Compositions and methods for epigenetic modulation of nav1.7

Abstract

Described herein are epigenetic modulators that modulate the expression of voltage-gated sodium channels such as Na V 1.7 without editing the genetic sequence. The epigenetic modulators can include nucleic acid binding agents that bind to a target sequence within a gene encoding the voltage-gated sodium channel and expression modulators that modulate the expression of the gene. Also described herein are methods of using epigenetic modulators of Na V 1.7 to treat inflammation, pain, or both.

Description

[0001] Cross-referencing

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 588,260, filed October 5, 2023, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC MODULATION OF NAV1.7”; U.S. Provisional Application No. 63 / 543,029, filed October 6, 2023, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC MODULATION OF NAV1.7”; and U.S. Provisional Application No. 63 / 692,441, filed September 9, 2024, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC MODULATION OF NAV1.7”, each of which is incorporated herein by reference in its entirety for all purposes.

[0003] Government support

[0004] This invention was made with government support under patent U44NS122114 granted by the National Institutes of Health and patent DISC2-13013 granted by the California Institute for Regenerative Medicine. The government holds certain rights in this invention.

[0005] sequence list

[0006] This application contains a sequence list that has been electronically submitted in Extensible Markup Language (XML) format, and which is hereby incorporated herein by reference in its entirety. The XML copy created on October 4, 2024, is named “423157-711021_SL.xml” and has a size of 189,732 bytes. Background Technology

[0007] Chronic pain affects between 19% and 50% of the world's population, with over 100 million people affected in the United States alone. Despite the side effects and limited efficacy of opioids, they have become the preferred treatment for chronic pain in recent years for private and VA-prescribing physicians. However, opioids are highly addictive, and more than 130 Americans die every day from overdoses. Therefore, opioid overdoses pose a serious threat to public health. Although chronic pain is more prevalent than cancer, diabetes, and cardiovascular disease combined, drug development for chronic pain has not achieved the remarkable progress seen in these other treatment areas. Despite decades of research, broad-spectrum, long-acting, non-addictive, and effective treatments for chronic pain remain elusive. Summary of the Invention

[0008] In various aspects, this disclosure provides a zinc finger protein comprising (a) at least 90% sequence identity with SEQ ID NO: 2, (b) at least 98% sequence identity with SEQ ID NO: 3, and / or (c) at least 90% sequence identity with SEQ ID NO: 4.

[0009] In some aspects, the zinc finger protein includes a sequence having at least 95% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4. In some aspects, the zinc finger protein includes a sequence having at least 97% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4. In some aspects, the zinc finger protein includes a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In some aspects, the zinc finger protein has an affinity for SCN9A. In some aspects, the zinc finger protein has an affinity for a polynucleotide sequence having at least 90% identity with any one of SEQ ID NO: 73 to SEQ ID NO: 97.

[0010] In various aspects, this disclosure provides an epigenetic regulator comprising a zinc finger protein linked to a repressor domain.

[0011] In some aspects, the epigenetic regulator includes a zinc finger protein having a sequence having at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO: 169. In some aspects, the zinc finger protein includes a sequence having at least 95%, at least 97%, at least 98%, or 99% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO: 169. In some aspects, the epigenetic regulator includes a zinc finger protein with affinity for SCNA9. In some aspects, the epigenetic regulator includes a zinc finger protein having an affinity for a polynucleotide sequence having at least 90% sequence identity with any of SEQ ID NO: 73 to SEQ ID NO: 97. In some aspects, the epigenetic regulator includes a zinc finger protein having an affinity for a polynucleotide sequence of any of SEQ ID NO: 73 to SEQ ID NO: 97. In some aspects, the epigenetic regulator includes a repressive domain comprising ZIM3, SID, KOX1, ZNF554, ZNF264, ZNF324, MeCP2, MBD2b, SID, HP1a, SIRT5, SETD8, HDT1, SUPR, FOG1, DNMT3A, DNMT3L, DMT3, or combinations thereof. In some aspects, the repressive domain comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 5 to SEQ ID NO: 20. In some aspects, the repressive domain includes any one of SEQ ID NO: 5 to SEQ ID NO: 20. In some aspects, the repressive domain includes SEQ ID NO: 5 or SEQ ID NO: 9. In some aspects, the zinc finger protein in the epigenetic regulator is linked to the repressive domain via a peptide linker. In some aspects, the epigenetic regulator includes a second repressive domain. In some aspects, the second repressive domain includes ZIM3, SID, KOX1, ZNF554, ZNF264, ZNF324, MeCP2, MBD2b, SID, HP1a, SIRT5, SETD8, HDT1, SUPR, FOG1, DNMT3A, DNMT3L, DMT3, or combinations thereof. In some aspects, the second repressive domain has a sequence having at least 90% sequence identity with any one of SEQ ID NO: 5 to SEQ ID NO: 20.In some aspects, the second repressive domain comprises a sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 20. In some aspects, the second repressive domain comprises a sequence of either SEQ ID NO: 6 or SEQ ID NO: 7. In some aspects, the epigenetic regulator comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 25 to SEQ ID NO: 36. In some aspects, the epigenetic regulator comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 25 to SEQ ID NO: 36. In some aspects, the epigenetic regulator comprises a sequence having at least 95% sequence identity with any one of SEQ ID NO: 25 to SEQ ID NO: 36. In some aspects, the epigenetic regulator comprises the sequence of SEQ ID NO: 25. In some aspects, the epigenetic regulator includes the sequence of SEQ ID NO: 26. In some aspects, the epigenetic regulator includes the sequence of SEQ ID NO: 27. In some aspects, the epigenetic regulator includes the sequence of SEQ ID NO: 28.

[0012] In various aspects, this disclosure provides a polynucleotide encoding the zinc finger protein described herein. In some aspects, the polynucleotide includes a promoter.

[0013] In various aspects, this disclosure provides a polynucleotide encoding the epigenetic regulators described herein. In some aspects, the polynucleotide includes a promoter.

[0014] In some aspects, the promoter comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 37 to SEQ ID NO: 47 and SEQ ID NO: 98. In some aspects, the promoter comprises a sequence of SEQ ID NO: 37 to SEQ ID NO: 47 and / or SEQ ID NO: 98.

[0015] In various aspects, this disclosure provides a delivery vector encapsulating the polynucleotides of the present disclosure. In some aspects, the delivery vector comprises a viral vector. In some aspects, the delivery vector comprises a delivery-enhancing peptide. In some aspects, the delivery-enhancing peptide comprises a protoxin, jingzhaotoxin, theraphotoxin, phlotoxin, Grammostola porteri toxin, huwentoxin, Ceratogyrus cornuatus toxin, heteropodatoxin, heteroscodratoxin, and / or a penetration-enhancing peptide. In some aspects, the delivery-enhancing peptide comprises a sequence having at least 90% sequence identity with any of SEQ ID NO: 48 to SEQ ID NO: 72. In some aspects, the viral vector comprises an AAV vector, a lentiviral vector, a herpes simplex virus (HSV), or a rabies virus vector. In some respects, delivery vectors include lipid nanoparticles that encapsulate DNA, mRNA, or circular RNA encoding epigenetic regulators.

[0016] In various aspects, this disclosure provides a method for downregulating Na+ in cells. V Methods 1.7. In some aspects, the methods relate to expressing the epigenetic regulators described herein in cells.

[0017] In various aspects, this disclosure provides a method for treating a disorder or disease in a subject. In some aspects, the method includes expressing the epigenetic regulator described herein in the subject. In some aspects, the disorder is related to Na... V 1.7 Relevance. In some respects, the condition is pain, inflammation, and / or cancer. In some respects, the condition is small fiber neuropathy, back pain, rheumatoid arthritis, osteoarthritis, spinal stenosis, chronic cough, migraine, trigeminal neuralgia, erythromelalgia, and paroxysmal excruciating pain. In some respects, inflammation is associated with arthritis. In some respects, arthritis is rheumatoid arthritis or osteoarthritis. In some respects, treating inflammation includes preventing inflammation. In some respects, treating inflammation includes reducing inflammation. In some respects, treating pain includes preventing pain. In some respects, treating pain includes reducing pain. In some respects, pain is associated with neuropathy, chemotherapy, or inflammation.

[0018] In various aspects, this disclosure provides a method for delivering an epigenetic regulator to cells in the form of a purified protein. In some aspects, the method includes administering the disclosed epigenetic regulator to cells. In some aspects, the epigenetic regulator is modified to enhance cellular uptake. In some aspects, the modification includes fusion with a cell-penetrating peptide. In some aspects, the cell-penetrating peptide comprises TAT, polyarginine, or a combination thereof.

[0019] In various aspects, this disclosure provides a composition for direct protein delivery, wherein the composition comprises the disclosed epigenetic regulator and a pharmaceutically acceptable carrier. In some aspects, the composition further comprises a cell-penetrating peptide fused to the epigenetic regulator. In some aspects, the composition further comprises a liposome encapsulating the epigenetic regulator.

[0020] In various aspects, this disclosure provides a method for treating a disorder in a subject, wherein the method includes administering a therapeutically effective amount of the disclosed epigenetic modulator to the subject via direct protein delivery. In various aspects, the epigenetic modulator is administered via a route selected from the group consisting of intravenous injection, subcutaneous injection, intramuscular injection, aerosol administration, and local application to target tissues (such as the trigeminal ganglion or dorsal root ganglion). In various aspects, the disorder is selected from the group consisting of: pain, inflammation, cancer, chemotherapy-induced peripheral neuropathy, small fiber neuropathy, back pain, rheumatoid arthritis, osteoarthritis, spinal stenosis, chronic cough, migraine, trigeminal neuralgia, erythromelalgia, and paroxysmal excruciating pain.

[0021] By incorporating references

[0022] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0023] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the invention and are illustrated in the accompanying drawings:

[0024] Figures 1A to 1C The inhibition of SCN9A in human cell lines was demonstrated to identify the primary candidate. Figure 1A Eleven zinc finger proteins were transfected into HuH7 cells, and the NaV1.7 inhibition level was measured by qPCR using the ΔΔCt method. Figure 1BThe same ZF array was tested in an IMR-90, and the repression level was measured by qPCR using the ΔΔCt method. Figure 1C Data obtained by NCATS (an agency of the NIH) using our primary ZF candidate. Repression levels obtained by ddPCR.

[0025] Figures 2A to 2B An example of isolating the NaV1.7 specific promoter is shown. Figure 2A Seven NaV1.7 promoters driving mCherry expression were transfected into the human cell line HuH7, which highly expresses NaV1.7. Two promoters with high transgenic expression and short sequences, capable of being packaged in AAV, were further investigated in vivo. Figure 2B A schematic diagram of pain reversal in the CIPN model.

[0026] Figures 3A to 3B Results from studies involving five promoters are presented, demonstrating the efficacy of different promoters. Data in each group represent individual biological replicates, with a sample size of 7 to 8 individuals per group. Statistical analysis was performed using two-way ANOVA followed by Dunnett's post-hoc test. Figure 3A The statistical significance was demonstrated when compared to the mCherry control group. Figure 3B This demonstrates the statistical significance compared to the untreated group. A p-value less than 0.05 (p<0.05) is considered statistically significant. The error bars represent the standard error (SEM) of the mean.

[0027] Figure 4A Genotyping results for the 1197th base pair are shown for A) an SCN9A mutant iPSC line and B) a healthy H1 embryonic stem cell (ESC) line with a wild-type SCN9A genome sequence. The SCN91 mutant iPSC line shows a persistent conflict between guanine and adenine base readbacks, while the ESCs consistently read back guanine without conflict.

[0028] Figure 4B An experimental protocol for generating SCN9A-positive sensory neuron-like cells was demonstrated. iPSCs differentiated into immature sensory neurons by changing the culture medium daily for seven consecutive days, with each medium containing a unique mixture of differentiation-inducing factors.

[0029] Figures 5A to 5F This image shows confocal images of iPSC-derived sensory neurons from a patient diagnosed with IEM. Sensory neurons were transduced using mCherry fluorescent protein. The iPSCs underwent sensory neuron differentiation over 19 days. Figure 5A IEM iPSCs are used to lay plate cells for differentiation into sensory neurons. Figure 5BDay 2 of the differentiation process. Figure 5C (Day 6 of differentiation. Sensory neuron-like cells transduced with 499 mCherry were imaged on day 19.) Figure 5D Open passageway. Figure 5E mCherry channel. Figure 5F The superposition of bright field and mCherry.

[0030] Figure 6 This demonstrates the inhibition of Nav1.7 in IEM-iPSC. Points represent individual biological replicates; n=2.

[0031] Figures 7A to 7B An isolated human DRG experiment was demonstrated. Figure 7A This illustrates a schematic method for isolated human DRG experiments and ( Figure 7B The image shown is a representative RNAscope image.

[0032] Figure 8 RNAscope quantification of SCN9A in isolated human DRG is presented. Dots represent individual biological replicates; n = 10 to 33.

[0033] Figure 9 Transgenic expression in in vitro cultures of mouse DRGs was demonstrated.

[0034] Figure 10A A schematic diagram showing the measurement of AAV9 Nav1.7-1 transduction throughout the DRG via confocal imaging is presented.

[0035] Figure 10B The expression of mCherry in mice after intrathecal injection of AAV9-Nav1.7-1-mCherry was demonstrated.

[0036] Figure 10C This demonstrates the lack of transgene expression in the liver via immunohistochemistry using the Nav1.7 specific promoter (499 promoter).

[0037] Figure 10D Transgenic expression in isolated human DRG cultures was demonstrated.

[0038] Figures 11A to 11E This study demonstrates the efficacy of ZF4-KRAB delivered via AAV9 in target binding and pain relief in a carrageenan model of inflammatory pain. Figure 11A A diagram illustrating the overall strategy. Figure 11B A schematic diagram of a carrageenan-induced inflammatory pain model. Figure 11C The in vivo NaV1.7 inhibition level was determined by qPCR. (n=5; error bar: SEM; Student's t-test; ***p = 0.0008). Figure 11D For paws injected with carrageenan and saline, the total withdrawal latency was calculated using the area under the curve (AUC). Mice treated with ZF4-KRAB showed a significantly increased withdrawal latency in paws injected with carrageenan (n=10; error bar at SEM; Student's t-test, p < 0.0001). Figure 11E At 3, 6, 12, and 44 weeks following intrathecal injection of AAV9-mCherry and AAV9-ZF4-KRAB, the AUC of total pwL in the paws of mice injected with carrageenan and saline was calculated. A significant increase in pwL was observed in the carrageenan-injected paws of mice injected with AAV9-ZF4-KRAB (points represent individual biological replicates; n = 5 to 8; error bar is SEM; Student's t-test, ****P < 0.0001).

[0039] Figure 12 A schematic diagram of a paclitaxel-induced neuropathic pain model is shown. Mice were injected intraperitoneally (ip) with AAV9-mCherry, AAV9-ZF4-KRAB, or saline. Following a baseline von Frey threshold test on day 14, mice were subsequently injected intraperitoneally (ip) with 8 mg / kg paclitaxel (32 mg / kg cumulative dose) at days 14, 16, 18, and 20 post-IT injection. Tactile aberrant pain was assessed via the von Frey ciliary test at days 21 (b, c) and 105 (d, e) post-IT injection, and cold aberrant pain was assessed via the application of acetone.

[0040] Figures 13A to 13D This study demonstrates the in vivo efficacy of ZF4-KRAB in a chemotherapy-induced neuropathic pain model. Figure 13A Compared with the untreated group, ZF4-KRAB reduced paclitaxel-induced tactile paresthesia (n=8; error bar at SEM; Student's t-test; ***p = 0.0007, ***p = 0.0004). Figure 13B Compared with the untreated group, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain (n=8; error bar: SEM; Student's t-test; ****p < 0.0001, **p = 0.008). Figure 13C Compared with the untreated group, ZF4-KRAB reduced paclitaxel-induced tactile paresthesia 105 days after the last paclitaxel injection (n=5–8; error bar at SEM; Student's t-test; ****p < 0.0001, ***p = 0.0001). Figure 13DCompared with the untreated group, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain (n=5–8; error bar at SEM; Student's t-test; p < 0.0001). Similar proof-of-concept data were obtained using CRISPR-dCas9 (data not shown).

[0041] Figures 14A to 14D This study demonstrates the in vivo efficacy of ZF4-KRAB in a chemotherapy-induced neuropathic pain model. Figure 14A Compared with the mCherry group, ZF4-KRAB reduced paclitaxel-induced tactile paresthesia (n=8; error bar at SEM; Student's t-test; ***p = 0.0007, ***p = 0.0004). Figure 14B Compared with the mCherry group, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain (n=8; error bar at SEM; Student's t-test; ****p < 0.0001, **p = 0.008). Figure 14C Compared with the mCherry group, ZF4-KRAB reduced paclitaxel-induced tactile paresthesia 105 days after the last paclitaxel injection (n=5–8; error bar at SEM; Student's t-test; ****p < 0.0001, ***p = 0.0001). Figure 14D Compared with the mCherry group, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain (n=5–8; error bar: SEM; Student's t-test; p < 0.0001).

[0042] Figures 15A through 15H illustrate the safety assessment in mice. (Figure 15A) Plots the body weight of mice injected with AAV9-mCherry and AAV9-ZF4-KRAB (dots represent individual biological replicates; n=8 for both mCherry and ZF4-KRAB groups; error bar at SEM; Student's t-test; ns = not significant). (Figure 15B) Plots the body temperature of mice injected with AAV9-mCherry and AAV9-ZF4-KRAB (error bar at SEM; Student's t-test). (Figure 15C) A rotarod study used to determine motor coordination and balance in mice injected with AAV9-ZF4-KRAB targeting NaV1.7 (error bar at SEM; two-way ANOVA and Bonferroni post-hoc test). (Fig. 15D) Grip strength of mice injected with AAV9-mCherry and AAV9-ZF4-KRAB (error bar: SEM; two-way ANOVA and Bonferroni post-hoc test). (Fig. 15E) Marble burying behavior of mice injected with AAV9-mCherry and AAV9-ZF4-KRAB. (Fig. 15F) Nesting behavior of mice injected with AAV9-mCherry and AAV9-ZF4-KRAB. (Fig. 15G) No significant changes in olfactory detection were observed in mice injected with AAV9-ZF4-KRAB compared to the control group (error bar: SEM; one-way ANOVA and Bonferroni post-hoc test; ns = not significant). (Figure 15H) The new object recognition test showed that mice injected with AAV9-ZF4-KRAB had comparable memory retention compared with the control group (error bar is SEM; one-way ANOVA and post-hoc test with bonferroni; ns = not significant). Detailed Implementation

[0043] This article describes compositions and methods for epigenetically modifying gene expression without editing the genome. It also describes methods for treating pain, inflammation, or both using epigenetic regulation of gene expression. Compositions for epigenetic regulation may comprise a nucleic acid binder (e.g., a nucleic acid-binding protein) or a polynucleotide encoding such a binder that binds to a target sequence within the genome. The target sequence may be a region (e.g., a coding or regulatory region) of a gene (such as a gene associated with pain or inflammation). In some embodiments, the binding of the nucleic acid binder to the target sequence may regulate (e.g., downregulate or upregulate) gene expression. In some embodiments, the binding of the nucleic acid binder to the target sequence may deliver an expression regulator (e.g., a transcriptional repressor, transcriptional activator, or epigenetic editor) to the gene, thereby regulating gene expression. The gene may encode a pain-related voltage-gated sodium channel (e.g., Na+). V1.7). For example, the gene could be SCN9A. Nucleic acid binders (e.g., SCN9A binders) could include nucleic acid-binding proteins, such as zinc finger proteins. The nucleic acid binders could be expressed together with or linked to expression regulators (e.g., ZIM3, SID, KOX1, ZNF554, ZNF264, ZNF324, MeCP2, MBD2b, SID, HP1a, SIRT5, SETD8, HDT1, SUPR, FOG1, DNMT3A, DNMT3L, DMT3, or combinations thereof) that regulate gene expression.

[0044] Using the compositions disclosed herein (e.g., adjusting Na) V The composition of expression in 1.7 can be used to epigenetically regulate gene expression for the treatment of pain, inflammation, or both in a subject. Pain or inflammation may be associated with a condition (e.g., arthritis or a neurological disorder) or with the treatment of a condition (e.g., chemotherapy for cancer). In some embodiments, the method of regulating gene expression may include delivering a polynucleotide encoding a nucleic acid binder and an expression regulator targeting a target gene to the subject's cells and expressing the nucleic acid binder and expression regulator in the cells, thereby regulating gene expression. Various methods can be used to deliver the polynucleotide to the subject's cells. For example, a viral vector (e.g., adeno-associated virus (AAV) or lentiviral vector) or a plasmid can be used to deliver the polynucleotide. In some embodiments, the method of regulating gene expression may include delivering a nucleic acid binder and an expression regulator to the subject's cells, thereby regulating gene expression. For example, the nucleic acid binder and expression regulator may be delivered on or within nanoparticles (such as lipid nanoparticles).

[0045] Epigenetic regulators

[0046] The compositions disclosed herein may comprise or encode an epigenetic regulator. The epigenetic regulator can modulate the expression of a target gene without editing the genomic sequence. In some embodiments, the epigenetic regulator may comprise a nucleic acid binder, an expression regulator, or both. The nucleic acid binder may be linked to the expression regulator (e.g., expressed as a fusion protein) such that binding of the nucleic acid binder to the target sequence delivers the expression regulator to the target sequence. Once in proximity to the target sequence, the expression regulator can modulate the expression of the gene containing the target sequence.

[0047] Zinc finger protein

[0048] Zinc finger proteins (ZFPs) contain DNA-binding domains composed of Cys2His2 zinc fingers. ZFPs constitute the largest individual family of transcriptional regulators encoded by the genomes of higher organisms.

[0049] In some embodiments, the nucleic acid composition comprises a sequence encoding a ZFP. The ZFP may comprise a natural or modified sequence. Non-limiting examples of ZFP sequences are provided in Table 1.

[0050] Table 1 – Exemplary Zinc Finger Proteins

[0051]

[0052] In some cases, targeting Na V ZFP 1.7 comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 1 to SEQ ID NO: 4. In some cases, targeting Na V ZFP 1.7 contains the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In some cases, it targets Na. V ZFP 1.7 contains a sequence having at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1. In some cases, targeting Na V ZFP 1.7 contains the sequence of SEQ ID NO: 1. In some cases, targeting Na... V ZFP 1.7 contains a sequence having at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2. In some cases, targeting Na... V ZFP 1.7 contains the sequence of SEQ ID NO: 2. In some cases, it targets Na. VZFP 1.7 contains a sequence having at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 3. In some cases, targeting Na... V ZFP 1.7 contains the sequence of SEQ ID NO: 3. In some cases, it targets Na. V ZFP 1.7 contains a sequence having at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4. In some cases, targeting Na... V ZFP 1.7 contains the sequence of SEQ ID NO: 4.

[0053] Zinc finger proteins can bind to target sequences. In some embodiments, the target sequence is a gene (such as one encoding Na+). V It is part of the gene (1.7). Table 2 provides examples of zinc finger protein target sequences.

[0054] Table 2 – Exemplary Target Sequences

[0055]

[0056] In some cases, it is used to regulate Na V 1.7 The expressed ZFP target sequence comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 73 to SEQ ID NO: 97. In some cases, it is used to regulate Na V 1.7 The expressed ZFP target sequence includes any of the sequences of SEQ ID NO: 73 to SEQ ID NO: 97.

[0057] Expression regulators

[0058] Expression regulators can modulate the expression of target molecules. In some embodiments, expression regulators include activators that activate expression. In some embodiments, expression regulators include repressors that inhibit expression. Expression regulators may include transcriptional regulatory domains having transcriptional repressive activity (e.g., a repressor domain) or transcriptional activating activity (e.g., an activator domain). In some cases, the repressor domain includes ZIM3. In some cases, the repressor domain includes a Krupper-associated box (KRAB) domain (which recruits histone methyltransferases and deacetylases). Non-limiting examples of repressor domains are provided in Table 3.

[0059] Table 3 – Exemplary Suppression Domains

[0060]

[0061] The compositions disclosed herein may comprise or encode a repressor domain of any of SEQ ID NO: 5 to SEQ ID NO: 20, or a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 5 to SEQ ID NO: 20. The repressor domain may be a variant or combination of the repressor domains of any of SEQ ID NO: 5 to SEQ ID NO: 20. In some embodiments, the repressor domain comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with ZIM3 (SEQ ID NO: 8). In some embodiments, the repressor domain includes sequence identity with KOX1 or a portion thereof (e.g., SEQ ID NO: 5 or SEQ ID NO: 9) of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%.

[0062] In some embodiments, expression regulators include VP64 (recruiting transcription activators), p65 (recruiting transcription activators), p300 catalytic domain (histone acetyltransferase), TET1 catalytic domain (DNA demethylase), TDG (DNA demethylase), Ldb1 self-association domain (recruiting enhancer-associated endogenous Ldb1), SAM activator (VP64, p65, HSF1) (recruiting transcription activators), VPR (VP64, p65, Rta) (recruiting transcription activators), Sin3a (recruiting histone deacetylase), LSD1 (histone demethylase), SUV39H1 (histone methyltransferase), G9a (EHMT2) (histone methyltransferase), DNMT3a (DNA methyltransferase) or DNMT3a-DNMT3L (DNA methyltransferase), p16, p300, CD, SunTag, FOG1, DNMT3A, DNMT3L, DMT3, or variants or combinations thereof.

[0063] In some embodiments, expression regulators include KRAB (also known as KOX), SID, MBD2, MBD3, HP1a, the DNMT family (including DNMT1, DNMT3A, DNMT3B, DNMT3L, DNMT2A), Sin3a, Rb, MeCP2 (methyl-CpG binding protein 2), ROM2, AtHD2A, LSD1, SUV39H1, or G9a (EHMT2) or variants or combinations thereof. Variants of the KRAB domain include ZIM3, ZNF554, ZNF264, ZNF324, ZNF354A, ZNF189, ZNF543, ZNP82, ZNF669, ZNF582, KOX1-MeCP2, ZNF30, ZNF680, ZNF331, ZNF33A, ZNF528, ZNF320, ZNF350, ZNF175, ZNF214, ZNF184, ZNF8, ZNF596, KOX1, ZNF37A, ZNF394, ZNF610, ZNF273, ZNF34, ZNF250, ZNF98, ZNF675, ZNF213, NLuc, ZFP28-2, ZNF224, or ZNF257, or variants or combinations thereof.

[0064] In some embodiments, the expression regulator includes domains that recruit transcription activators, histone acetyltransferases, DNA demethylases, enhancer-associated endogenous Ldb1, histone methyltransferases and deacetylases, histone deacetylases, histone demethylases, DNA methyltransferases, acetylation domains or deacetylation domains, or combinations thereof.

[0065] connector

[0066] In some embodiments, the transcription regulator is linked to a nucleic acid binder. The transcription regulator may be located at the N-terminus or C-terminus of the nucleic acid binder. Domains may be linked via peptide linkers. Domains may be linked via disulfide bonds.

[0067] In some embodiments, the epigenetic regulator is linked to a nucleic acid-binding domain via a peptide linker. Non-limiting examples of peptide linkers include (GGS)n (SEQ ID NO: 170) and (GGGS). n (SEQ ID NO: 171), (GGGGS) n (SEQ ID NO: 172), (G) n (SEQ ID NO: 173), (EAAAK) n (SEQ ID NO: 174), A(EAAAK) n ALEA(EAAAK) n A (SEQ ID NO: 175), PAPAP (SEQ ID NO: 176), AEAAAKEAAAKA (SEQ ID NO: 177), (Ala-Pro) x (SEQ ID NO: 178), LE, GlySer-polyPro(Glyc)-polyPro(Glyc)-polyPro(Glyc), GlySer-polyPro-polyPro(Glyc)-polyPro, GlySer-polyPro-GlySer(Glyc)- polyPro, GlySer-polyPro-polyPro-polyPro, GlySer-polyPro-β2m-polyPro, GlySer-polyPro-β2m-GlySer, polyPro-β2m-GlySer-β2m-GlySer, G lySer-polyPro-β2m-GlySer-β2m-polyPro, GlySer-polyPro-Ub-GlySer, GlySer-polyPro-ZAG-polyPro, GlySer-GlySer-ZAG-GlySer-ZAG-polyP ro, GlySer(Glyc)-GlySer(Glyc)-polyPro, (G4S)3-cTPR3-(G4S)3, (G4S)3-cTPR6-(G4S)3, (G4S)3-cTPR9-(G4S)3, (G4S)3-cTPR12-(G4S)3 and (G4S) n(SEQ ID NO: 172); wherein n is independently selected from 1 to 10, x is 10-34, polyPro is a proline-rich hinge sequence from IgA1, polyPro(Glyc) is a proline-rich hinge sequence from IgA1 with an embedded potential N-linked glycosylation site (Asn-Ser-Ser), β2m is β2-microglobulin, Ub is ubiquitin, ZAG is Zn-α2-glycoprotein, and cTPRX is a common tetrapeptide repeat sequence with repeat count X. Other examples of peptide linkers include amino acid sequences of MGS, GSS, GS, GGGSGT (SEQ ID NO: 179), GTGGGS (SEQ ID NO: 180), or GGGSGGGS (SEQ ID NO: 181).

[0068] Epigenetic regulators

[0069] Compositions for epigenetic regulation of a target may comprise an epigenetic regulator. The epigenetic regulators of this disclosure may comprise a zinc finger protein that binds a target sequence (e.g., a region of SCN9A) and a repressive domain (e.g., KRAB). In some embodiments, the zinc finger protein may comprise any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO: 169. In some embodiments, the repressive domain may comprise a KRAB repressive domain (e.g., SEQ ID NO: 5 or SEQ ID NO: 9). The repressive domain may be located at the N-terminus of the zinc finger protein, or the repressive domain may be located at the C-terminus of the repressive domain. In some embodiments, the repressive domain and the zinc finger protein are separated by a linker (e.g., a linker comprising glycine and serine). In some embodiments, the epigenetic regulator may comprise a second repressive domain (e.g., the Sin3 interaction domain (SID) of SEQ ID NO: 6 or SEQ ID NO: 7). In some embodiments, the epigenetic regulator includes a nuclear localization signal (e.g., SEQ ID NO: 21 (PKKKRKV) or SEQ ID NO: 22 (PKKKRKVLEPKKKRKVPGMAPKKKRKV)). The nuclear localization signal can localize the epigenetic regulator to the cell nucleus. In some embodiments, the epigenetic regulator includes an affinity tag (e.g., a FLAG tag of SEQ ID NO: 23 (DYKDDDDK) or SEQ ID NO: 24 (MDYKDHDGDYKDHDIDYKDDDDK).

[0070] Table 4 provides examples of epigenetic regulators that include zinc finger proteins and repressive domains.

[0071] Table 4 – Exemplary Epigenetic Regulators

[0072]

[0073] In some embodiments, the epigenetic regulator may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 25 to SEQ ID NO: 36. In some embodiments, the epigenetic regulator comprises a sequence of any of SEQ ID NO: 25 to SEQ ID NO: 36.

[0074] Polynucleotide composition

[0075] The compositions disclosed herein may comprise a polynucleotide encoding an epigenetic regulator or a component of an epigenetic regulator. For example, the polynucleotide may encode a nucleic acid binder, an expression regulator (e.g., a repressive domain, an activation domain, or an epigenetic editor), or a combination thereof. In some embodiments, the nucleic acid binder and the expression regulator may be expressed as a fusion protein.

[0076] Encoded sequence

[0077] The polynucleotide may comprise one or more coding sequences. In some embodiments, the coding sequence may encode an epigenetic regulator or a portion thereof of the present disclosure. For example, the coding sequence may encode a nucleic acid binding agent (e.g., a zinc finger protein of any of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO: 169) or an expression regulator (e.g., a repressor domain of any of SEQ ID NO: 5 to SEQ ID NO: 20) or a combination thereof. In some embodiments, the polynucleotide encodes a polypeptide of any of SEQ ID NO: 25 to SEQ ID NO: 36. In some embodiments, the polynucleotide encodes a polypeptide comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 25 to SEQ ID NO: 36.

[0078] promoter

[0079] The polynucleotide may further include a promoter operatively linked to a coding sequence of a component encoding an epigenetic regulator (e.g., a nucleic acid binder, an expression regulator, or a combination thereof). The promoter regulates transcription of the coding sequence. In some embodiments, the promoter may be a ubiquitous promoter that activates transcription of the coding sequence regardless of tissue type. In some embodiments, the promoter may be a cell-specific promoter (e.g., a neuron-specific promoter) that activates transcription of the coding sequence in a target cell type. For example, the promoter may be for expressing Na+. V 1.7 Cell specificity. Table 5 provides examples of promoters that may be included in the polynucleotides disclosed herein.

[0080] Table 5 – Exemplary Promoters

[0081]

[0082] In some embodiments, the polynucleotide may comprise a promoter having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 37 to SEQ ID NO: 47 or SEQ ID NO: 98. The promoter may regulate the transcription of one or more coding sequences of the polynucleotide.

[0083] In some embodiments, the promoter is specific to the target molecule, such that the nucleic acid composition is specific to or expressed only in those cells that express the target to increase therapeutic selectivity. As a non-limiting example, the target molecule is SCN9A.

[0084] In some embodiments, the promoter further increases the specificity of AAV tropism to cells expressing the target molecule. As a non-limiting example, the target molecule is SCN9A. Downregulating or upregulating the target molecule only in cells expressing it can reduce off-target effects and general toxicity. This is important for preventing the expression of effectors (e.g., epigenetic regulators) in immune cells such as glial cells, microglia, macrophages, astrocytes, etc., which can mediate immune responses against the gene therapies proposed herein.

[0085] In some embodiments, the promoter pairs SCN9A (Na V1.7) It has specificity and is used to drive Na V 1.7 or other gene products (e.g., SEQ ID NO: 37 to SEQ ID NO: 43). In some embodiments, the promoter is specific to the gene target described herein. In some embodiments, the promoter is a ubiquitous promoter (e.g., SEQ ID NO: 45 to SEQ ID NO: 47 or SEQ ID NO: 98).

[0086] In some embodiments, some promoters can be induced by small molecules or other means. These inducible expression promoters include tetracycline-responsive promoters, glucocorticoid-responsive promoters, RU-486-responsive promoters, peroxide-induced promoters, and tamoxifen-induced promoters.

[0087] Furthermore, when pathology (such as injury or inflammation) occurs, there are promoters that can be induced. Injury-induced promoters include the glycopyridine promoter, which is specific to nociceptive afferent neurons. The inflammation-induced promoter NF-κB can also be used for inflammation-related pain.

[0088] Tandem promoters and combinations of promoters can also be used to prevent immune responses and generate more cell type-specific expression.

[0089] In some embodiments, the expression of epigenetic regulators and / or transcriptional regulatory domains occurs under natural or physiological induction by the promoter.

[0090] In some embodiments, pan-neuronal gene promoters are used to regulate gene expression in neurological disorders and to suppress pain-related genes. Non-limiting examples of promoters include promoters for microtubule-associated protein 2 (MAP-2), neuron-specific enolase (NSE), choline acetyltransferase (ChAT), protein gene product 9.5 (PGP9.5) (also known as ubiquitin C-terminal hydrolase 1 (UCHL-1)), human synaptic protein 1 (hSYN1) gene, NeuN gene (Fox-3, Rbfox3, or hexanucleotide-binding protein-3), α-calcium / calmodulin-dependent protein kinase II (CaMKIIα)), Rheb gene (a brain-enriched ras homolog), and TRKA promoter (tyrosine kinase A). In some embodiments, promoters are neuron-specific, such as the pol II promoter, including Thy1 and H1xb9. Small latency-associated promoters from herpesvirus pseudorabies virus can also be used for panneuronal expression of effectors (ZFPs) fused with repressor domains.

[0091] Other promoters include cytomegalovirus (CMV), SV40, elongation factor 1-α (EF1a) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, jET promoter, and herpes simplex virus (HSV).

[0092] In some embodiments, the promoter comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID NO: 37 to SEQ ID NO: 47 or SEQ ID NO: 98.

[0093] In some embodiments, the promoter is the SCN9A promoter.

[0094] In some embodiments, the promoter is naturally associated with genes related to: channel diseases, Dravet syndrome, epilepsy syndromes, familial hemiplegic migraine, Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, sodium channel myotonia, autism, long QT syndrome, Brugada syndrome, or progressive cardiac conduction disorders (also known as Lenègre disease), pain (e.g., inflammatory pain, visceral pain, migraine pain, erythromelalgia pain, fibromyalgia pain, idiopathic pain, somatic pain), neurological disorders, dementia, Alzheimer's disease, Parkinson's disease, ALS, multiple sclerosis, central nervous system diseases, or combinations thereof.

[0095] In some embodiments, the promoter is a promoter selected from the following genes: SNCA, GBA, LRRK2, SOD1, ataxia-protein-2, SCA2, BFD1, FUS, C9orf72, brain-derived neurotrophic factor, nerve growth factor, neurotrophic factor, BCL11A, FMR1, DNM2, PrP, UBE3A, GYS1, and GFAP.

[0096] In some embodiments, the promoter is the pol II promoter (e.g., Thy1 and H1xb9), a short latency-associated promoter (e.g., from herpesvirus pseudorabies virus), a cytomegalovirus promoter, SV40, elongation factor 1-α (EF1a) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, or herpes simplex virus (HSV) promoter.

[0097] Other components

[0098] Polynucleotides may further include enhancers, introns, nuclear localization signals, inverted terminal repeats (ITRs), terminator sequences, or combinations thereof. Polynucleotides may be included in delivery media such as vectors.

[0099] This document also provides nucleic acid compositions comprising adeno-associated viruses with sequences modified to encode peptides that are specific to the protein products of target molecule. Such compositions can be used to target nucleic acids to cells expressing target molecule for targeted therapeutic purposes. For example, the peptide specifically binds to the protein product of the target molecule. Non-limiting examples of specific binding include peptides that bind to the protein product of the target molecule with high affinity (e.g., affinity in the nanomolar range).

[0100] Delivery carrier

[0101] In some embodiments, the compositions herein are delivered in a delivery vector. The delivery vector can be used to deliver epigenetic regulators, components of epigenetic regulators, or polynucleotides encoding epigenetic regulators. In some embodiments, the delivery vector encapsulates a protein or polynucleotide. In other embodiments, the compositions are delivered in combination with a cationic molecule.

[0102] In some embodiments, the composition is delivered to the subject via a medium. The medium may be a liposome, lipid nanoparticle, nanocapsule, or exogenous body.

[0103] In some embodiments, the composition is delivered via a viral vector. Non-limiting viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVhu68, AAVrh.10, AAVrh74, AAVDJ), etc. In some cases, the vector is recombinant adeno-associated virus (AAV). In some cases, the AAV is AAV9. AAV9 may be chosen because it is effective against pain-related proteins (such as Na+). V 1.7) Highly expressed dorsal root ganglion (DRG) neurons exhibit the highest tropism. Furthermore, AAV9 has been shown to be safe.

[0104] All delivery vehicles (viral or non-viral vectors) can exhibit enhanced tropism for cells expressing the protein product of the target molecule. For example, the vehicle may contain a peptide that binds to the protein product of the target molecule. As a non-limiting example, a peptide bound to Na+... V 1.7 Combining to target the expression of Na+ nucleic acids V 1.7 cells.

[0105] Targeted portion

[0106] In some embodiments, the compositions herein are delivered together with a nucleic acid sequence encoding a peptide targeting portion via a viral vector (such as the AAV capsid described herein).

[0107] In some embodiments, the compositions herein are delivered via a nonviral delivery medium, such as, but not limited to, liposomes, lipid nanoparticles, nanocapsules, or exogenous bodies. In some such embodiments, the medium may include a targeting portion (such as a small molecule or peptide targeting portion) and / or be linked to the targeting portion.

[0108] In some embodiments, the targeting portion includes a peptide targeting portion that binds to the protein product of the target molecule to target the nucleic acid to specific cells. In some cases, the target molecule is present on the target cells. In some cases, the target cells are associated with a disease or ailment of the subject.

[0109] Non-limiting examples of peptide targeting moieties for use with viral and non-viral delivery methods include JNJ63955, m3-tiger tarantula toxin-IV, flortoxin 1 (PhlTx1), protoxin-II (ProTx-II), keratoxin-1 (CcoTx1), tiger tarantula toxin-IV (HwTx-IV), μ-TRTX-Pn3a, Jz-Tx-V, GsAFI, Tp1a (protoxin-III), GpTx-1, HpTx1, Hm1a, and their variants and combinations. The peptide may be derived from one or more of the following organisms: green velvet wolf spider (Thrixopelma pruriens), American steel blue (Pamphobeteus nigricolor), jingzhao hairy spider (Chilobrachys jingzhao), Grammostola antracist, Phlogiellusgenus, green velvet wolf spider, charcoal tarantula (Grammostola anthracina), tiger tarantula (Selenocosmia huwena), right-angled baboon spider (Ceratogyrus cornuatus), white-fronted giant crab spider (Heteropoda venatoria) and / or gorgeous rainforest baboon spider (Heteroscodra maculate).

[0110] Table 6 provides examples of peptides that can be used for targeting.

[0111] Table 6 - Exemplary Peptide Targeting Sections

[0112]

[0113] In some cases, the peptide includes a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 48 to 72. The peptides in Table 6 may be related to Na… V 1.7 Combining and therefore being usable for expressing Na V Cells with a molecular weight of 1.7 provide AAV tropism. In some cases, the peptides include ProTx-II, ProTx-III, ProTx-II variant JNJ63955, HwTx-IV variant m3-HwTx-IV, CcoTx1 variant 2670, PhlTx1 variant D7A-PhlTx1, or JxTx-V variant AM-0422.

[0114] AAV carrier

[0115] Recombinant adeno-associated viruses (AAVs) are among the most commonly used vectors for in vivo gene delivery. To enter a host cell, the virus 1) binds to receptors and glycan co-receptors on the cell surface, 2) is endocytosed, 3) travels through the endosome compartment, 4) escapes the endosome, and 5) is transported to the nucleus. Once inside the nucleus, the virus sheds its capsid, and its single-stranded genome is converted into a double-stranded genome, which the host cell can now use as a template for gene expression. The AAV receptor (AAVR, KIAA0319L) has been reported to be crucial for AAV entry into cells; however, some recombinant AAVs can enter cells independently of AAVR. Glycosylation is also known to play a role in viral transduction efficiency. Altering the capsid composition of AAVs alters their ability to enter cells. Currently, at least four main techniques are used to modify and improve AAV tropism: rational engineering, directed evolution, phylogenetic analysis, and chemical conjugation.

[0116] One way to modify viral tropism is to generate a large library of peptides to be added to the surface of an AAV (Anaerobic Antiviral Agent), and then characterize the resulting variants to determine their tropism. This method is labor-intensive, requiring a great deal of screening work, because the library of peptides to be screened is more or less randomly generated, so success is based on a numbers game. In a similar technique called directed viral evolution, organs are collected from the first round of viral infection and screened to select viral variants with the desired tropism (e.g., brain specificity) for use in subsequent rounds of infection to further select for more specific tropisms. However, some of these screening methods cannot be translated between species.

[0117] This article describes a rational design approach to improve AAV tropism, rather than using random library screening. Rational design strategies for AAV capsid engineering include peptide domain insertion and chemical biology approaches. Peptides known to interact specifically with target cells can be added. By using binding peptides that bind to proteins encoding target molecules, the AAVs described herein exhibit increased tropism for cells expressing target molecules, resulting in a more targeted strategy.

[0118] In some embodiments, peptides used to increase tropism with Na V 1.7 Combination. Used to increase Na. V 1.7 The tropism peptides include: JNJ63955, m3-tiger tarantula toxin-IV, flortoxin 1 (PhlTx1), protoxin-II (ProTx-II), keratoxin-1 (CcoTx1), tiger tarantula toxin-IV (HwTx-IV), and those described elsewhere herein, such as any one of SEQ ID NO: 48 to SEQ ID NO: 72 or a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the same sequence as any one of SEQ ID NO: 48 to SEQ ID NO: 72.

[0119] target molecules

[0120] In some embodiments, the compositions described herein regulate the expression of one or more target molecules associated with a disease or ailment of a subject. In some cases, the expression of the target molecules is activated. In some cases, the expression of the target molecules is repressed.

[0121] One or more target molecules may include DNA or RNA. In some embodiments, the target molecule includes DNA. In some embodiments, the target molecule includes a coding region of a gene. In some embodiments, the target molecule includes DNA complementary to non-coding RNA. Non-coding RNA may be associated with the diseases or disorders described herein, such as pain. For example, non-coding RNA is associated with neuropathic pain, such as spinal nerve ligation, nerve-preserving injury, chronic compression injury, or diabetic neuropathy, or combinations thereof.

[0122] In some embodiments, the non-coding RNA includes SCN9A natural antisense transcript (NAT), Kcna2 antisense RNA, H19, Gm21781, MRAK009713, uc.48+, NONRATT021972, BC168687, Speer7-ps, Uc007pbc.1, XLOC_041439, Mlxipl, Rn50_X_0739.1, CCAT1, rno circ 0004058, rno_circRNA_007512, or Egr2 antisense RNA, or combinations thereof.

[0123] In some embodiments, one or more target molecules include nucleic acids associated with the diseases or disorders described herein. In some embodiments, one or more target molecules include nucleic acids associated with pain. Pain includes neuropathic pain, inflammatory pain, visceral pain, migraine pain, erythromelalgia pain, fibromyalgia pain, idiopathic pain, and somatic pain. For example, the target molecule may be encoding Na+. V 1.7 SCN9A gene. In some embodiments, the target molecule includes the sequence of any one of SEQ ID NO:73 to SEQ ID NO:97.

[0124] In some embodiments, one or more target molecules include nucleic acids associated with channel disorders. In some cases, one or more target molecules include nucleic acids encoding channels. Channels can be ion channels, such as sodium channels, potassium channels, calcium channels, and / or chloride channels.

[0125] In some embodiments, one or more target molecules include nucleic acids associated with neurological disorders. In some embodiments, one or more target molecules include nucleic acids associated with dementia. In some embodiments, one or more target molecules include nucleic acids associated with Alzheimer's disease. In some embodiments, one or more target molecules include nucleic acids associated with Parkinson's disease. In some embodiments, one or more target molecules include nucleic acids associated with Huntington's disease. In some embodiments, one or more target molecules include nucleic acids associated with schizophrenia. In some embodiments, one or more target molecules include nucleic acids associated with amyotrophic lateral sclerosis (ALS). In some embodiments, one or more target molecules include nucleic acids associated with multiple sclerosis. In some embodiments, one or more target molecules include nucleic acids associated with central nervous system disorders. In some embodiments, one or more target molecules include nucleic acids associated with Drave syndrome, epilepsy syndrome, familial hemiplegic migraine, Ōtahara syndrome, West syndrome, Lennox-Gasto syndrome, sodium channel myotonia, autism, long QT syndrome, Brugada syndrome, or progressive cardiac conduction disorders (also known as Lenegel's disease), or combinations thereof.

[0126] Genes involved in pain

[0127] The human genome encodes genes that could confer protection against unwanted pain. Genetic research has identified a hereditary loss-of-function mutation in human voltage-gated sodium channels called Na... V 1.7 (SCN9A) is associated with a rare genetic disorder that causes pain insensitivity without other neurodevelopmental alterations. Therefore, this sodium channel has become an attractive target for developing therapies for chronic pain. However, due to Na... V The high sequence identity between subtypes hinders the development of selective small molecule inhibitors, and in fact, many inhibitors targeting Na+... V Small molecule drugs (1.7) fail due to a lack of specificity. Antibodies face a similar situation because there is a trade-off between selectivity and potency due to the specific (open or closed) conformation of the channel they bind to. In fact, even commercially available antibodies targeting human channels perform poorly in Western blotting. Interfering RNA (RNAi) has also been used to target Na+. V 1.7. However, as an exogenous system, RNAi competes with endogenous mechanisms such as microRNAs or RISC complexes. Therefore, RNAi can compete with and impair the fundamental homeostatic mechanisms of RNA synthesis and degradation. Furthermore, due to the high RNA turnover rate, RNAi methods exhibit poor pharmacokinetic prospects and require higher doses. Primarily due to these drawbacks, targeting Na+ based on these methods... V None of the treatments mentioned in section 1.7 have successfully reached the final stages of clinical trials. In contrast, this article discloses the use of nucleic acid-binding domains (e.g., zinc finger proteins) and epigenetic modulators (e.g., KRAB repressors) to inhibit the transcription of SCN9A and / or other pain-related genes. Permanent genome editing using such methods is not intended to permanently eliminate pain. Instead, these epigenomic engineering methods can transiently modulate Na+. V 1.7 Gene Expression. Furthermore, compared to other methods, this method may have a lower risk of off-target effects. This method does not pharmacologically target proteins or RNA, but rather targets Na+ at the DNA level. V1.7. This may produce more durable results than methods targeting proteins or RNA. Through this approach, highly specific, durable, and reversible pain treatments can be designed. Treatment duration is important because many pain states caused by chronic inflammation and nerve damage are persistent and often require continuous re-administration. This genetic approach provides continuous, controlled regulation of aberrant pain management. Furthermore, because the disclosed method can be readily designed to target several genes, it represents a new paradigm for pain management, as it provides a synergistic approach of targeting single or multiple sodium channels to more effectively alleviate pain.

[0128] Treatment

[0129] Various embodiments provide methods for treating a subject's disease or ailment with the compositions described herein. In some embodiments, the composition comprises an epigenetic regulator or a polynucleotide encoding an epigenetic regulator. The composition may be delivered via AAV or a non-viral medium.

[0130] In an exemplary embodiment, the disease or ailment includes pain. Pain includes neuropathic pain, inflammatory pain, visceral pain, migraine pain, erythromelalgia pain, fibromyalgia pain, idiopathic pain, and somatic pain. In some embodiments, the pain is induced by chemotherapy (e.g., paclitaxel-induced). Inflammatory pain includes rheumatoid arthritis pain. The disease or ailment also includes sodium. V 1.7 or other diseases or disorders that may be targeted by genes involved in pain. In some embodiments, the disease or disorder is related to Na V 1.7 Related. In some embodiments, the treatment method includes treating a subject for inflammation with a composition comprising an epigenetic regulator or a polynucleotide encoding an epigenetic regulator. Inflammation may be associated with a disease or condition such as arthritis.

[0131] In some embodiments, the disease or ailment is cancer. In some embodiments, the disease or ailment includes small fiber neuropathy, back pain, rheumatoid arthritis, osteoarthritis, spinal stenosis, chronic cough, migraine, trigeminal neuralgia, erythromelalgia, and paroxysmal excruciating pain.

[0132] In some embodiments, the disease or ailment includes channel diseases. Channel diseases include Delaware syndrome, epilepsy syndrome, familial hemiplegic migraine, Ōtahara syndrome, West syndrome, Lennox-Gasto syndrome, sodium channel myotonia, autism, long QT syndrome, Brugada syndrome, progressive cardiac conduction disorders (also known as Renegler's disease), etc. In some embodiments, the ailment may be pain, inflammation, or both.

[0133] In some embodiments, the disease or disorder includes a neurological disease or disorder. Neurological diseases or disorders include dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. The disease or disorder may include a central nervous system disease.

[0134] In some embodiments, the disease or ailment includes an inflammatory disease or ailment. As a non-limiting example, an inflammatory disease or ailment is rheumatoid arthritis.

[0135] In some embodiments, disease or illness includes infection.

[0136] In some embodiments, the disease or disorder includes β-thalassemia, Fragile X, central nucleomyopathy, prions, Angelman Syndrome, Lafora disease, or Alexander disease, or a combination thereof.

[0137] In some embodiments, the treatment method includes administering the nucleic acid composition described herein and one or more additional active agents. For example, the additional active agent may be used to supplementally treat a disease or ailment.

[0138] In some embodiments, the subject refers to any animal, including but not limited to humans, non-human primates, rodents, and domesticated and hunted animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and hunted animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a human.

[0139] In various embodiments, the subject may be a subject who has been previously diagnosed with or identified as having or possessing a disease or ailment requiring treatment. In various embodiments, a subject who has been previously diagnosed with or identified as having or possessing such a disease or ailment may or may not have received treatment for the ailment. In other embodiments, the subject may also be a subject who has not previously been diagnosed with a disease or ailment, and the treatment agent is for preventative (prophylactic) purposes.

[0140] Drug composition, administration and dosage

[0141] In various embodiments, the compositions herein are formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to intrathecal, epidural, intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods, and / or via a single dose or multiple doses. Exemplary routes of administration for the nucleic acids described herein include lumbar puncture, intracerebellomedullary cistern administration, and intraganglionic administration.

[0142] In one exemplary embodiment, the composition is delivered into the intrathecal space of the spinal sheath using any suitable delivery method. When targeting Na... V This method may be particularly useful at 1.7, because Na V 1.7 plays a role in receiving nociceptive afferents, and their cell bodies are located in the corresponding segmental dorsal root ganglion (DRG) neurons. Therefore, delivery into the intraspinal space can effectively target Na+. V The composition of 1.7 is delivered to DRG neurons, which can minimize the possibility of off-target biodistribution and reduce the viral load required for transduction.

[0143] It should be understood that the actual dosage may vary depending on the route of administration; the delivery system used (e.g., AAV or liposomes); the target cells; the organ or tissue; the subject; and the degree of effect sought. The size and weight of the tissue, organ, and / or patient may also affect dosing. The dosage may further include additional agents, including but not limited to carriers. Non-limiting examples of suitable carriers are those known in the art: for example, water, physiological saline, ethanol, glycerol, lactose, sucrose, dextran, agar, pectin, plant-derived oils, phosphate-buffered saline, and / or diluents. The pharmaceutical composition may also contain any pharmaceutically acceptable carrier.

[0144] Pharmaceutical compositions can be delivered in therapeutically effective amounts. The precise therapeutically effective amount is the quantity of the composition that will produce the most effective outcome in terms of therapeutic efficacy in a given subject. This amount will vary depending on a variety of factors, including but not limited to the characteristics of the nucleic acid (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. When an intrathecal route is recommended, the pharmaceutical product may be diluted ex vivo with the subject's cerebrospinal fluid to achieve an isobaric solution prior to administration.

[0145] Reagent test kit

[0146] A kit for performing the methods described herein is further provided. The kit is an assembly of components, including at least one of the compositions described herein. Thus, in some embodiments, the kit includes a nucleic acid encoding a nucleic acid-binding domain and an epigenetic regulator. The nucleic acid may be combined or complexed with another component, such as a delivery medium, or may be used for direct delivery without modification. In some cases, the nucleic acid is complexed with a cationic molecule. In some cases, the nucleic acid is configured for delivery via a viral delivery medium, such as an AAV capsid protein.

[0147] The kit may include instructions for use of the components. Optionally, the kit may also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, aspiration or measuring tools, bandaging materials, or other useful instruments readily identifiable by those skilled in the art.

[0148] Materials or components assembled in the kit can be provided to practitioners in any convenient and suitable manner to maintain their operability and usability. For example, components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room temperature, refrigerated, or frozen temperatures. Components are typically contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the present invention. Packaging materials are constructed using well-known methods, preferably to provide a sterile, uncontaminated environment. The packaging materials used in the kit are those commonly used in gene expression assays and therapeutic administration. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of containing a single kit component. Thus, for example, packaging can be a glass vial or a pre-filled syringe for containing an appropriate amount of the composition containing the nucleic acids described herein. Packaging materials typically have an external label indicating the contents of the kit and / or the purpose and / or the components of the kit.

[0149] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural references.

[0150] As used herein, the terms “about” and “approximately” when referring to numbers are used to include numbers that fall within the range of 10%, 5%, or 1% in either direction (greater or less) unless otherwise stated or otherwise apparent from the context (unless such numbers exceed 100% of the possible value).

[0151] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term percentage “identity” can refer to two or more sequences or subsequences having a specified percentage of identical nucleotide or amino acid residues, when compared and aligned to obtain maximum correspondence, such as using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or measured by visual inspection. Depending on the application, percentage “identity” may be present in regions of the sequences being compared, such as in functional domains, or alternatively, across the entire length of the two sequences to be compared.

[0152] For sequence comparisons, a sequence typically serves as a reference sequence to be compared with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, and the sequence algorithm program parameters are also specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0153] For the purposes of this paper, percentage identity and sequence similarity can be performed using the BLAST algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0154] The composition can be applied alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated.

[0155] As used herein, the term “subject” broadly refers to any animal, including but not limited to humans and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0156] As used herein, the term "effective amount" refers to an amount of composition sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more applications, doses, or administrations and is not intended to be limited to a particular formulation or route of administration.

[0157] As used in this article, the term "therapeutic effective dose" is the amount that effectively relieves the symptoms of a disease. A therapeutic effective dose can also be a "preventive effective dose," since prevention can be considered a form of therapy.

[0158] As used herein, the term "administration / administering" refers to the act of giving a drug, prodrug, or other agent or therapeutic treatment to a subject or their cells, tissues, and organs, whether in vivo, in vitro, or ex vivo. Exemplary routes of administration to the human body may include through the subarachnoid space of the brain or spinal cord (intrathecal), joints (intra-articular), eyes (ocular), mouth (oral), skin (local or transdermal), nose (nasal cavity), lungs (inhalation), oral mucosa (cheek or tongue), ear, rectum, vagina, or by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.).

[0159] As used herein, the term “treatment” refers to a method of achieving a beneficial or anticipated clinical outcome. Beneficial or anticipated clinical outcomes may include the reduction of symptoms, a decrease in the severity of the disease, suppression of the underlying cause of the disease or disorder, stabilization of the disease in a non-late stage, delay of disease progression, and / or improvement or reduction of the disease condition.

[0160] As used herein, the term "pharmaceutical composition" refers to a combination of an active ingredient with an inert or active carrier, making the composition particularly suitable for therapeutic or diagnostic purposes in vitro, in vivo, or ex vivo.

[0161] As used herein, the terms “pharmaceutical acceptable” or “pharmacologically acceptable” mean a composition that, when administered to a subject, substantially does not produce adverse reactions (e.g., toxicity, anaphylaxis, or immune response).

[0162] As used herein, the term “pharmaceutically acceptable carrier” means any of the standard drug carriers, including but not limited to phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), glycerol, liquid polyethylene glycol, aprotic solvents (e.g., dimethyl sulfoxide, N-methylpyrrolidone, and mixtures thereof), and various types of wetting agents, solubilizers, antioxidants, fillers, protein carriers (e.g., albumin), any and all solvents, dispersion media, coatings, sodium dodecyl sulfate, isotonic agents and absorption delay agents, disintegrants (e.g., potato starch or sodium carboxymethyl starch), etc. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 21st edition, Mack Publ. Co., Easton, PA (2005), which is incorporated herein by reference in its entirety.

[0163] Example

[0164] The present invention is further illustrated by the following non-limiting examples.

[0165] Example 1

[0166] Encoding Na V 1.7 AAV9 vector for epigenetic regulators

[0167] This example describes the encoding Na. V 1.7 AAV9 vector for epigenetic regulators. The payload polynucleotide encoding the epigenetic regulator is encapsulated in an AAV9 capsid under the transcriptional control of the promoter. When expressed from the payload polynucleotide, the epigenetic regulator regulates Na+. V 1.7 Expression. Na V 1.7 Epigenetic regulators contain a target Na+ domain linked to a repressor domain (e.g., any one of SEQ ID NO: 5 to SEQ ID NO: 20). V 1.7 (e.g., any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO: 169) of the zinc finger protein. Optionally, the epigenetic regulator comprises the sequence of any one of SEQ ID NO: 25 to SEQ ID NO: 36. The promoter is Na V 1.7 A specific promoter (e.g., any one of SEQ ID NO: 37 to SEQ ID NO: 43), a neuron-specific promoter (e.g., the human synapse protein promoter of SEQ ID NO: 44), or a ubiquitous promoter (e.g., any one of SEQ ID NO: 45 to SEQ ID NO: 47). The promoter is SEQ ID NO: 98.

[0168] Example 2

[0169] Through Na V Epigenetic regulation of pain treatment 1.7

[0170] This example describes the process via Na V Epigenetic regulation for pain treatment (1.7). A vector encoding any one of SEQ ID NO: 25 to SEQ ID NO: 36 is administered to a subject experiencing pain or inflammation. Optionally, the vector is the AAV9 vector described in Example 1. Following administration, the epigenetic regulator is expressed in the subject and inhibits Na+ expression. V 1.7 Expression in subjects. The subject's pain, inflammation, or both were reduced.

[0171] Example 3

[0172] In vitro human optimization of zinc finger design targeting human NaV1.7

[0173] This example illustrates the repression of SCN9A in human cell lines to identify leading zinc finger candidates. To design zinc fingers targeting the human SCN9A (Nav1.7) DNA sequence, novel ZF constructs that bind to the human SCN9A DNA sequence were designed and tested in vitro to determine which had the highest target binding. For genome repression, we and others have found that targeting close to the TSS (-50 to +300 bp relative to TSS) enhances efficacy (Gilbert et al., 2013). This limitation complicates the design but, on the other hand, reduces the likelihood of off-target effects, as DNA binding does not necessarily imply gene regulation. Therefore, we designed eleven novel ZF constructs that recognize the human SCN9A gene and tested them in vitro to determine which showed higher efficacy in target binding, using a strategy similar to that previously shown in Neuro2a mouse cells (Figure 1). We selected HuH7 (JCRB0403; JCRB cell bank) and IMR-90 (ATCC® CCL-186™) for initial screening due to their high NaV1.7 expression levels. As part of an ongoing collaboration to help end long-term addiction (HEAL) initiatives, the National Center for Advanced Translational Sciences (NCATS), a branch of the NIH, validated our best ZF candidates (ZF5, ZF7, and ZF9) in nociceptor-like cells differentiated from induced pluripotent stem cells (iPSCs). Eleven zinc finger proteins were transfected into HuH7 cells, and NaV1.7 repression levels were measured by qPCR using the ΔΔCt method. Figure 1A The same ZF array was tested in the IMR-90, and the inhibition level was measured by qPCR using the ΔΔCt method. Figure 1B Data obtained by NCATS (a branch of NIH) using our primary ZF candidate (). Figure 1C The level of inhibition obtained by ddPCR.

[0174] Example 4

[0175] Isolation of NaV1.7 specific promoter

[0176] This example describes an in vivo promoter study to determine a NaV1.7-specific promoter using a chemotherapy-induced peripheral neuropathy (CIPN) model. To reduce potential toxicity, we propose using a minimally specific promoter that drives ZF expression only in NaV1.7-expressing cells. Potentially, using a NaV1.7-specific promoter, the expression of the ZF effector will increase when external conditions increase NaV1.7 expression (as described in various pain disorders), and therefore, the dose level can be autoregulated according to environmental needs. Therefore, we investigated several DNA regions surrounding the TSS of human SCN9A and tested the expression of the fluorescent protein mCherry (HuH7;) in NaV1.7-expressing cells. Figure 2A As a negative control, we used the MCF-7 cell line, which does not express NaV1.7, to determine the absence of "leakage" expression. Subsequently, we tested preclinical in vivo efficacy using the CIPN pain model. Figure 2B , Figure 3A and Figure 3B In this context, we used not only the two best NaV1.7 promoters, 499 and 623, from our in vitro work, but also the weakly synthetic promoter pJET (Bailey et al., 2018), which had previously been used with intrathecal AAV9, and the human synaptic protein 1 promoter, which drives expression only in neurons (Schoch et al., 1996; McLean et al., 2014; Jackson et al., 2016). Although the 623 promoter showed slightly slower efficiency than the others, the 499 promoter showed as much efficiency as the others. Therefore, we chose the 499 promoter sequence for further investigation.

[0177] Example 5

[0178] Analysis of zinc finger protein profiles in patients with hereditary erythema limba

[0179] This example describes the zinc finger profile in a patient with hereditary erythromelalgia. The zinc finger protein profile was determined in induced pluripotent stem cells (iPSCs) derived from patients diagnosed with hereditary erythromelalgia (IEM) due to an SCN9A gene mutation. Genotyping was performed on the 1197th bp of both the SCN9A mutant iPSC line and the healthy H1 embryonic stem cell (ESC) line with the wild-type SCN9A genome sequence. The SCN9A mutant iPSC line exhibited persistent conflicts between guanine and adenine bases, while the ESC line read back guanine without conflicts, such as... Figure 4AThe results showed a point mutation at the 1197th bp of the open reading frame in the SCN9A mutant iPSC line, representing a heterozygous guanine-to-alanine transition. This point mutation resulted in a change at the 400th amino acid of SCN9A, converting valine to methionine (Cao, Lishuang et al., Science translational medicine, Vol. 8, 335 (2016): 335ra56. doi:10.1126 / scitranslmed.aad7653).

[0180] Differentiation of iPSCs from patients with IEM (IEM-PSC)

[0181] based on Figure 4B The experimental protocol described herein was used to differentiate iPSCs derived from patients with IEM, generating sensory neuron-like cells expressing SCN9A. iPSCs differentiated into immature sensory-like neurons by changing the culture medium daily for seven consecutive days, each medium containing a unique mixture of differentiation-inducing factors. On day 8 of the differentiation process, cells were isolated and replate at 100,000 cells per cm² surface area. From day 8 to day 19, cells were maintained in the maturation medium Senso-MM, with the medium changed twice daily. On day 12, cells were transduced with different titers of AAV9 for 48 hours and harvested on day 19.

[0182] Confirmation of IEM-PSC differentiation

[0183] The differentiation process of sensory neurons in iPSCs lasted 19 days. On day 7 of the differentiation process, iPSCs were isolated and re-laminated. On day 9 of the differentiation process, iPSCs were transduced with AAV9, a zinc finger candidate encoding mCherry control or inhibiting SCN9A. Confocal images of iPSC-derived sensory neurons from patients diagnosed with IEM are depicted on [image / description]. Figures 5A to 5F In the middle. Sensory neuron differentiation in IEM-iPSCs on days 0, 2, and 6 was shown to be... Figure 5A , Figure 5B and Figure 5C middle. Figure 5D Display the bright field channel (for comparison). Figure 5E Display the mCherry channel (for comparison), and Figure 5F Display the overlay of the bright field channel and the mCherry channel (comparison).

[0184] Suppression of Nav 1.7 in IEM-iPSC

[0185] The expression levels of Nav1.7 in the presence of various zinc finger proteins (ZF191, ZF8-P, and ZF8-PB) targeting SCN9A were determined by qPCR. All zinc finger proteins targeting SCN9A significantly inhibited the expression level of Nav1.7, such as… Figure 6 As shown in the image.

[0186] Example 6

[0187] Nav1.7 was downregulated using human dorsal root ganglion (DRG) ex vivo culture.

[0188] Optimization of AA9 transduction in human DRG

[0189] This example describes the use of human DRG ex vivo culture to downregulate NaV1.7. To investigate the downregulation of NaV1.7, human dorsal root ganglia (DRGs) were transduced in vitro with AAV9 at different AAV9 infection multiples (MOIs) to optimize transduction, such as... Figure 7A As depicted in [the image / description]. Human DRG cells were plated at a density of 100,000 cells per well, containing approximately 100 neurons. Two to three days after platening, human DRG cells were transduced with various MOIs of AAV9 (e.g., MOI 1E+6, MOI 2E+6, etc.). Twelve to thirteen days after transduction with AAV9, RNAscope was used to determine the gene expression level of Nav1.7. The number of Nav1.7 mRNA transcripts determined by RNAscope was based on the fluorescent spot count of approximately 100 randomly selected cells in the microscopic image. The copy number per cell was determined by dividing the total spot count by the number of cells counted. Representative RNAscope images are shown in [the image / description]. Figure 7B middle.

[0190] RNAscope quantification of SCN9A in isolated human DRG

[0191] RNAscope quantification of SCN9A gene expression levels was performed. The results showed that the zinc finger protein ZF9 significantly inhibited the expression of SCN9A in vitro. Compared with human DRG cultures treated with mCherry (negative control), human DRG cultures treated with ZF9 showed 91% inhibition of SCN9A gene expression, such as... Figure 8 As depicted in the text.

[0192] The efficacy of the Nav1.7 specific promoter in transgene expression in mouse DRG ex vivo cultures

[0193] Transgenic expression in in vitro cultures of mouse DRGs with either the Nav1.7-specific promoter or the CMV promoter was measured. Both the CMV and Nav1.7-specific promoters resulted in strong mCherry expression in in vitro cultures following AAV9-mCherry transduction, such as... Figure 9 As shown in the image.

[0194] AAV9 Nav1.7-1 transduction throughout the DRG

[0195] Confocal imaging was used to assess the transduction of AAV9 Nav1.7-1 in the entire DRG. A schematic method for immunofluorescence of the entire mouse DRG is depicted in [the figure]. Figure 10A In the middle. 7.5 × 10¹ was administered to each mouse via intrathecal (IT) injection. 0 One vector genome (vg). After harvesting, tissues were collected and fixed for one day. After blocking and permeabilization, the tissues were cultured with primary antibodies at a concentration of 1:1000 (rabbit anti-neurofirhinone, rat anti-FLAG, and chicken anti-mCherry). Subsequently, the tissues were cultured with secondary antibodies at a concentration of 1:250 (goat anti-rat Alexa Fluor 647, goat anti-rabbit Alexa Fluor 488, and goat anti-chicken Alexa Fluor 594). Afterward, the tissues were counterstained with Hoechst staining and then mounted on slides for image analysis using a confocal microscope (Leica SP8). Figure 10B As shown, the Nav1.7-1 promoter induces mCherry expression in mice. Immunohistochemistry (IHC) of mouse liver showed that the Nav1.7-specific promoter did not induce transgene expression, as... Figure 10C As shown in the image. Infection of isolated human DRG cultures containing AAV-499-mCherry resulted in transgene expression, as shown in the image. Figure 10D As shown in the image.

[0196] Example 7

[0197] Efficacy of ZF4-KRAB delivered via AAV9 in target adhesion and pain relief

[0198] This example describes a chemotherapy-induced neuropathic pain model. After establishing in vivo efficacy in an inflammatory pain model, an epigenomic suppression strategy for neuropathic pain using the chemotherapy drug paclitaxel was tested in a multineuropathy model. To establish this model, mice were first injected with 1 × 10⁻⁶ oz. 12 Mice were administered AAV9-mCherry (n=8), AAV9-ZF4-KRAB (n=8), or saline (n=16) via vg / mice. A baseline of tactile thresholds (von Frey cilia) was established 14 days after administration and before paclitaxel administration. Paclitaxel was subsequently administered to mice at days 14, 16, 18, and 20 at a dose of 8 mg / kg (total cumulative dose 32 mg / kg). A group of mice injected with saline did not receive any paclitaxel (n=8) to establish tactile aberrations induced by chemotherapy drugs, such as... Figure 11AAs shown in the figure. Tactile aberration pain in mice was assessed via von Frey cilia at 21 and 105 days. One group of mice (n=8) injected with saline solution received an intraperitoneal injection of gabapentin (100 mg / kg) one hour prior to the test. Figure 11B As depicted in Figure 11C. The 50% tactile threshold was calculated. Significantly reduced tactile aneurysm was observed in mice injected with AAV9-zinc finger-4-KRAB at days 21 (P = 0.0007) and 105 (P < 0.0001) post-AAV9 injection. Additionally, mice in the AAV9-mCherry (n=8) group showed increased tactile aneurysm at day 105 compared to day 21, and responded to the lowest von Frey cilia examined (0.04 g). In contrast, the withdrawal threshold was increased in mice receiving AAV9-zinc finger-4-KRAB, suggesting that in situ NaV1.7 inhibition produces long-term prevention of chemotherapy-induced tactile aneurysm. In addition, an increased number of withdrawal responses was observed in mice tested for cold abnormal pain in the AAV9-mCherry negative control group, while a decreased number of withdrawal responses were observed in the AAV9-zinc finger-4-KRAB group (P<0.0001 for days 21 and 105), indicating that in situ inhibition of NaV1.7 also produces long-term prevention of chemotherapy-induced cold abnormal pain. Figure 11B , Figure 11C The key to the success of this treatment is the duration of action (or the half-life of the treatment) to avoid frequent lumbar punctures, and the results show that the treatment is durable (preventing pain for at least 44 weeks according to the inflammatory pain model and at least 15 weeks in the CIPN model). Figure 11D and Figure 11E ).

[0199] In vivo efficacy of ZF4-KRAB in the CIPN model

[0200] To determine the reversal of pain after an abnormal pain state is induced in a chemotherapy-induced peripheral neuropathy (CIPN) mouse model. Figure 12 As shown, downregulating NaV1.7 with ZF4-KRAB reverses abnormal pain induced by cycles of paclitaxel chemotherapy. A schematic diagram of a paclitaxel-induced neuropathic pain model is depicted in... Figure 11A In mice, AAV9-mCherry, AAV9-ZF4-KRAB, or saline were administered via intrathecal (IT) injection. Following baseline von Frey threshold testing on day 14, mice were subsequently administered paclitaxel (8 mg / kg paclitaxel, 32 mg / kg cumulative dose) via intraperitoneal (ip) injection at days 14, 16, 18, and 20 after IT injection. 21 days after IT injection... Figure 13A , Figure 13B , Figure 14A and Figure 14B ) Tianhe 105 ( Figure 13C , Figure 13D , Figure 14C and Figure 14D On [date], tactile aberrations in mice were assessed using the von Frey ciliary test and cold aberrations using the acetone test. Compared to the control, ZF4-KRAB reduced paclitaxel-induced tactile aberrations (…). Figure 13A ; Figure 14A Compared to the control, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain ( ). Figure 13B ; Figure 14B Compared to the control group, ZF4-KRAB reduced paclitaxel-induced tactile paresthesia 105 days after the last paclitaxel injection. Figure 13C ; Figure 14C Compared to the control, ZF4-KRAB reduced paclitaxel-induced cold abnormal pain ( ). Figure 13D ; Figure 14D Figure 13 shows the statistical analysis compared to the untreated group (no paclitaxel), and Figure 14 shows the statistical analysis compared to the mCherry group. Similar data were obtained using the CRISPR-dCas9 system (data not shown).

[0201] Safety assessment in mice

[0202] Preliminary safety assessments were established in mice. To determine the potential safety and side effects of NaV1.7 epigenetic repression via ZF-KRAB, we conducted a series of toxicity / side effect test groups to examine the general health and behavior of mice. These tests were sensitive to changes in self-care, increased pain / stress, and morbidity. We administered 1 × 10⁻⁶ ozontally to mice. 12Mice were given either AAV9-mCherry (n=8) or AAV9-ZF4-KRAB (n=8) via vg / molecular injection. We then examined the mice's body weight and temperature 8–12 weeks after the IT injection (Figs. 15A and 15B). AAV9-ZF4-KRAB did not produce any indicators of functional impairment. To determine if there were any changes in motor function, we performed a rotarod balance test, which requires various proprioceptive, vestibular, and fine-tuning motor abilities, as well as motor learning abilities (Carter et al., 2001). Mice were placed on a rotarod and subjected to an acceleration test strategy, where the rotarod started at 0 rpm and was subsequently accelerated at 10 rpm (Roberts et al., 1993; Finn et al., 1997). Fall time (seconds) was recorded and mice were tested in 3 trials across 3 groups, and we found no significant change in fall time compared to the control group (Fig. 15C). We also measured grip strength, marble burial, and nesting, but found no significant changes in mice injected with AAV9-ZF4-KRAB (Figs. 15D to 15F). Importantly, individuals with CIP exhibit olfactory loss due to NaV1.7 loss-of-function mutations (Cox et al., 2006; Weiss et al., 2011), and we performed an olfactory test to examine the mice's ability to locate desired food buried under their bedding. To determine if the mice had lost their sense of smell, we performed a test to examine their ability to locate desired food buried under their bedding. We found no significant changes in olfaction in mice injected with AAV9-ZF4-KRAB (Fig. 15G), which may indicate that AAV9 does not transduce neurons in the olfactory bulb. Finally, we performed cognitive tests to determine if any cognitive side effects were found using a novel object recognition test. This test analyzes and identifies memories while maintaining the spatial integrity of objects, and is believed to involve the hippocampus, nasal cortex, and raphe nuclei (Winters et al., 2004; Mumby et al., 2005; Lieben et al., 2006). The basic principle is that animals explore new environments, and with repeated exposure, exploratory behavior decreases (Berlyne, 1950) (i.e., habituation). We found no significant changes in cognition in mice injected with AAV9-ZF4-KRAB (Figure 15H). These results indicate that epigenetic repression of Nav1.7 via the zinc finger has no general effect on non-noxious behavior. Overall, ZF4-KRAB demonstrates safety and efficacy in treating chronic inflammatory and neuropathic pain in mice.

[0203] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A zinc finger protein comprising: (a) At least 90% sequence identity with SEQ ID NO: 2; (b) At least 98% sequence identity with SEQ ID NO: 3; or (c) At least 90% sequence identity with SEQ ID NO:

4.

2. The zinc finger protein according to claim 1, comprising at least 95% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

4.

3. The zinc finger protein according to claim 1, comprising at least 97% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

4.

4. The zinc finger protein according to claim 1, comprising the sequence of any one of SEQ ID NO: 1 to SEQ ID NO:

4.

5. The zinc finger protein according to any one of claims 1 to 4, wherein the zinc finger protein has an affinity for SCN9A.

6. The zinc finger protein according to any one of claims 1 to 5, wherein the zinc finger protein has an affinity for a polynucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO:73 to SEQ ID NO:

97.

7. The zinc finger protein according to any one of claims 1 to 6, wherein the zinc finger protein has an affinity for the polynucleotide sequence of any one of SEQ ID NO: 73 to SEQ ID NO:

97.

8. An epigenetic regulator comprising a zinc finger protein according to any one of claims 1 to 7 linked to a repressor domain.

9. An epigenetic regulator comprising a zinc finger protein linked to a repressor domain, wherein the zinc finger protein comprises at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO:

169.

10. The epigenetic regulator of claim 9, wherein the zinc finger protein comprises at least 95%, at least 97%, at least 98%, or 99% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO:

169.

11. The epigenetic regulator according to claim 9 or claim 10, wherein the zinc finger protein comprises any one of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 99 to SEQ ID NO:

169.

12. The epigenetic regulator according to any one of claims 8 to 11, wherein the zinc finger protein has an affinity for SCNA9.

13. The epigenetic regulator according to any one of claims 8 to 12, wherein the zinc finger protein has an affinity for a polynucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 73 to SEQ ID NO:

97.

14. The epigenetic regulator according to any one of claims 8 to 13, wherein the zinc finger protein has an affinity for the polynucleotide sequence of any one of SEQ ID NO: 73 to SEQ ID NO:

97.

15. The epigenetic regulator according to any one of claims 8 to 14, wherein the repressive domain comprises ZIM3, SID, KOX1, ZNF554, ZNF264, ZNF324, MeCP2, MBD2b, SID, HP1a, SIRT5, SETD8, HDT1, SUPR, FOG1, DNMT3A, DNMT3L, DMT3, or a combination thereof.

16. The epigenetic regulator according to any one of claims 8 to 15, wherein the repressive domain comprises at least 90% sequence identity with any one of SEQ ID NO: 5 to SEQ ID NO:

20.

17. The epigenetic regulator according to any one of claims 8 to 16, wherein the repressive domain comprises any one of SEQ ID NO: 5 to SEQ ID NO:

20.

18. The epigenetic regulator according to any one of claims 8 to 17, wherein the repressive domain comprises SEQ ID NO: 5 or SEQ ID NO:

9.

19. The epigenetic regulator according to any one of claims 8 to 18, wherein the zinc finger protein is linked to the repressor domain via a peptide linker.

20. The epigenetic regulator according to any one of claims 8 to 19, further comprising a second repressive domain.

21. The epigenetic regulator of claim 20, wherein the second repressive domain comprises ZIM3, SID, KOX1, ZNF554, ZNF264, ZNF324, MeCP2, MBD2b, SID, HP1a, SIRT5, SETD8, HDT1, SUPR, FOG1, DNMT3A, DNMT3L, DMT3, or a combination thereof.

22. The epigenetic regulator of claim 20 or claim 21, wherein the second repressive domain comprises at least 90% sequence identity with any one of SEQ ID NO: 5 to SEQ ID NO:

20.

23. The epigenetic regulator according to any one of claims 20 to 22, wherein the second repressive domain comprises any one of SEQ ID NO: 5 to SEQ ID NO:

20.

24. The epigenetic regulator according to any one of claims 20 to 22, wherein the second repressive domain comprises SEQ ID NO: 6 or SEQ ID NO:

7.

25. The epigenetic regulator according to any one of claims 8 to 24, comprising at least 80% sequence identity with SEQ ID NO: 26 or SEQ ID NO:

34.

26. The epigenetic regulator according to any one of claims 8 to 25, comprising at least 90% sequence identity with SEQ ID NO: 26 or SEQ ID NO:

34.

27. The epigenetic regulator according to any one of claims 8 to 26, comprising at least 95% sequence identity with SEQ ID NO: 26 or SEQ ID NO:

34.

28. The epigenetic regulator according to any one of claims 8 to 27, comprising SEQ ID NO: 26 or SEQ ID NO:

34.

29. The epigenetic regulator according to any one of claims 8 to 28, comprising SEQ ID NO:

25.

30. The epigenetic regulator according to any one of claims 8 to 28, comprising SEQ ID NO:

26.

31. The epigenetic regulator according to any one of claims 8 to 28, comprising SEQ ID NO:

27.

32. The epigenetic regulator according to any one of claims 8 to 28, comprising SEQ ID NO:

28.

33. A polynucleotide encoding a zinc finger protein according to any one of claims 1 to 7.

34. A polynucleotide encoding an epigenetic regulator according to any one of claims 8 to 32.

35. The polynucleotide of claim 33 or claim 34, further comprising a promoter.

36. The polynucleotide of claim 35, wherein the promoter comprises at least 90% sequence identity with any one of SEQ ID NO: 37 to SEQ ID NO: 47 and SEQ ID NO:

98.

37. The polynucleotide of claim 35 or claim 36, wherein the promoter comprises any one of SEQ ID NO:37 to SEQ ID NO:47 and SEQ ID NO:

98.

38. A delivery vector encapsulating a polynucleotide according to any one of claims 33 to 37.

39. The delivery vector of claim 38, comprising a viral vector.

40. The delivery vector of claim 39, wherein the viral vector comprises a delivery-enhancing peptide.

41. The delivery carrier of claim 40, wherein the delivery-enhancing peptide comprises protoxin, tarantula toxin, tarantula venom, flotoxin, Chilean rose tarantula venom, tiger tarantula venom, right-angled baboon tarantula venom, heteropod tarantula venom, ornate rainforest baboon tarantula venom or penetration-enhancing peptide.

42. The delivery vector of claim 40 or claim 41, wherein the delivery-enhancing peptide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 48 to SEQ ID NO:

72.

43. The delivery vector according to any one of claims 39 to 42, wherein the viral vector comprises an AAV vector, a lentiviral vector, a herpes simplex virus (HSV) or a rabies virus vector.

44. The delivery carrier according to claim 38, comprising lipid nanoparticles.

45. The delivery vector of claim 44, wherein the lipid nanoparticles encapsulate DNA, mRNA or circular RNA encoding an epigenetic regulator according to any one of claims 8 to 32.

46. ​​A method for downregulating Na in cells V 1.7 The method comprising expressing an epigenetic regulator according to any one of claims 8 to 32 in the cells.

47. A method of treating a disease in a subject, the method comprising expressing an epigenetic regulator according to any one of claims 8 to 32 in the subject.

48. The method of claim 47, wherein the disease is related to Na V 1.7 related.

49. The method according to any one of claims 47 to 48, wherein the ailment is pain, inflammation, and / or cancer.

50. The method according to any one of claims 47 to 49, wherein the disease is small fiber neuropathy, back pain, rheumatoid arthritis, osteoarthritis, spinal stenosis, chronic cough, migraine, trigeminal neuralgia, erythromelalgia, and paroxysmal excruciating pain.

51. The method of claim 49, wherein the inflammation is associated with arthritis.

52. The method of claim 51, wherein the arthritis is rheumatoid arthritis or osteoarthritis.

53. The method according to any one of claims 47 to 52, wherein treating inflammation includes preventing said inflammation.

54. The method according to any one of claims 47 to 53, wherein treating inflammation includes reducing said inflammation.

55. The method according to any one of claims 47 to 54, wherein treating pain includes preventing said pain.

56. The method according to any one of claims 47 to 55, wherein treating pain includes relieving said pain.

57. The method according to any one of claims 47 to 56, wherein the pain is associated with neurosis, chemotherapy, or inflammation.

58. A method for delivering an epigenetic regulator in the form of a purified protein into cells, the method comprising: The epigenetic regulator according to any one of claims 8 to 32 is administered to the cells.

59. The method of claim 58, wherein the epigenetic regulator is modified to enhance cellular uptake.

60. The method of claim 59, wherein the modification comprises fusion with a cell-penetrating peptide.

61. The method of claim 60, wherein the cell-penetrating peptide comprises TAT, polyarginine, or a combination thereof.

62. A method for delivering an epigenetic regulator in the form of RNA encapsulated in lipid nanoparticles, exogenous bodies, or liposomes, the method comprising: The epigenetic regulator is administered to the cells according to any one of claims 8 to 32.

63. A composition for direct protein delivery, comprising: (a) the epigenetic regulator according to any one of claims 8 to 32; and (b) Pharmaceutically acceptable carrier.

64. The composition of claim 63, further comprising a cell-penetrating peptide fused with the epigenetic regulator.

65. The composition of claim 63, further comprising liposomes encapsulating the epigenetic regulator.

66. A method for treating a disease in a subject, the method comprising: A therapeutically effective amount of the epigenetic regulator according to any one of claims 8 to 32 is administered to the subject via direct protein delivery.

67. The method of claim 66, wherein the epigenetic regulator is administered via a route selected from the group consisting of intravenous injection, subcutaneous injection, intramuscular injection, aerosol administration, and local application to a target tissue.

68. The method of claim 67, wherein the target tissue is selected from the group consisting of the trigeminal ganglion, the dorsal root ganglion, and combinations thereof.

69. The method according to any one of claims 66 to 68, wherein the disease is selected from the group consisting of pain, inflammation, cancer, small fiber neuropathy, back pain, rheumatoid arthritis, osteoarthritis, spinal stenosis, chronic cough, migraine, trigeminal neuralgia, erythromelalgia, and paroxysmal excruciating pain.

70. The method according to any one of claims 66 to 68, wherein the disorder is trigeminal neuralgia.

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