains a guide RNA spacer sequence having a sequence selected from SEQ ID NOs: 11-106 and 107.

[0174] Aspect 54: The epigenetic method according to any one of aspects 44-53, wherein the construct of the zinc finger-inhibitory factor and / or the construct of the dCas9-inhibitory factor domain provides non-permanent gene suppression of voltage-gated sodium channels.

[0175] Aspect 55: The epigenetic method according to aspect 54, wherein the voltage-gated sodium channel is selected from NaV1.7, NaV1.8, and NaV1.9.

[0176] Aspect 56: The epigenetic method according to aspect 55, wherein the voltage-gated sodium channel is NaV1.7.

[0177] Aspect 57: The epigenetic method according to any one of aspects 44-56, wherein the construct of the zinc finger-inhibitory factor and / or the construct of the dCas9-inhibitory factor domain are packaged and delivered by a recombinant virus or vector.

[0178] Aspect 58: The epigenetic method according to aspect 57, wherein the recombinant virus is an adenovirus, a gamma-retrovirus, an adeno-associated virus (AAV), a herpes simplex virus (HSV), or a lentivirus.

[0179] Aspect 59. The epigenetic method according to aspect 57, wherein the recombinant virus is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vector (scAAV).

[0180] Aspect 60. The epigenetic method according to any one of aspects 44 to 59, wherein the construct of the zinc finger-suppressor and / or the construct of the dCas9-suppressor domain is administered intravenously, intraperitoneally, intrathecally, intraganglionically, intraneurally, intracranially, or intramuscularly.

Examples

[0181] The following examples are intended for illustration but do not limit the present disclosure. These are typical ones that can be used, but other methods known to those skilled in the art may also be used.

Example 1

[0182] Design and construction of vectors Cas9 and zinc-finger AAV vectors were constructed by sequentially assembling the corresponding gene blocks (Integrated DNA Technologies) into a custom-synthesized rAAV2 vector backbone. Oligonucleotides (IDT) encoding the spacers were cloned into the AgeI cloning site via Gibson assembly to insert the gRNA sequence into the dNCas9 plasmid. gRNAs were designed to predict gRNAs73 using in silico tools.

[0183] Culture of mammalian cells Neuro2a cells were grown in EMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics - antimycotics at 37 °C and 5% CO 2 atmosphere.

[0184] Lipid-mediated gene transfer into cells One day before gene transfer, Neuro2a cells were seeded into 24-well plates at a cell density of 1 or 2E+5 cells per well. 0.5 mL of each plasmid was added to 25 μL of Opti-MEM medium, followed by addition of 25 μL of Opti-MEM containing 2 μL of Lipofectamine 2000. The mixture was incubated at room temperature for 15 minutes. Then the total volume of the solution was added to the cells in the 24-well plate, and the plate was gently swirled to mix. After 24 hours, the medium was changed, and the plate was incubated at 37 °C in a 5% CO 2 incubator for 72 hours. The cells were harvested, centrifuged, and frozen at -80 °C.

[0185] Production of AAV Viruses were prepared by the Gene Transfer, Targeting, and Therapy (GT3) Core at the Salk Institute for Biological Studies (La Jolla, CA), or prepared in-house using the GT3 core protocol. Briefly, HEK293T cells were used to generate AAV2 / 1, AAV2 / 5, and AAV2 / 9 virus particles by a triple gene transfer method and purified by iodixanol gradient. The culture density at the time of gene transfer was 80% - 90%. Two hours before gene transfer, the medium was replaced with pre-warmed medium. Each virus was produced in five 15 cm plates and transfected with 10 μg of pXR-capsid (pXR-1, pXR-5, and pXR-9), 10 μg of recombinant transcription vector, and 10 μg of helper vector per plate using polyethyleneimine (PEI; 1 mg / mL linear PEI in DPBS solution, [pH 4.5], using HCl) at a PEI:DNA mass ratio of 4:1. The mixture was incubated at room temperature for 10 minutes and then dropped into the medium. After 72 hours, the virus was harvested and purified using ultracentrifugation on an iodixanol density gradient. The final volume was then brought to approximately 100 μL using a 50-kDa filter (Millipore), and the virus was dialyzed against 1x PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher Scientific), and quantified by qPCR using primers specific for the ITR region against the standard (ATCC VR-1616): AAV-ITR-F: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 127) and AAV-ITR-R: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 128).

[0186] Animal experiments All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. All mice were obtained from the Jackson Laboratory. Adult male C57BL / 6 mice (25 - 30 g) at 2 months of age were housed with free access to food and water under a 12 - hour light - dark cycle with a maximum of 5 mice per cage. All behavioral tests were performed during the light cycle period.

[0187] Intrathecal injection of AAV Until the loss of the right - eye reflex was observed, an equal volume of O 2 and 2.5% isoflurane delivered in room air were used to induce anesthesia. The lower back of the mice was shaved and 70% ethanol was applied. Then, as described above (104), using a Hamilton syringe and a 30G needle, 5 μL of AAV at a total of 1E+12 vg / mouse was intrathecally injected (i.t.) into the mice between vertebrae L4 and L5. Tail flicks were considered an indicator of proper needle placement. After injection, all mice resumed movement as observed prior to i.t. injection.

[0188] Pain model Intra - plantar injection of carrageenan: Carrageenan - induced inflammation is a classical model of edema formation and hyperalgesia (105 - 107). Twenty - one days after AAV pretreatment, anesthesia was induced as described above. λ - Carrageenan (Sigma Aldrich; 2% (W / V), dissolved in 0.9% (W / V) NaCl solution, 20 μL) was subcutaneously injected into the plantar (ventral) surface of the same - side foot with a 30G needle. An equal volume of isotonic saline was injected into the opposite foot. As an indicator of edema / inflammation, the foot thickness was measured with calipers before and 4 hours after carrageenan / saline injection. Hargreaves tests were performed before injection (t = 0) and at t = 30, 60, 120, 240 minutes, and 24 hours after injection. The experimenter was blinded to the treatment - group composition. After 24 hours, the mice were euthanized.

[0189] Paclitaxel-induced neuropathy: Paclitaxel (Tocris Biosciences, 1097) was dissolved in a 1:1:18 (v / v) mixture of ethanol, Cremophor EL (Millipore, 238470), and sterile 0.9% (w / v) NaCl solution. Paclitaxel injection (8 mg / kg) was administered intraperitoneally (i.p.) at a volume of 1 mL / 100 g body weight every other day for a total of 4 injections to induce neuropathy (32 mg / kg), resulting in a cumulative dose equivalent to 28.4 - 113.5 mg / m2 in humans as described above. Behavioral tests were performed 24 hours after the final administration.

[0190] Intrathecal injection of BzATP: BzATP (2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt) was purchased from Millipore Sigma and dissolved in physiological saline (0.9% NaCl) to a final concentration of 30 nmol based on previous tests. The saline was also used as a solvent control, and both were injected in a volume of 5 μl. Using a 30-gauge needle attached to a Hamilton syringe, intrathecal injection was performed by lumbar puncture under isoflurane (2.5%) anesthesia.

[0191] Behavioral tests Before the test, mice were acclimated to the behavior and the laboratory for at least 30 minutes. As a positive control, gabapentin (Sigma, G154) was dissolved in physiological saline and injected i.p. at 100 mg / kg / mouse.

[0192] Heat withdrawal latency (Hargreaves test): To measure the acute nociceptive heat threshold, the Hargreaves test was performed using a plantar test apparatus (Ugo Basile, Italy) for the plantar surface. Forty minutes before the test, the animals were allowed to move freely on a glass floor in a transparent plastic storage container (6 cm in diameter × 16 cm in height). Subsequently, a mobile radiant heat source was placed under the glass floor and focused on the hind paw. The withdrawal latency was measured with a 30-second cut-off time. An IR intensity of 40 was used. Thermal stimuli were repeated three times on each hind paw at 10-minute intervals, and the mean of the paw withdrawal latency was obtained. The experimenter was blinded to the composition of the treatment groups.

[0193] Allodynia: For the BzATP pain model, the tactile threshold (allodynia) was evaluated at 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours after BzATP injection. For the paclitaxel model, the tactile threshold (allodynia) was evaluated at 24 hours and 29 days after the final injection of paclitaxel. Forty-five minutes before the test, the mice were placed in a cage with a transparent plastic wire mesh bottom for acclimation. As described above, the 50% probability of the withdrawal threshold was evaluated using up-and-down methods with von Frey filaments of 2.44 - 4.31 (0.04 - 2.00 g) (Semmes Weinstein von Frey anesthesiometer; Stoelting Co., Wood Dale, IL, USA).

[0194] Cold allodynia A droplet of acetone was applied to the plantar surface of the hind paw, and cold allodynia was measured. The mice were placed on the elevated platform of an individual plastic cage and allowed to acclimate for at least 30 minutes until exploratory behavior ceased. A syringe barrel without a needle tip was loaded with acetone. Then, one drop of acetone (about 20 μL) was applied to the plantar surface of the hind paw through a mesh platform. Care was taken to gently apply the acetone foam to the skin on the paw so as not to induce a physical stimulus through contact between the syringe barrel and the paw. The withdrawal time of the paw during the 60-second observation period after acetone application was recorded. The behavior of paw withdrawal was associated with secondary animal responses such as rapid twitching of the paw, tremors, biting, and / or licking of the paw. The test order was alternated between the left and right paws until 5 measurements were made for each paw. A 5-minute interval of cross-stimulation was given between the tests of the left and right paws.

[0195] Tissue collection Twenty-four hours after carrageenan administration, the spinal cord was collected by hydraulic extraction (2 mL of ice-cold saline was injected through a short blunt 20-gauge needle placed in the spinal canal after decapitation). After collecting the spinal cord tissue, the DRGs of L4-L6 on each side were combined and the spinal cord was frozen. The samples were placed in 1.5 mL centrifuge tubes without Dnase / Rnase, rapidly frozen with dry ice, and stored at -80 °C for future analysis.

[0196] Gene expression analysis and qPCR RNA from Neuro2a cells was extracted using the Rneasy Kit (QIAGEN; 74104), and RNA from DRG was extracted using the Rneasy Micro Kit (QIAGEN; 74004). cDNA was synthesized from the RNA using the Protoscript II Reverse Transcriptase Kit (NEB; E6560L). Real-time PCR (qPCR) reactions with gene-specific primers were performed technically in three replicates and biologically (Neuro2a cells) in three replicates using the KAPA SYBR Fast qPCR Kit (Kapa Biosystem, KK4601). Relative mRNA expression was normalized to the GAPDH level, and fold changes were calculated using the comparative CT (ΔΔCT) method, normalized to GAPDH. Mean fold changes and SD were calculated using Microsoft Excel.

[0197] Western blot Neurons 2a cells were thawed and protein extraction was performed using RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Thermo Fisher 89900) supplemented with protease inhibitor (Sigma P8849). Total protein was quantified using a BCA protein assay kit (Thermo Fisher 23225), and 40 μg of protein was loaded onto a 4–15% polyacrylamide gel (BioRad 4561085). The protein was transferred to a PVDF membrane (Thermo Fisher IB401001), and the membrane was blocked with 5% (w / v) blotting-grade blocker (Biorad 1706404) dissolved in TBS-T (Thermo Fisher, 28358, supplemented with 0.1% (v / v) Tween-20, BioRad 1610781). Next, the membrane was incubated overnight at 4 °C with primary antibodies (anti-NaV1.7 (Abcam; ab85015) diluted 1:1000 and anti-GAPDH (Cell Signaling, 2118) diluted 1:4000). The membrane was then washed three times with TBS-T and incubated for 1 h at room temperature with a secondary antibody, anti-rabbit horseradish peroxidase conjugate (Cell Signaling, 7074) diluted 1:20000. After washing with TBST, the blot was visualized using SuperSignal West Femto chemiluminescent substrate (Thermo Fisher) and visualized on X-ray film.

[0198] RNAscope ISH assay mCherry, NaV1.7, and NeuN probes were designed by Advanced Cell Diagnostics (Hayward, CA). The mCherry probe (ACD catalog number 404491) was designed to detect 1480 - 2138 bp (KF450807.1, Channel C1), the NaV1.7 (ACD catalog number 313341) was designed to detect 3404 - 4576 bp of the Rattus norvegicus NaV1.7 mRNA sequence (NM_018852.2, Channel C3), and the NeuN probe (ACD catalog number 313311) was designed to target 1827 - 3068 bp of the Rattus norvegicus NeuN gene (NM_001039167.1, Channel C2). Before sectioning, DRGs were placed in 4% PFA for 2 h at room temperature and then incubated overnight at 4 °C in 30% sucrose. Tissues were sectioned (12 μm thick) and placed on positively charged microscope glass slides (Fisher Scientific). All hybridization and amplification steps were performed according to the ACD RNAscope V2 Fixed Tissue protocol. Stained slides were covered with a fluorescence mounting medium (ProLong Gold antifade reagent P36930; Life Technologies) and scanned into digital images at 20× magnification using a Zeiss 880 Airyscan Confocal. Data were processed using ZEN software (provided by the manufacturer).

[0199] Statistical analysis Results are presented as mean ± standard error (SE). Statistical analysis was performed using GraphPad Prism (Ver. 8.0, GraphPad Software, San Diego, CA, USA). Results were analyzed using two-way ANOVA with Student's t-test (for differences between two groups), one-way ANOVA (for multiple groups), or two-way ANOVA with Bonferroni post hoc test (for time-course experiments with multiple groups). Differences between groups with p < 0.05 were considered statistically significant.

[0200] Design and construction of split vectors The split-Cas9 / dCas9 AAV vectors were constructed by sequentially assembling the corresponding gene blocks (integrated DNA technology) into a custom-synthesized rAAV2 vector backbone. Oligonucleotides (IDT) encoding the spacers were cloned into the AgeI cloning site via Gibson assembly to insert the gRNA sequences into the NCas9 or dNCas9 plasmids.

[0201] Production of AAV Briefly, HEK293T cells were used to produce AAV2 / 9 and virus particles by the triple transfection method and purified by iodixanol gradient. The culture density at the time of transfection was 80% - 90%. Two hours before transfection, the medium was replaced with pre-warmed medium. Each virus was produced in 5 x 15 cm plates, where polyethyleneimine (PEI; 1 mg / mL linear PEI in 1 DPBS solution, [pH 4.5], using HCl) was used to transfect each plate at a PEI:DNA mass ratio of 4:1 with 7.5 mg of pXR-capsid (pXR-9), 7.5 mg of the recombinant transfer vector, and 22.5 mg of the pAd5 helper vector. The mixture was incubated at room temperature for 10 minutes and then dropped into the medium. After 72 hours, the virus was harvested and purified using ultracentrifugation on an iodixanol density gradient. Next, the final volume was brought to 1 mL using a 100-kDa filter (Millipore), and the virus was dialyzed against 1 PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher Scientific).

[0202] Injection of AAV AAV injection was performed by intrathecal injection in adult C57BL / 6J mice (10 weeks old) using either 1E+12 vg / mouse of split Cas9 (total virus 2E+12 vg / mouse) or 1E+12 vg / mouse of ZF.

[0203] To establish strong Nav1.7 inhibition, the in vitro Nav1.7 inhibition effects were compared using KRAB-dCas9 and ZFP-KRAB constructs. For this purpose, ten guide RNAs (gRNAs; Table 3) designed by an in silico tool that predicts highly effective gRNAs based on chromatin location and sequence features were cloned into the split dCas9 platform. Also, since higher efficiency can be achieved by using multiple gRNAs, the two most predicted efficient gRNAs (SCN9A-1 and SCN9A-2) were also cloned into a single construct. Next, four ZFP-KRAB constructs targeting the Nav1.7 DNA sequence were designed (Table 4). These constructs were transfected into a mouse neuroblastoma cell line expressing Nav1.7 (Neuro2a), and Nav1.7 was suppressed by qPCR relative to GAPDH. Six out of the ten gRNAs suppressed Nav1.7 transcripts by more than 50% compared to the non-target gRNA control. Among them, gRNA-2 was the single gRNA with the highest suppression (56%), and the suppression level of the double gRNA was 71% (p<0.0001). These were then utilized in subsequent in vivo studies (Figure 5A). Among the ZFP-KRAB designs, the zinc-finger-4-KRAB construct showed the highest suppression (88%; p<0.0001) compared to the negative control (mCherry) selected for subsequent in vivo studies (Figure 5A). Western blotting confirmed a decrease in the corresponding protein levels in both the zinc-finger-4-KRAB group and the KRAB-dCas9-double gRNA group (Figure 5B).

[0204] Table 3: CRISPR-Cas9 guide RNA spacer sequences JPEG2025084803000014.jpg68137

[0205] Table 4: Zinc finger protein genomic target sequences JPEG2025084803000015.jpg31135

[0206] After establishing Nav1.7 inhibition in vitro, the efficacy of the best ZFP-KRAB and KRAB-dCas9 constructs from in vitro screening (zinc finger-4-KRAB and KRAB-dCas9-dual gRNA) in a carrageenan-induced inflammatory pain model was tested. AAV9-mCherry (negative control; n = 10), AAV9-zinc finger-4-KRAB (n = 10), AAV9-KRAB-dCas9 without gRNA (negative control; n = 10), and AAV9-KRAB-dCas9-dual gRNA (n = 10) at 1E+12 vg / mouse were injected intrathecally (i.t.) into mice. Intrathecal delivery of AAV9 with significant neuronal tropism helps to efficiently target the DRG (Figure 6A). After 21 days, thermal pain sensitivity was measured to establish the baseline response threshold. For all four groups of mice, carrageenan (ipsilateral) was injected into one hind paw to induce inflammation, and saline was injected into the other hind paw (contralateral) as an internal control in the mice. Next, the thermal pain sensitivity of the mice was tested 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours after carrageenan injection (Figure 1B). Twenty-four hours after carrageenan administration, the mice were euthanized and the DRG (L4-L6) was extracted. The expression level of Nav1.7 was measured by qPCR, and significant inhibition of Nav1.7 was observed in mice injected with AAV9-zinc finger-4-KRAB (67%; p = 0.0008) and in mice injected with AAV9-KRAB-dCas9-dual gRNA (50%; p = 0.0033) compared with mice injected with AAV9-mCherry and compared with mice injected with AAV9-KRAB-dCas9 without gRNA (Figure 1C). The mean paw withdrawal latency (PWL) was calculated for both paws injected with carrageenan and saline (Figure 6B-C), and the area under the curve (AUC) of the total mean PWL was calculated. As expected, the paw injected with carrageenan developed thermal hyperalgesia, measured by a decrease in PWL after application of a thermal stimulus, compared with the paw injected with saline (Figure 1D). Also, a significant increase in PWL was observed in mice injected with either AAV9-zinc finger-4-KRAB or AAV9-KRAB-dCas9-dual gRNA.This indicates that suppression of Nav1.7 in mouse DRG leads to a decrease in heat hyperalgesia in an inflammatory pain state. There was no significant difference in the heat latency of the control (non-inflamed foot) among the AAV treatment groups, indicating that knockdown of Nav1.7 had minimal impact on normal heat sensitivity. As an indicator of edema / inflammation, the ipsilateral and contralateral feet were measured with calipers before carrageenan injection and 4 hours after injection, which was the time point of the highest heat hyperalgesia. Significant edema formation was observed in both the experimental and control groups, indicating that Nav1.7 suppression does not affect inflammation (Figure 6D).

[0207] To verify the effectiveness of ZFP-KRAB in improving heat hyperalgesia in the carrageenan model of inflammatory pain, another experiment was conducted, and the small molecule drug gabapentin was tested as a positive control. Mice were injected with AAV9-mCherry (n = 5), AAV9-zinc-finger-4-KRAB (n = 6), or saline (n = 5) at a dose of 1E+12 vg / mouse. After 21 days, heat pain sensation was measured for all mice as described above. One hour before carrageenan administration, gabapentin (100 mg / kg) was intraperitoneally (i.p.) injected into mice administered intrathecal saline as a positive control. The drug is known to reduce carrageenan-induced heat hyperalgesia in rodents by binding to the spinal α2δ subunit of voltage-dependent calcium channels. Twenty-four hours after carrageenan administration, the mice were euthanized and DRG (L4-L6) was extracted. The expression level of Nav1.7 was measured by qPCR, and significant suppression of Nav1.7 was observed in the AAV9-zinc-finger-4-KRAB group (***p = 0.0007) and the gabapentin group (*p = 0.0121) (Figure 7A). Before and 4 hours after carrageenan injection, the contralateral and ipsilateral feet were measured with calipers, and significant edema formation was confirmed in the injected feet of all groups compared to the non-injected feet of all groups (Figure 7B). The mean PWL was calculated for the feet injected with both carrageenan and saline (Figure 2B-C). Next, for the AAV9-zinc-finger-4-KRAB group and the gabapentin group, the paw withdrawal latency of the carrageenan-injected paw was compared with that of the AAV9-mCherry carrageenan-injected control at each time point using two-way ANOVA calculations to measure whether there was a significant reduction in thermal hyperalgesia (Figure 7C). When comparing the hind paws injected with carrageenan, it was observed that only the AAV9-zinc-finger-4-KRAB group showed a significantly higher PWL at all time points after carrageenan injection compared to the AAV9-mCherry control group. Also, significance in PWL was observed in the gabapentin positive control group at the 30-minute, 1-hour, and 4-hour time points, but not at the 24-hour time point. This result reflects the half-life of gabapentin (3 - 5 hours). Next, the area under the curve (AUC) was calculated for thermal hyperalgesia. A significant increase was observed in the PWL of the carrageenan-injected gabapentin group (p = 0.0208) (Figure 2B) and the zinc-finger-4-KRAB group (115% improvement, p = 0.0021) (Figure 2C) compared to the carrageenan-injected AAV9-mCherry control group. Furthermore, the AAV9-zinc-finger-4-KRAB group had a PWL 31% higher than that of the gabapentin positive control group. There was no significant difference in the thermal withdrawal latency of the contralateral non-inflammatory paw among the groups.

[0208] After establishing the in vivo efficacy in an inflammatory pain model, experiments were conducted using a chemotherapy-induced peripheral neuropathy model with paclitaxel, and an epigenetic suppression strategy for neuropathic pain was verified. To establish this model, mice were first injected with AAV9-mCherry (n = 8), AAV9-zinc-finger-4-KRAB (n = 8), AAV9-KRAB-dCas9-dual gRNA (n = 8), AAV9-KRAB-dCas9 without gRNA (n = 8), or saline (n = 16) at a dose of 1E+12 vg / mouse. Baselines for tactile thresholds (von Frey filaments) were established before and 14 days after paclitaxel administration. Next, on days 14, 16, 18, and 20, mice were administered paclitaxel at a dose of 8 mg / kg (total cumulative dose 32 mg / kg). To establish tactile allodynia induced by the chemotherapeutic agent, a group of mice (n = 8) injected with saline without paclitaxel was used. Twenty-one days after the first injection and 1 hour before the test, gabapentin (100 mg / kg) was injected i.p. into a group of mice (n = 8) injected with saline. Next, the mice were tested for tactile allodynia with von Frey filaments and cold allodynia with acetone (Figure 3A). The 50% tactile threshold was calculated. A decrease in tactile threshold was observed in mice administered AAV9-mCherry and AAV9-KRAB-dCas9 without gRNA, while the withdrawal threshold increased in mice administered gabapentin, AAV9-zinc-finger-4-KRAB, and AAV9-KRAB-dCas9-dual gRNA. This indicates that in situ Nav1.7 suppression leads to improvement in chemotherapy-induced tactile allodynia (Figure 3B). Similarly, an increase in the number of withdrawal responses was seen in mice tested for cold allodynia in the negative control groups (AAV9-mCherry and AAV9-KRAB-dCas9 without gRNA), while the withdrawal response decreased in both the AAV9-zinc-finger-4-KRAB group and the AAV9-KRAB-dCas9-dual gRNA group. This indicates that in situ Nav1.7 suppression also leads to a decrease in chemotherapy-induced cold allodynia (Figure 3C).

[0209] Next, experiments were conducted to verify whether in situ suppression of Nav1.7 via KRAB-dCas9 could improve neuropathic pain induced by BzATP. This molecule activates P2X receptors at the central terminal, causing central sensitization of pain. Mice were first injected with AAV9-mCherry (n = 6), AAV9-KRAB-dCas9 without gRNA (n = 5), and AAV9-KRAB-dCas9-dual gRNA (n = 6) at a dose of 1E+12 vg / mouse. After 21 days, tactile thresholds were measured using von Frey filaments, and mice were injected i.t. with BzATP (30 nmol). Next, tactile allodynia was measured 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours after BzATP administration (Figure 3D). In mice injected with AAV9-KRAB-dCas9-dual gRNA, significant reduction of tactile allodynia was observed at 30 minutes, 1 hour, and 2 hours, and an overall increase in tactile threshold was observed at all time points (Figure 3E).

[0210] To verify whether Nav1.7 suppression in situ is effective in the long term, the carrageenan inflammatory pain model was repeated for thermal hyperalgesia 21 and 42 days after i.t. injection of AAV (n = 8 / group) (Figure 4A). On both day 21 (Figure 7D) and day 42 (Figure 4B), a significant improvement in PWL was observed in the carrageenan-injected paw of the zinc finger-4-KRAB group. This indicates the durability of the approach. To verify whether Nav1.7 suppression in situ was also effective in the long term in a model of neuropathic pain, tactile allodynia and cold allodynia were measured 49 days after the first injection of AAV and 29 days after the last injection of paclitaxel (total cumulative dose 32 mg / kg; Figure 4C). Compared to previous time points (Figure 3B-C), mice in both the AAV9-mCherry group (n = 8) and the AAV9-KRAB-dCas9-dual gRNA group (n = 8) had increased tactile allodynia at day 49 compared to day 21 and responded to the lowest von Frey filament tested (0.04 g). In contrast, mice administered AAV9-zinc-finger-4-KRAB and AAV9-KRAB-dCas9-dual gRNA had increased withdrawal thresholds. This indicates that Nav1.7 suppression in situ leads to long-term improvement in chemotherapy-induced tactile allodynia (Figure 4C). As before, an increase in the number of withdrawal responses was seen in mice tested for cold allodynia in the negative control groups (without AAV9-mCherry and AAV9-KRAB-dCas9-gRNA), while the withdrawal responses decreased in both the AAV9-zinc-finger-4-KRAB group and the AAV9-KRAB-dCas9-dual gRNA group. This indicates that Nav1.7 suppression in situ also leads to long-term improvement in chemotherapy-induced cold allodynia (Figure 4E).

[0211] The present disclosure also demonstrates that the methods and compositions restore a state of chronic pain. In these experiments, mice were first treated with paclitaxel to induce chronic pain. After confirming organic allodynia with von Frey filaments, the mice were injected with a gene inhibitor, and after 2 and 3 weeks, organic allodynia recovered in the group of mice that received the ZF gene inhibitor (Figure 10A-B).

[0212] It will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A zinc-finger repressor construct comprising an engineered zinc finger DNA binding domain linked to a transcriptional repressor, wherein the zinc-finger DNA binding domain comprises one to six zinc-finger sequences, the zinc-finger sequences being (a) SCN9A or (b) SCN10A or (c)TRPV1 / 2 / 3 / 4, P2XR3, TRPM8, TRPA1, P23X2, P2RY, BDKRB1 / 2, Hlr3A, ACCNs, TRPV4, TRPC / P, ACCN1 / 2, SCN 1 / 3 / 8A / 9A, SCN10A, SCN11A, KCNQ, BDNF, OPRD1 / K1 / M1, CNR1, GABRs, TNF, PLA2, IL1 / 6 / 12 / 18, COX-2, NTRK1 , NGF, GDNF, TNF, LIF, CCL1, CNR2, TLR2 / 4, P2RX47, CCL2, CX3CR1, BDNF, NR1 / 2, GR1A1-4, GRC1-5, NK1R, CACNA1A-S, and CACNA2D1, thereby inhibiting expression of the gene product.

2. The zinc-finger inhibitor construct of claim 1, wherein the zinc-finger inhibitor comprises a nuclear localization signal (NLS), a linker, or both.

3. The transcriptional repressors include mSin3 interacting domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl-CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krueppel-related box ( 2. The zinc-finger inhibitor construct of claim 1, wherein the zinc-finger inhibitor is selected from the group consisting of: KRAB.

4. The zinc-finger repressor construct of claim 1, wherein the transcription repressor domain is a KRAB domain.

5. A polynucleotide encoding one or more components of the zinc-finger inhibitor construct of claim 1.

6. A vector comprising the polynucleotide of claim 5, preferably wherein the polynucleotide is operably linked to a promoter.

7. The vector of claim 6, wherein the promoter is selected from the group consisting of human cytomegalovirus (CMV) promoter, CAG promoter, Rous sarcoma virus (RSV) LTR promoter / transcriptional enhancer, SV40 promoter, EF1-α promoter, CMV immediate / early gene transcriptional enhancer / CBA promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, TRPV1 promoter, synapsin promoter, calcium / calmodulin-dependent protein kinase II promoter, tubulin αI promoter, neuronal-specific enolase promoter, and glial fibrillary acidic protein (GFAP) promoter.

8. 7. The vector of claim 6, further comprising a regulatory control sequence, preferably the regulatory control sequence being a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

9. 9. The vector according to claim 8, wherein the vector is a recombinant adeno-associated virus vector (rAAV vector), preferably the rAAV is selected from the group consisting of AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F), AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV210, AAV22, AAV23, AAV24, AAV25, AAV26, AAV27, AAV28, AAV29, AAV30, AAV31, AAV32, AAV33, AAV34, AAV35, AAV36, AAV37, AAV38, AAV39, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV46, AAV47, AAV48, AAV49, AAV49, AAV41, AAV42, AAV43, AAV44, AAV45, AAV45, AAV46, AAV47, AAV48, AAV49 ... (VP3 mutant Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 mutant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and a self-complementary vector (scAAV), optionally wherein the polynucleotide comprises one or more inverted repeats (ITRs), or wherein the polynucleotide comprises a polyA sequence.

10. The vector of claim 6, wherein the polynucleotide is engineered to be expressed in a cell, or the vector is a lentiviral vector, a gammaretroviral vector, or a herpes simplex viral vector, or the vector comprises a nucleic acid encoding the KRAB sequence of SEQ ID NO:

7.

11. 10. A zinc-finger inhibitor construct according to claim 1 for use in treating or controlling chronic pain in a subject.

12. the pain is selected from neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus associated neuropathy, nerve injury, rheumatoid arthritis pain, arthritic pain, burns, back pain, eye pain, visceral pain, cancer pain, bone cancer pain, migraine, carpal tunnel syndrome pain, fibromyalgia, neuritis pain, cystic pain, pelvic hypersensitivity pain, pelvic pain, post-herpetic neuralgia pain, post-operative pain, post-stroke pain, and menstrual pain; or the pain is associated with a disease or disorder selected from the group consisting of peripheral neuropathic neuropathy, diabetic neuropathy, postprandial neuralgia, trigeminal neuralgia, back injury, cancer neuropathy, HIV neuropathy, limb loss, carpal tunnel syndrome, stroke, alcoholism, hyperthyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, and epilepsy; or The zinc-finger inhibitor construct of claim 11, wherein said treatment is treatment of chronic pain.

13. The transcriptional repressors include mSin3 interacting domain (SID) protein, methyl-CpG binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl-CpG binding protein 2 (Mecp2), friend of GATA 1 (Fog1), regulator of MAT2 (ROM2), Arabidopsis HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and Krüppel-related box ( 12. The zinc-finger inhibitor construct of claim 11, wherein the zinc-finger inhibitor is selected from the group consisting of: KRAB.

14. The zinc-finger repressor construct of claim 11, wherein the repressor domain is KRAB.

15. The zinc-finger inhibitor construct is packaged and delivered by a recombinant virus, preferably the recombinant virus is an adenovirus, a gammaretrovirus, an adeno-associated virus (AAV), a herpes simplex virus (HSV), or a lentivirus, preferably the recombinant virus is an AAV1, AAV1(Y705+731F+T492V), AAV2, AAV2(Y444+500+730F+T491V), AAV3, AAV3(Y705+731F) 12. The zinc-finger inhibitor construct of claim 11, selected from the group consisting of AAV4, AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 mutants Y705F / Y731F / T492V), AAV7, AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 mutant Y731F), AAV10, AAV10(Y733F), AAV-ShH10, AAV11, AAV12, and self-complementary vectors (scAAV).

Images