Voltage-gated sodium channel specific peptide aptamers and uses thereof

Abstract

Disclosed herein are polypeptide aptamers comprising peptide aptamers that functionally interact with Nav1.7 voltage gated sodium channels, polynucleotides and infectious particles encoding the polypeptide aptamers, and methods of treating diseases and conditions, such as neuropathic pain, using the same.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 495,946, filed April 13, 2023, the entire contents of which are incorporated herein by reference. Statement on Federally Funded Development Research

[0002] not applicable. sequence list

[0003] This application includes an ordered list, which is submitted as an XML file named "650053_01054.xml", measuring 93,688 bytes, created on April 15, 2024. This order list was submitted electronically through the Patent Center and is incorporated herein by reference in its entirety. Background Technology

[0004] Voltage-gated sodium channel (Na V Na+ is a key regulator of neuronal excitability and pain perception (1). Mammals possess nine types of Na+. V Subtype, of which Na v 1.7 、 Na v 1.8 and Na v 1.9 It is mainly expressed in the primary sensory neurons (PSN) of the dorsal root ganglion (DRG) (2). The important role of these NaV subtypes in human pain has been verified, for example, Na v Loss-of-function mutations at 1.7 (SCN9A) result in congenital insensitivity to pain (CIP); conversely, gain-of-function mutations result in hereditary erythromelalgia (IEM) and paroxysmal severe pain disorder (PEPD), which have symptom spectra opposite to CIP (2). V 1.6, Na V 1.1 and Na V 1.3 is also expressed in PSN and has been reported as a potential target for analgesics (3, 4). Currently, Na... V 1.7 is Na V The primary target for developing analgesic therapies (5).

[0005] Over the past few decades, significant efforts have been made to develop selective and efficient Na+ technology. V 1.7 Blockers have been used to treat pain (6), but with limited success. Most tests are conducted on small molecule sodium for pain treatment. V1.7 Blockers have shortcomings in terms of target binding, lack of target specificity or selective bioavailability of pain axes, and their systemic distribution can lead to cardiotoxicity, dyskinesia, and central nervous system (CNS) side effects (6, 7). Developing targeted Na V 1.7 biologicals are another growing trend in the analgesic field (8-10). Na V 1.7 neutralizing monoclonal antibodies have analgesic efficacy, but their results are not always consistent (11). Tarantula peptides Na V 1.7 blockers (10, 12) have analgesic effects, but poor membrane permeability, Na V 1.7 lack of selectivity and short half-life (6). It has been suggested to use Na v 1.7-RNAi (6) and CRISPR-dCAS9 or ZEN epigenetic Na V 1.7 inhibitory gene therapy for analgesia (13), but these interventions at the mRNA and epigenetic levels lack the specificity of direct channel interventions, reduce safety and allow off-target effects (6, 14, 15), and CAS9 immunity poses additional challenges for CRISPR gene therapy (16). Thus, there is a need in the art for improved analgesics and improved strategies to treat pain in affected subjects. SUMMARY

[0006] In one aspect of the disclosure, a polypeptide aptamer is provided. In some embodiments, the polypeptide aptamer comprises a sequence selected from the group consisting of SEQ ID NOs: 1-9, 50, and 52, or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9, 50, and 52. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v 1.7 channel protein.

[0007] In another aspect of the disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human NaVl.7 channel protein. v 1.7 Channel protein.

[0008] In another aspect of the disclosure, a polynucleotide is provided. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human NaVl.7 channel protein. v1.7. Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or enhancer, wherein the nucleotide sequence is operably linked to the promoter or enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

[0009] In another aspect of the disclosure, an infectious particle is provided. In some embodiments, the infectious particle comprises a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9, or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v 1.7. Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or enhancer, wherein the nucleotide sequence is operably linked to the promoter or enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6).

[0010] In another aspect of the disclosure, pharmaceutical compositions are also provided. In some embodiments, the pharmaceutical composition comprises an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9, or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v 1.7 Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or an enhancer, wherein the nucleotide sequence is operably linked to the promoter or the enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6).

[0011] In another aspect of the disclosure, methods are provided. In some embodiments, the method comprises contacting a cell with a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9 or an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6 or a sequence having at least 85% identity to one of SEQ ID NOs: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1 or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence and the order from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v 1.7 Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or an enhancer, wherein the nucleotide sequence is operably linked to the promoter or the enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6). In some embodiments, the method reduces the peak Na v 1.7 Current density by at least 50%. In some embodiments, the method does not change the Na v 1.7 Channel's steady-state inactivation properties.

[0012] In some embodiments, a method of reducing or inhibiting stimulation of a neuron is provided. In some embodiments, the method comprises contacting the neuron with a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9 or an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6 or a sequence having at least 85% identity to one of SEQ ID NOs: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1 or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v 1.7 Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or an enhancer, wherein the nucleotide sequence is operably linked to the promoter or the enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6). In some embodiments, the method reduces the peak Na v 1.7 Current density by at least 50%. In some embodiments, the method does not change the Na v 1.7 Channel's channel-steady-state inactivation properties.

[0013] In some embodiments, other methods are provided. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9.

[0014] In some embodiments, methods of treating neuropathic pain in a subject in need thereof are provided. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a pharmaceutical composition to treat neuropathic pain in the subject, the pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NOs: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order of arrangement from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v1.7 Channel proteins. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or enhancer, wherein the nucleotide sequence is operably linked to the promoter or enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6). In some embodiments, the administering comprises local delivery of the pharmaceutical composition. In some embodiments, the local delivery of the pharmaceutical composition comprises delivery to a dorsal root ganglion of the subject. In some embodiments, the method reduces mechanical allodynia or cold allodynia in the subject. In some embodiments, the subject is a human subject. In some embodiments, the subject has chronic pain. In some embodiments, the subject has neuropathic pain or neurogenic pain. In some embodiments, the subject has been diagnosed with osteoarthritis.

[0015] In some embodiments, a method of treating pain resulting from a traumatic nerve injury in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition to treat pain resulting from a traumatic nerve injury, the pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NO: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds human Na v1.7 Channel proteins. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or enhancer, wherein the nucleotide sequence is operably linked to the promoter or enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the adeno-associated virus (AAV) is an AAV type 6 (AAV6). In some embodiments, the administering comprises local delivery of the pharmaceutical composition. In some embodiments, the local delivery of the pharmaceutical composition comprises delivery to a dorsal root ganglion of the subject. In some embodiments, the method reduces mechanical sensitization or cold sensitization in the subject. In some embodiments, the subject is a human subject. In some embodiments, the subject has chronic pain. In some embodiments, the subject has been diagnosed with osteoarthritis.

[0016] In another aspect of the disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide encoding a human Na v 1.8 Protein 。 In some embodiments, the human Navl.8 protein has the amino acid sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the human Na v 1.8 Protein v In some embodiments, the heterologous polynucleotide encoding the human Na v b2 Protein v In some embodiments, the polynucleotide encoding the human Na

[0017] In another aspect of the disclosure, a kit, system, or platform is provided. In some embodiments, the kit, system, or platform comprises a polypeptide aptamer or a polynucleotide and reagents for performing an electrophysiology experiment; the polypeptide aptamer comprises a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9; the polynucleotide comprises a sequence encoding a polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. In some embodiments, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NOs: 1, 4, or 6. In some embodiments, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the sequence is SEQ ID NO: 1. In some embodiments, the polypeptide aptamer further comprises a detectable marker. In some embodiments, the detectable marker comprises a fluorescent protein. In some embodiments, the fluorescent protein comprises a green fluorescent protein. In some embodiments, the polypeptide aptamer further comprises a linker. In some embodiments, the linker comprises SEQ ID NO: 10. In some embodiments, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, the linker, and the sequence are a single contiguous peptide sequence, and the order from N-terminus to C-terminus is the detectable marker, the linker, the sequence. In some embodiments, the polypeptide aptamer binds to human Na v 1.7 Channel protein. In some embodiments, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19. In some embodiments, the polynucleotide further comprises a promoter or an enhancer, wherein the nucleotide sequence is operably linked to the promoter or the enhancer. In some embodiments, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter. In some embodiments, the polynucleotide further comprises at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. In some embodiments, the electrophysiology experiment comprises patch clamp electrophysiology.

[0018] In some embodiments, the kit, system, or platform comprises a polypeptide aptamer or a polynucleotide, and instructions for treating a subject having neuropathic pain, the polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9, the polynucleotide comprising a sequence encoding a sequence selected from the group consisting of SEQ ID NOs: 1-9 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1A 、 1B , 1C, 1D, 1E, 1F, 1G, 1H, 1I and 1J. Na v 1.7 - Computer prediction and candidate Na V 1.7iPA (1.7iPA) design. Rat Na v 1.7 protein with Na v 1.7 DI-DIV (A) , the lower red bar shows the location of the predicted iPA (B). DEPICTER consensus prediction for IDR (C). DEPP predicted phosphorylation sites (D). Nine candidate iPA and their amino acid (aa) sequences, location in Na v 1.7, and IDR scores, from top to bottom, correspond to SEQ ID NO: 1-9 (E). Map showing the individual components of the AAV plasmid encoding GFP-iPA, with the black line pointing to the iPA (F). Structure analysis of GFP-fused 1.7iPA1 by I-TASSER, with the upper panel showing the structure of free 1.7iPA1 (G). Images (GFP, left; phase, middle; merged picture, right) show expression of constructs carrying 1.7iPA1-4 and 6 after transfection of HEK cells (H). Scale bar: 25 μm. After transfection of HEK cells with 1.7iPA1-4 and 6, GFP and Gapdh western blot analysis was performed on cell lysates (I). Nine iPA on I Na were initially screened by whole-cell patch-clamp recordings. * and *** indicate p<0.05 and 0.001, respectively; one-way ANOVA and Tucky post-hoc test (J).

[0020] Figure 2A 、 2B , 2C, 2D, 2E and 2F. Confirmation of 1.7iPA1, 4 and 6 inhibition of I Na 1.7. (A) I Na 1.7 representative traces. Inset: recording protocol and current / time scale. Summary of confirmation tests of candidate iPA expression in HEK1.7 cells, with comparison of the average peak current density-voltage (I / V) relationships for the different constructs shown in (B), and the average peak I Na1.7 Quantitative analysis of density; *p < 0.001, one-way ANOVA followed by Tukey’s post-hoc test. Expression of GFPiPA1, GFPiPA4 and GFPiPA6 had no effect on steady-state activation (D, inset: V1 / 2 activation) and fast inactivation (E, inset: V1 / 2 inactivation) compared to naive cells and GFP or NP-transfected HEK 1.7 cells. v iPA1 is highly conserved in rat, mouse and human (SEQ ID NOs: 30-32 from top to bottom) (F). Black and yellow stars at the bottom indicate positively and negatively charged amino acids; red and blue stars at the top indicate known lysine acetylation and serine phosphorylation sites, IDR score and percentage of positively (+) and negatively (-) charged amino acids are shown at the right of the alignment.

[0021] Figure 3A 、 3B , 3C, 3D, 3E, 3F and 3G. Na V Sodium channel specificity of iPA1 inhibition. 1.7iPA1 with rat species TTXs Na V 1.6, Na V 1.3, Na V 1.1 (SEQ ID NOs: 1 and 33-35) (A), and TTXr Na V 1.5, Na V 1.8 and Na V 1.9 (SEQ ID NOs: 1 and 36-38) (B). Amino acid sequence alignment of the corresponding sequences. Homologous amino acids (identity and similarity) are highlighted in dark or light grey shading and the percentage of identical or similar amino acids is shown at the right of the alignment. (C-G) Group plots from left to right showing I Na Comparison of the traces (inset: pulse protocol and scale); peak I Na Density (p < 0.001, one-way ANOVA and Turkey’s post-hoc test), I / V curve, steady-state activation (inset: V1 / 2 activation) and fast inactivation kinetics (inset: V1 / 2 inactivation).

[0022] Figure 4A 、 4B , 4C, 4D, 4E, 4F, 4G and 4H. Na V Effect of iPA1 on I Na of rat DRG neurons (male) and hiPSC-SNs (female). (A-C) Group plots from top to bottom showing total I Na (A), TTXs I Na(B) and TTXr I Na Representative trajectory and average peak value of (C) Na Density data were derived from sensory neurons (≤35 μm in diameter) isolated from primordial male rats treated as follows: (from left to right) sham treatment (surgical exposure only, but no injection), and L4 / L5 DRG injection of AAV6-encoded GFP, GFP-NP, and GFP-NaViPA1 4 weeks later. Insets: Representative PSN images for each group (scale bar: all 25 μm), current / time scale, and recording pulse protocol. (D, E) Representative mosaic ICC images showing LV-GFPNP (D) and LV-GFPNaViPA1 at the same MOI=5. V hiPSC-SN at DIV25 after iPA1(E) transduction. (FH) shows hiPSC-SN from NP expression and Na expression. V The total I recorded by hiPSC-SN (DIV25) of iPA1 Na (F), TTXs I Na (G) and TTXr I Na Representative trajectory and average peak value of (H) Na Density. Illustration: Current / time scale and recording pulse scheme. *, ** and *** indicate p<0.05, 0.01 and p<0.001, respectively; one-way ANOVA and Turkey post-hoc test.

[0023] Figure 5A , 5B 5C, 5D, 5E, 5F, 5G, 5H, 5I, and 5J. Na V iPA1 binds to full-length Na v 1.7 Protein and phosphoinositol. (A) Immunoblotting (IB) shows the Na+ content of cell lysates from primordial HEK cells as well as HEK1.5, HEK1.7, HEK1.6, HEK1.1, HEK1.3, CHO1.8 cells and 50B11 cells. v 1.7 Antibody selectivity. Representative immunohistochemical images (BE) show Na+ concentrations in SDH (red), sciatic nerve (green), DRG neurons (red), and cutaneous nerve fibers (red). v 1.7 Detection. Scale bar: 100 μm. (F) From pseudo-treatment (no plasmid transfection), GFP, GFPNa V Membrane samples and Na+ in the cytoplasm of HEK1.7 cells transfected with iPA1 and GFP1.7iPA2 were collected. v 1.7, GFP, NKA, NKA1a, Gapdh's IB. (G) Use "non-denaturing" lysis buffer to lyse GFP, GFPNaV Input (cell lysate, 20 mg each lane) and pull-down beads (10 mL each lane) from iPA1 and GFP1.7 iPA2 transfected HEK 1.7 cells. Na v 1.7 IB (left) and silver staining (right). From top to bottom are SEQ ID NOs: 39-46. (H) Mass spectrometry analysis of the 100-300 kDa stained gel pieces excised from GFPNa V iPA1 (G, red asterisk indicates Na v 1.7 site) shows the presence of unique human Na V iPA1 pull-down sample (red). In GFPNa v 1.7 peptides (red). In GFPNa V 1D SDS-PAGE gel silver staining (I) and results of PIP strip analysis (J) of GFP-affinity pulldown beads using denaturing RIPA buffer prepared from cell lysates of iPA1 and GFP transfected NG108-15 cells. LPA, lysophosphatidic acid; LPC, lysophosphatidyl choline; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol (3) phosphate; PtdIns(4)P, phosphatidylinositol (4) phosphate; PtdIns(5)P, phosphatidylinositol (5) phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1 -phosphate; PtdIns(3,4)P2, phosphatidylinositol (3,4) bisphosphate; PtdIns(3,5)P2, phosphatidylinositol (3,5) bisphosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5) bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5) triphosphate; PA, phosphatidic acid; PS, phosphatidylserine.

[0024] Figure 6A , 6B , 6D, 6E, 6F, 6G, and 6H. Na vMultiple basic NLS and adjacent serines in iPA1. (A) Sequence alignment of NaviPA1, mutant 1 (mt) with ten serine residues replaced by alanine, mt2 with predicted NLS domain arginine / lysine (R / K) replaced by alanine, and mt3-6 with double or triple serine residues replaced by alanine at different serine sites as indicated. From top to bottom are SEQ ID NO: 1 and 47-52. (B-E) ICC comparison of GFP signal 48 hours after plasmid transfection of HEK 1.7 cells with NaviPA1, GFPNP, mt1, and mt2. (F) Representative immunoblots of HEK 1.7 and GFPNP, NaviPA1, mt1, and mt2 in cytosolic, membrane, and nuclear loads indicated by GAPDH, NKA1a, and LamB1, respectively. (Fl) Quantitative comparison of membrane binding and nuclear entry of NaviPA1, mt1, and mt2 after transfection, * and ** indicate p<0.05 and 0.01, one-way ANOVA and Tukey’s post-test. (G) Representative I v 1.7 and GFPNP, NaviPA1, mt1, and mt2. Cytosolic, membrane, and nuclear loads are indicated by GAPDH, NKA1a, and LamB1, respectively. (Fl) Quantitative comparison of membrane binding and nuclear entry of NaviPA1, mt1, and mt2 after transfection, * and ** indicate p<0.05 and 0.01, one-way ANOVA and Tukey’s post-test. (G) Representative I Na 1.7 traces. (H) Quantitative summation of peak I Na densities; *, **, and *** indicate p<0.05, <0.01, and <0.001; one-way ANOVA and Tukey’s post-test.

[0025] Figure 7A 、 7B , 7C, 7D, 7E, 7F, 7G, 7H, 7I, and 7J. Established neuropathic pain was treated by DRG AAV6-NaViPA1 (male rats). Purified AAV was prepared according to the animal experimental protocol in the schematic (B) (A, silver staining). DRG was injected with AAV6-Na V vF, pinprick (Pin), heat (Heat), and cold (Cold) tests before and after iPA1 (n=7) or AAV6-NP (control, n=8) (C-F). For treatment data, we used the measurement at day 14 after TNI and before AAV treatment (tBL) as the peak pain intensity (100%), and normalized the measurements of each sensory modality after treatment to the measurement at tBL, and calculated the percentage of pain relief for each modality at multiple time points (Figures 1C1-F1). * p<0.05, ** p<0.0,1, and *** p<0.001 compared to tBL within group, and # p<0.05, ## p<0.01, and ###p < 0.001. Repeated measures two-way ANOVA for vF and heat tests with Tukey post-hoc analysis within groups and Bonferroni post-hoc analysis between groups; non-parametric Friedman ANOVA for needle and cold tests with Dunn’s post-hoc. The average pain relief over the 6 weeks of treatment showed a 52%, 49%, 69% and 67% reduction in mechanical and thermal pain behavior for vF, needle, cold and heat test stimuli, respectively (Figure G). ** p < 0.01 and p < 0.001, unpaired two-tailed Student’s t-test. (H) AAV-Na V iPA1 (n = 7) vs. AAV-NP (control, n = 8) pre-conditioning chamber and GBP paired chamber CPP scores (seconds, s), *** p < 0.001 (unpaired, two-tailed Student’s t-test).

[0026] Figure 8A 、 8B , 8C, 8D, 8E, 8F and 8G. IHC and target gene expression of GFP-NaViPA1. (A-D) Representative IHC montages (GFPNa V iPA1 vs. Tubb3) showing AAV-Na V iPA1 injection for 6 weeks (A), GFP-Na V iPA1 vs. Na V 1.7 and Na V 1.6 positive neurons (B, C), but not colocalized with GFAP positive perineuronal glial cells (D, square area magnified, montage shown as D1). (E-G) Representative IHC montages showing GFPNa V iPA1 (green) and Na V 1.7 (red) (E), GFPNa V iPA1 (green) and Tubb3 (red) (F) in the sciatic nerve, and GFPNa V iPA1 (green) and NF200 (red) (G) in the skin section at the peripheral end of PSN. Scale bars (pm): A, 200; B, C, D and D1, 100; E, 200; G and G, 50 pm.

[0027] Figure 9A 、 9B , 9C, 9D, 9E and 9F. Na VEffect of iPA1 expression on rat PSN (male) neuronal excitability. (A, B) Representative AP traces evoked by 180 pA (A) and 280 pA (B) depolarizing current (same cell) at RMP, recorded from DRG neurons of sham, TNI only, and TNI rats treated with AAV6-NP or AAV6-Na V GFP-expressing neurons in iPA1 -treated TNI rats. (C) Comparison of responses (number of APs evoked by 250 ms stimulus) of DRG neuron populations in different groups over a range of step current injections from 100 to 280 pA; ***p < 0.001, two-way ANOVA on group main effects and Bonferroni post-test. Bar-scatter plots showing analysis of rheobase (D) measured from RMP and number of APs evoked by 180 pA (E) and 280 pA (F) input current, respectively. Numbers in each group are the number of neurons analyzed in each group. * and *** indicate p < 0.05 and p < 0.001, respectively, one-way ANOVA and Turkey post-test.

[0028] Figure 10A , 10B , 10C, 10D, 10E, 10F, 10G and 10H. DRG-AAV6-Na V Analgesic effect of iPA1 treatment in female TNI rats. Similar to Figure 7, showing DRG delivery of AAV6-Na VSignificant analgesic effect (A-D) and percentage of pain reduction (A1-D1) were observed after iPA1. *, ** and *** indicate p < 0.05, 0.01 and 0.001 for intra-group comparisons compared to treatment baseline (tBL); # p < 0.05, ## p < 0.01 and ### p < 0.001 for inter-group comparisons. Repeated measures parametric two-way ANOVA was performed for vF and heat tests followed by Tukey post-hoc analysis for intra-group and Bonferroni post-hoc analysis for inter-group; non-parametric Friedman ANOVA with Dunn’s post-test was performed for pinprick and cold tests. Right panel A2-D2 shows the average pain relief for each modality over the 3.5 months of treatment, ** p < 0.01 and *** p < 0.001 for inter-group tAUC comparisons (unpaired, two-tailed Student’s t-test). Difference score (s) in CPP between pre-conditioning chamber and GBP paired chamber for AAV-NaViPA1 (n = 8) and AAV-NP (control, n = 8), * p < 0.01 (unpaired, two-tailed Student’s t-test) (E). Representative montage IHC images of GFP-NaViPA1 colocalization with Tubb3 (F), NaV1.7 (G) and NaV1.6 (H) show expression profile of neurons 6 weeks after AAV-NaViPA1 injection. Scale bar: 100 pm for F-H.

[0029] Figure 11A , 11B, 11C, 11D, and 11E. In silico DEPICTER and I-TASSER. (A) IDR predicted by DEPICTER (3) which aggregates results from multiple servers (IUPred-L and -S: Predicting intrinsically disordered proteins; SPOT-Disorder-Single: Accurate single-sequence prediction of intrinsically disordered proteins; Disco Protein Binding: Prediction of IDR protein binding; fMoRFpred: Fast prediction of molecular recognition features; DisoRDPbind: Predictor of disordered RNA, DNA, and protein binding regions; Anchor 2: Potential binding sites in disordered regions; DiscoRNA (DNA) Bind: Prediction of potential RNA (DNA) binding regions; Disordered: Flexible linker predictor; DiscoMultipleFunction: Annotation of disordered multifunctional residues), each algorithm is indicated on the left. (B-E) Crystal structure analysis of GFP1.7 iPA2, 3, 4, and 6 by I-TASSER as indicated.

[0030] Figure 12A , 12B , 12C, 12D, and 12E. HEK cell-based Na V1.8 Establishment of stable expression system. (A) Comparison of I / V curves of HEK 1.7 and HEK 1.8 cells. (B and C) Voltage-dependent activation and steady-state fast inactivation curves recorded from HEK 1.8 cells, HEK 1.7 cells, and HEK 1.5 cells. Inset: recording protocol. (D) I Na 1.8 Inhibition of (D) by TTX (1-30 mM) in HEK 1.8 cells Na 1.7 Comparison of inhibition of (E). ** and *** indicate p<0.01 and 0.001, Student's t test (F and L), and one-way ANOVA and Tucky post-hoc analysis (K).

[0031] Figure 13A 、 13B , 13C, 13D, and 13E. Na V Effect of iPA1 on transiently expressed I Na 1.8 No effect. From Na V 1.8 + GFPNP and Na V 1.8 + Na V Representative I Na 1.8 currents (A). Na v 1.8 + GFPNP (n=21) and Na v Peak current density scatter plots of HEK 1.8 cells transfected with iPA1 (n=22) (B). Na V 1.8 + GFPNP and Na V I / V curves of HEK 1.8 cells transfected with iPA1 (n=22) (C). Na V 1.8 + GFPNP and Na V Comparison of activation and inactivation voltage dependence of HEK 1.8 cells transfected with iPA1 (n=22) (D). Na V 1.8 + GFPNP and Na V 1.8 + Na V Comparison of inactivation recovery of HEK 1.8 cells transfected with iPA1 (n=22) (E). In this set of experiments, pipette solution contained (mM): CsCl 140, NaCl 10, EGTA 1.1, HEPES 10, pH 7.30; bath solution contained (mM): NaCl 140, TEA-Cl 20, KCl 3, CaCl2 1, MgCl2 1, HEPES 10, pH 7.30, and 1 μM TTX.

[0032] Figure 14A 、 14B , 14C, 14D, 14E, 14F, 14G, and 14H. Na ViPA1 has effects on NG108 and F11 cells. Na 1.7, NG108 cells I Kv HVA I of DRG neurons Ca The effect. Representative I at 0 mV recorded from cells transfected with pseudo-treatment, GFPNP, and NaViPA1. Na 1.7 Single Trajectory, Merged I Na 1.7, I / V curves and TTX (0.1mM) curves, and peak I values ​​for NG108-15 cells (AC) and F11 cells (DF). Na 1.7 Density. Illustration: Scheme and current / time scale. ***p<0.001, one-way ANOVA and Turkey's post-hoc test. (G) Representative I of sham-treated NG108 cells. Kv The figure shows I defined by the outward current blocked by tetraethylammonium (TEA, 5mM). Kv Alternatively, use GFP, 1.7 NP, and Na. V Representative I of iPA1-transfected NG108 cells Kv (G1-G5). Illustration: Recording scheme and current / time scale. I Kv Density-voltage (I / V) curve (G6) and peak I Kv Quantitative analysis of density (G7), p>0.05, one-way ANOVA and Tukey post-hoc test. (H)DRG neuron HVA I Ca Record. Typical HVA I in primary rat miniature neurons. Ca The trajectory showed an activation threshold of approximately −30 mV, a maximum current amplitude of activation of -10 mV, and minimal inactivation (H). Sham-treated rats (H1) and rats injected with AAV6-GFP (H2), -1.7NP (H3), and -Na... V Typical HVA I recorded at -10 mV in primitive rat neurons of iPA1(H4) Ca Trajectory. HVA I Ca Density-voltage (I / V) curve (H5) and average peak HVA I Ca Density (H6), p>0.05, one-way ANOVA and Tukey post-hoc test.

[0033] Figure 15A and 15B TTXs and TTXrI in primary rat DRG neurons Na Recordings. (A-A2) Voltage-gated total, TTXr (1.0 mM TTX bathed in the same cells), and TTXs (total I) recorded from small DRG neurons. Na Subtract TTXr I Na )INa Representative trajectory. Illustration: Scale, voltage scheme, and peak I. Na Concentration quantification. (B-B2) Voltage-gated total, TTXs, and TTXr I recorded from small DRG neurons. Na 1.8 representative trajectories (manipulated via voltage scheme). TTXr and TTXs I Na The separation scheme: Apply a 500 ms pre-pulse of -120 or -50 mV, followed by a 50 ms test pulse of -100 to +40 mV in 10 mV steps (illustrated). TTXs and TTXr I Na Clearly visible after a -120 mV prepulse (top trajectory); only TTXr I is obtained after a -50 mV prepulse. Na (Bottom trajectory), TTXs components pass through total I Na Subtract TTXr I Na The numerical subtraction is used to obtain (intermediate trajectory). (B3) Total, TTXs and TTXr of small DRG neurons Na Average peak value I Na Density-voltage relationship. The smoothed line is the IV curve generated based on Boltzmann fitting parameters. Total, TTXs, and TTXr I Na Standardized peak I Na Density (B5) and average peak value I Na Density (B4).

[0034] Figure 16 Expressing Na V Lentiviral vector constructs of iPA and NP.

[0035] Figure 17A , 17B And 17C. hiPSC-SN's I Na BK I Kv and HVA I Ca Records. (A) Records from the original hiPSC-SN (DIV25, illustration: scheme and scale) total I Na TTXr I Na (1mM TTX in bath solution), TTXs I Na Representative trajectories and quantitative analysis of peak currents were performed. A subtraction scheme was used to separate TTXr and TTXs. Na (B) Recorded from the original hiPSC-SN (DIV21), TEA added to the bath solution, and Na expression. V iPA1 hiPSC-SN and NP-expressing hiPSC-SN BK I Kv Representative trajectory, and peak BK I KvQuantitative analysis of density (panel from left to right). No differences, one-way ANOVA and post-hoc test. V Representative HVA I of hiPSC-SNs of iPA1 and NP-expressing hiPSC-SNs Ca Trajectories, and peak HVA I Ca Quantitative analysis of density (panel from left to right). No differences, one-way ANOVA and post-hoc test.

[0036] Figure 18 . Na v I Na No effect. Representative I Na Trajectories and peak current density are shown in the graphs.

[0037] Figure 19A , 19B , 19C, 19D, 19E and 19F. Na V In vivo preliminary analgesic test of iPA1. (A-C) Mean sensitivity to vF, pinprick and cold tests groups after 3 weeks of TNI induction after DRG injection of AAV6-Na V iPA1 (n=5) or AAV6-NP (control, n=5) and time course of mean sensitivity to vF, pinprick and cold tests groups after 3 weeks of TNI induction after AAV injection. $$$ indicates p<0.001, tBL compared to 1 week after injection. **p<0.05 and ***p<0.001 (A) indicate intra-group comparisons with 1 week after AAV injection, ## p<0.01 and ### p<0.001 indicate inter-group comparisons. Repeated measures parametric two-way ANOVA followed by Tukey post-hoc analysis intra-group and Bonferroni post-hoc analysis inter-group for vF and heat tests; non-parametric Friedman ANOVA with Dunn’s post-hoc test for pinprick and cold tests. Right panel of A-C shows TNI tAUC calculated using measurements at 35 days after AAV; * p<0.05 and **p<0.01, unpaired two-tailed Student’s t-test for vF, Mann-Whitney U test for pinprick and cold tests. Representative montage IHC images of DRG sections (D, co-labelling of GFP with Tubb3 showing co-localization in merged images), ipsi. hindpaw skin sections (E) with dashed lines dividing dermis (De) and epidermis (Epi) borders and spinal cord sections (F) co-labelling with GFP and CGRP (red) showing co-localization in merged images (arrows pointing to reduced CGRP innervation in ipsi. DH) are shown.

[0038] Figure 20A , 20B, 20C, 20D, 20E, 20F, 20G, and 20H. HEK-Na V 1.8 Establishment of stable expression system. (A) pcDNA3.1(+)-SCN10A- Furin-P2A-SCN2B expression plasmid. (B-D) Immunoblotting of cell lysates confirms Na V 1.8a (B) and Na2b (C) stable expression, both highly enriched at the plasma membrane (D). Comparison of inward I Na1.8 Traces (E), A803467 responses (F), and single trace (+10 mV) comparison of HEK 1.8 and Cho 1.8 cell recordings (G). Comparison of I / V curves of HEK 1.7 and HEK 1.8 cells (H).

[0039] Figure 21A , 21B and 21C. 1.7 / 1.8 iPA inhibits I Na 1.7 and I Na 1.8. (A-C) Transfection of 1.7 / 1.8 iPA into NG108 cells has no effect on BK channels (bath contained 2 mM CaCl2). *, **, and *** indicate p<0.05, 0.01, and 0.001, one-way ANOVA and Tukey post-hoc test.

[0040] Figure 22A , 22B , 22C, 22D, and 22E. 1.7 / 1.8 iPA and sequence alignment. (A) Na V 1.7 (top) intracellular fragment IDR (bottom, gray area). Location of 1.7 / 1.8 iPA (red). (B-D) Conservation of AnkG and Pdzd2-I domains (red boxes) between (B) within 1.7 / 1.8 iPA (SEQ ID NO: 54-56), (C) between TTXs Na v (SEQ ID NO: 58-61), and (D) between Na V 1.7 and TTXr Na v (SEQ ID NO: 62-67); * at top of amino acid sequences indicates CK2 phosphorylation site that enhances binding to AnkG; alignment right shows IDR score, percent identity or similarity, and percent of negatively charged (-) amino acids. (E) Similar AnkG binding amino acid sequences in 1.7 / 1.8 iPA and Kv7.2 / 7.3, SEQ ID NO: 68-71.

[0041] Figure 23A , 23Band 23C. Kv7.2 / 7.3 M-current recordings (NG108 cells). M-currents were elicited by a 300 ms depolarizing step ranging from -80 mV to +120 mV in 10 mV increments with a 5 s interval between steps and a starting holding potential of -80 mV. 37、43 (A-C) M-current traces (A, XE991, selective I Kv 7.2 / 7.3 inhibitor), I / V curves (B) and peak I Kv 7.2 / 7.3 density (C) showing results with and without XE991, respectively. *p<0.05 and ***p<0.001, one-way ANOVA and Tukey post-hoc test.

[0042] Figure 24 Design of site-directed mutagenesis. 1.7 / 1.8 iPA and mutant 1.7 / 1.8 iPA sequences, with boxes indicating AnkG and Pdzd2-I domains; E, key amino acid for AnkG binding; * points to CK2 phosphorylation site, SEQ ID NOs: 57 and 72-75.

[0043] Figure 25 AAV plasmid expressing 1.7 / / 1.8 iPA. The DNA sequence of 1.7 / 1.8 iPA was cloned with a linker (GLRSRAQASNSAVDGTAGPGS) to form a chimeric single-GFP- linker-1.7 / 1.8 iPA configuration driven by the CBA promoter.

[0044] Figure 26A , 26B , 26C and 26D. (A) Na V 1.7 (top) intracellular fragment IDR (bottom, grey area). Position of 1.7 / 1.8 iPA (red). Expression of 1.7 / 1.8 iPA (GFP fusion) in HEK 1.7 (B) and HEK 1.8 (C) cells inhibited I Na 1.7 and I Na 1.8. (D) AnkG and Pdzd2-I domains (rectangles) within the sequence of 1.7 / 1.8 iPA SEQ ID NO: 53.

[0045] Figure 27 Sequence annotation of pCMV-cDNA3.1(+)-hSCN10A- Furin P2A-hSCN2B. DETAILED DESCRIPTION

[0046] Tetrodotoxin-sensitive (TTXs) voltage-gated sodium channels Na V 1.7 is encoded by the human SCN9A gene and is expressed on nociceptive neurons. The inventors designed NaV 1.7. The novel peptide aptamer inhibitors bind to Na V 1.7. In addition, the inventors have developed a novel method of pain treatment comprising administration of an infectious particle, such as an adeno-associated virus, encoding the disclosed peptide aptamer. The inventors have found that administration of the infectious particle effectively reduces allodynia and hypersensitivity in animals following tibial nerve injury (TNI), a rat model of neuropathic pain; see FIG. 7 and FIG. 9. Polypeptide aptamer

[0047] In one aspect of the disclosure, a polypeptide aptamer comprising a sequence is provided. As described above, the inventors designed peptide aptamers SEQ ID NOs: 1-9 (corresponding to peptides iPA1, iPA2, iPA3, iPA4, iPA5, iPA6, iPA7, iPA8, and iPA9 in Figure 1E FIG. 1) to bind to IDRs of Na V 1.7. In addition, the inventors have found that SEQ ID NO: 1 inhibits I Na 1.7, I Na 1.6, and I Na 1.3, but does not inhibit the anti-tetrodotoxin (TTXr) voltage-gated sodium channel I Na 1.5, and I Na 1.8; see, e.g., Figures 3C-3G and Table 2. It is believed that the disclosed polypeptide aptamers inhibit multiple Na V subtypes present on nociceptive neurons can provide a therapeutic advantage through a multi-faceted sensitivity reduction. Thus, the inventors believe that the disclosed polypeptide aptamers can be more advantageous than more specific Na V 1.7 targeting approaches for achieving analgesia.

[0048] As used herein, “aptamer” or “peptide aptamer” refers to a short peptide, e.g., less than 100 amino acids in length. Peptide aptamers are designed to bind to a target molecule, such as a target protein, e.g., Na V 1.7. While a peptide aptamer can comprise a portion of a full-length protein, a peptide aptamer does not comprise the full-length protein from which it is derived.

[0049] Surprisingly, the inventors have also found that iPA2 (SEQ ID NO: 2) increases the current density of Na V 1.7. Figure 1J

[0050] ​The disclosed polypeptide aptamer can comprise a sequence selected from the group consisting of SEQ ID NOs: 1-9, 50, and 52, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-9, 50, and 52. The sequence can be selected from SEQ ID NO: 1, 4, or 6, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO: 1, 4, or 6. The sequence can be selected from SEQ ID NO: 1, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0051] The disclosed polypeptide aptamer can consist essentially of a sequence selected from SEQ ID NOs: 1-9, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-9, 50, and 52. However, it should be understood that the disclosed polypeptide aptamer can comprise additional portions that do not interfere with the polypeptide aptamer’s ability to bind and / or modify TTS Na V other portions that do not interfere with the polypeptide aptamer’s ability to bind and / or modify TTS Na

[0052] The disclosed polypeptide aptamer can consist essentially of a sequence selected from SEQ ID NOs: 1-9, or a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-9, 50, and 52.

[0053] As used herein, “polypeptide” refers to a polymer of amino acids joined together by peptide bonds.

[0054] The term "sequence identity" as used herein refers to the extent to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. Identity of nucleic acid and protein sequences can be assessed by any method known in the art. For example, identity can be assessed using the Basic Local Alignment Search Tool ("BLAST"). The BLAST algorithm determines the homology of sequences by identifying similar segments between a query amino acid or nucleic acid sequence and a test sequence, preferably taken from a database of protein or nucleic acid sequences. The BLAST program can use default parameters or modified parameters provided by the user.

[0055] The term "percent sequence identity" is calculated as follows: the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) occurs in both sequences is determined over the comparison window, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percent sequence identity.

[0056] The inventors have demonstrated that the disclosed aptamers interact functionally when present in an isolated aptamer sequence, e.g., consisting of only one of SEQ ID NOs: 1-9 Figure 1J ), and when linked to a detectable marker (e.g., green fluorescent protein (GFP) Figure 4A ), the disclosed aptamer inhibits the function of, e.g., voltage-gated sodium channels (e.g., Na V 1.7 functionally interact. Thus, while the disclosed polypeptide aptamer can comprise only a sequence selected from SEQ ID NOs: 1-9, 50, and 52 or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9, 50, and 52, they can also comprise additional moieties and retain the ability to interact, e.g., inhibit the function of, voltage-gated sodium channels (e.g., Na V 1.7).

[0057] A "detectable marker" as used herein refers to a marker that is readily identifiable by virtue of a particular property of the detectable marker (e.g., electrochemiluminescence, chemiluminescence, fluorescence, radioactivity, nucleotide sequence, or amino acid sequence, etc.). In some embodiments, the detectable marker is a fluorescent protein, e.g., green fluorescent protein (GFP), e.g., SEQ ID NO: 23.

[0058] The disclosed polypeptide aptamer can comprise a linker, for example, SEQ ID NO: 10. The linker can be connected (e.g., covalently connected by a peptide bond) to the disclosed polypeptide aptamer. The disclosed polypeptide aptamer can comprise, in order from N-terminus to C-terminus: a detectable marker, for example, GFP, a linker, and a sequence. However, it should be understood that the disclosed polypeptide aptamer is not limited to any such arrangement, and the detectable marker or other moiety can be located at the N-terminus, C-terminus, or other location of the disclosed polypeptide aptamer.

[0059] The disclosed polypeptide aptamer can also comprise a "purification tag," which as used herein refers to a moiety that binds to a matrix (e.g., a column, a bead, a coated surface, etc.). Exemplary purification tags include, but are not limited to, a streptavidin tag, biotin, a FLAG tag, etc. Polynucleotide

[0060] In another aspect of the disclosure, a polynucleotide is provided, comprising a nucleotide sequence encoding the disclosed polypeptide aptamer.

[0061] The disclosed polynucleotide can also comprise a regulatory region, for example, a promoter, an enhancer, etc., operably linked to the nucleotide sequence encoding the disclosed polypeptide aptamer. A suitable promoter can be, for example, a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter.

[0062] As used herein, a polynucleotide is "operably linked" or "operatively bound" when the polynucleotide is in a functional relationship with a second polynucleotide sequence.

[0063] It is contemplated that the disclosed polynucleotide can be used to express the disclosed polypeptide aptamer in a cell (e.g., a human cell) or a human subject. Thus, the disclosed polynucleotide can be, for example, a plasmid, a minicircle, and can comprise regulatory regions that allow for viral delivery of the polynucleotide.

[0064] For example, the disclosed polynucleotide can comprise viral regulatory regions, for example, adeno-associated virus (AAV) inverted terminal repeat sequences (ITRs).

[0065] The term "inverted terminal repeat sequence" (ITR) as used herein refers to the DNA sequences that flank the AAV genome, which allow for genome insertion into a host cell. The ITR is the only cis-acting element required for AAV formation. Thus, other elements of the AAV genome can be added in trans to facilitate formation of a complete viral particle. Thus, the only viral genome portion that needs to be included in the DNA carried by the AAV is the 5' and 3' ITRs. Suitable ITRs are known in the art, for example, can include a 5' ITR having the sequence of SEQ ID NO: 28 and a 3' ITR having the sequence of SEQ ID NO: 29. Infectious particle

[0066] The inventors have demonstrated that delivery of the disclosed polypeptide aptamer via an infectious particle (i.e., an adeno-associated viral vector) is an effective strategy for inducing local anesthesia of nociceptive neurons; see, e.g., FIGS. 7 and 9. Accordingly, in another aspect of the disclosure, an infectious particle comprising a disclosed polynucleotide encoding a polypeptide aptamer of the disclosure is provided.

[0067] Suitable infectious particles, also referred to herein as “viral vectors,” are known in the art and commercially available. See, e.g., Deverman et al. (Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain, Nature Biotechnology, 34(2): 204-209, 2016) and Chan et al. (Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous system, Nature Neuroscience, 20(8): 1172-1179, 2017), which are incorporated by reference herein in their entireties. Those skilled in the art will be familiar with the elements and structures required to construct a vector encoding the constructs described herein. Exemplary infectious particles include an AAV particle, an adenoviral particle, a herpes viral particle, a baculoviral particle, or any other suitable viral particle.

[0068] The disclosed infectious particle can comprise an adeno-associated virus (AAV) comprising a previously described viral regulatory region that directs expression of a disclosed polynucleotide product in a host cell, the polynucleotide being comprised within or associated with the virus, wherein the expression product of the polynucleotide comprises one or more of the disclosed polypeptide aptamer. The expression product of the polynucleotide can comprise, e.g., the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or other polypeptide aptamer encompassed by the disclosure. In certain instances, the virus is selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, and the like. In exemplary embodiments, the AAV is AAV type 6; see, e.g., FIGS. 7 and 9. Pharmaceutical Compositions

[0069] It is contemplated that the disclosed polypeptide aptamer can be administered to a subject. Thus, in another aspect of the disclosure, a pharmaceutical composition comprising the disclosed polypeptide aptamer and a pharmaceutically acceptable carrier or excipient is provided.

[0070] As used herein, "effective amount" means the amount or dose of the disclosed composition that, when administered to a subject, either as a single dose or multiple doses, provides the desired effect in the diagnosis or treatment of the subject.

[0071] An effective amount can be readily determined by the attending diagnostician, as one of ordinary skill in the art, by using known techniques and observing results obtained under analogous circumstances. In determining the effective dose or amount of a compound to be administered, the attending diagnostician can consider a variety of factors, such as, species of subject; its size, age, and general health condition; the degree or severity of involvement with the disease or condition involved; individual subject's response; particular compound being administered; mode of administration; bioavailability of the formulation being administered; chosen dosing regimen; use of concomitant medication; and other relevant circumstances.

[0072] A typical dose can contain about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.05 mg / kg to about 50 mg / kg and / or about 0.1 mg / kg to about 25 mg / kg) of the disclosed polypeptide aptamer.

[0073] A typical dose of the disclosed pharmaceutical composition comprising viral particles can comprise about 5 μg to about 100 μg viral DNA, about 10 μg to about 50 μg viral DNA. A dose of the disclosed viral particles administered to a subject can comprise about 1 x 10 8 viral particles to about 1 x 10 12 viral particles. The dose can comprise 1 x 10 10 viral particles.

[0074] The composition comprising the disclosed polypeptide aptamer can be formulated into unit dosage form, each dosage containing from about 1 to about 500 mg of each polypeptide aptamer individually, or in single unit dosage form, such as, for example, about 5 to about 300 mg, about 10 to about 100 mg, and / or about 25 mg. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, diluent or carrier.

[0075] Direct injection into the dorsal root ganglion is one exemplary route for administering the compounds used in the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, transcutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, oral, or rectal routes. The route of administration can be varied as a matter of choice depending on the physical properties of the compound used, and the convenience of the subject and caregiver.

[0076] Those skilled in the art will appreciate that suitable formulations include those suitable for more than one route of administration. For example, a formulation can be one suitable for both intrathecal administration and intracerebral administration. Alternatively, suitable formulations include those suitable for only one route of administration, as well as those suitable for one or more routes of administration but not one or more other routes of administration. For example, a formulation can be one suitable for oral, transdermal, transcutaneous, intravenous, intramuscular, intranasal, inhalation, buccal, and / or intrathecal administration but not intracerebral administration.

[0077] The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The formulations used herein employ conventional formulation methods of pharmaceutical science. All commonly employed types of compositions can be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, lozenges, suppositories, transdermal patches, and suspensions. Generally, the compositions contain from about 0.5% to about 50% of the compound, depending on the desired dosage and the type of composition to be used. However, the amount of the compound is preferably defined as an "effective amount," i.e., an amount that provides the desired dosage of the compound to a patient in need of such treatment. It is believed that the activity of the compounds used in the compositions and methods disclosed herein is not greatly dependent on the nature of the composition, and thus the compositions can be selected and formulated primarily or solely for convenience and economy.

[0078] Capsules are prepared by mixing the compound with a suitable diluent and filling an appropriate amount of the mixture into capsules. Commonly used diluents include inert powdered substances such as starch, powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (e.g., fructose, mannitol, and sucrose), grain flours, and similar edible powders.

[0079] Tablets are prepared by direct compression, wet granulation, or dry granulation. The formulation of tablets generally includes diluents, binders, lubricants, and disintegrants (in addition to the compound). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (e.g., sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include starch, gelatin, and sugars (e.g., lactose, fructose, glucose, etc.). Natural and synthetic gums, including acacia, alginates, methylcellulose, polyvinylpyrrolidone, and the like, can also be used. Polyethylene glycols, ethylcellulose, and waxes can also be used as binders.

[0080] Tablets can be coated with sugar, for example, as a taste enhancer and sealant. Tablets also can be formulated with a large number of palatable substances, for example, mannitol, to improve the taste. Fast dissolve tablets also can be used, for example, to ensure that the dosage form is swallowed and to avoid difficulties some patients have in swallowing solid objects.

[0081] Lubricants can be used in tablet formulations to prevent sticking of the tablets and punches to the dies. Lubricants can be selected from the group of lubricating solids such as talc, magnesium and calcium stearate, stearic acid, hydrogenated vegetable oil, and the like.

[0082] Disintegrants also can be included in the tablets. Disintegrants are substances that swell upon contact with water to break apart the tablet and release the compound. Disintegrants include starches, clays, celluloses, alginates, and gums. By way of further example, corn and potato starch, methyl cellulose, agar, bentonite, lignin cellulose, powdered natural sponge, cationic exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethyl cellulose can be used.

[0083] Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acidic environment of the stomach. Such formulations can be prepared by coating the solid dosage form with a polymer film that is insoluble in acidic environments but soluble in basic environments. Exemplary films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate.

[0084] Transdermal patches also can be used to deliver the compounds. Transdermal patches can include a resinous composition in which the compound will dissolve or partially dissolve, and a film layer to protect the composition and maintain the resinous composition in contact with the skin. Other more complex patch compositions can be used, for example, patches in which a film is perforated with a plurality of holes through which the drug is pumped by osmotic action.

[0085] It also will be understood by those skilled in the art that formulations can be prepared using materials (e.g., active excipients, carriers (e.g., cyclodextrins), diluents, etc.) having particular properties (e.g., purity) such that the formulation is suitable for administration to humans. Alternatively, formulations can be prepared using materials having particular purity and / or other properties such that the formulation is suitable for administration to non-human subjects, but not suitable for administration to humans. Methods

[0086] The inventors have discovered that contacting one or more of the disclosed polypeptide aptamers with a cell (e.g., a neuron) can effectively reduce the Na V 1.7 Channel depolarization-induced neuronal excitation; see, e.g., Figure 1JThe disclosed polypeptide aptamer can be contacted with a cell, for example, by contacting the polypeptide aptamer directly with the cell; expressing a polynucleotide encoding the disclosed polypeptide aptamer in the cell or in another cell that secretes the polypeptide aptamer; or by contacting the cell with an infectious particle, e.g., an AAV comprising a disclosed polynucleotide configured to be expressed in the cell.

[0087] Thus, in another aspect of the disclosure, methods are provided. In some embodiments, the methods comprise contacting a cell with a disclosed polypeptide aptamer, a disclosed polynucleotide, or a disclosed infectious particle or cell comprising a disclosed polynucleotide.

[0088] As used herein, "contacting" refers to contacting a cell directly or indirectly in vitro, ex vivo, or in vivo (e.g., in a subject as defined herein). Contacting a cell can include adding a disclosed composition (e.g., a disclosed polypeptide aptamer, polynucleotide, or infectious particle or pharmaceutical composition) to a sample comprising a cell, tissue, etc., or administering the composition to a subject. Contacting can include administering the composition to a solution, cell, tissue, mammal, subject, patient, or human. For example, contacting a cell with a composition can include adding the composition to a cell culture.

[0089] The disclosed methods can comprise methods of reducing or inhibiting a stimulus to a neuron, comprising contacting a cell with a disclosed polypeptide aptamer, a disclosed polynucleotide, or a disclosed infectious particle comprising a disclosed polynucleotide.

[0090] The inventors have demonstrated that contacting a neuron with a disclosed polypeptide aptamer (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1, 4, or 6) can reduce the Na V 1.7 peak current density by at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%) in a neuron. Figure 2A In addition, the inventors have demonstrated that these methods do not alter the Na V 1.7 steady-state inactivation properties (e.g., at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%) in a neuron. Figure 2B -F).

[0091] The inventors have demonstrated that administration of an AAV carrying a viral genome encoding a disclosed polypeptide aptamer can effectively reduce allodynia and sensitivity in an animal with a tibial nerve injury (a model of neuropathic pain); see FIG. 7 and FIG. 9. Thus, in other aspects of the disclosure, methods comprise administering to a subject a disclosed pharmaceutical composition comprising an infectious particle.

[0092] In addition, the disclosed methods comprise methods of treating neuropathic pain in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed pharmaceutical composition to treat neuropathic pain in the subject.

[0093] The disclosed methods can also include methods of treating pain resulting from a traumatic nerve injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical composition to treat the subject's pain resulting from a traumatic nerve injury.

[0094] The inventors have demonstrated that direct administration of the disclosed pharmaceutical compositions to the dorsal root ganglion (DRG) of an animal can be effective in treating neuropathic pain in the animal. Thus, in the disclosed methods, the administration can comprise delivery to the dorsal root ganglion of the subject.

[0095] In certain cases, the disclosed methods inhibit nociceptor excitation. Nociceptors are specialized sensory neurons, e.g., nociceptive dorsal root ganglion neurons, that have Aδ and C fibers in peripheral nerves and sensory nonpeptidergic "free nerve endings" in innervated organs. Nociceptors transduce mechanical, thermal, and chemical stimuli into depolarizing receptive potentials. If the depolarization is strong enough, it opens voltage-gated ion channels and triggers the generation of action potentials that are conducted to the dorsal horn of the spinal cord or brainstem.

[0096] Targeted injection of the disclosed compositions can limit systemic exposure upon administration to a subject. For example, targeted delivery can comprise injection of an infectious particle comprising a polynucleotide encoding one or more of the disclosed polypeptide aptamers into or near a neural tissue of the subject. Examples of neural tissues into which the compositions described herein can be injected include, but are not limited to, ganglia (e.g., dorsal root ganglia), spinal nerves, preganglionic fibers, and paraganglia. Examples of neural tissues into which the compositions described herein can be injected near include, but are not limited to, the subarachnoid space surrounding the ganglia.

[0097] The disclosed polynucleotides can be administered using any suitable delivery vehicle. For example, in certain instances, a nucleic acid encoding a polypeptide aptamer for treating pain can be incorporated into a delivery vehicle capable of driving expression of the nucleic acid. Examples of delivery vehicles include, but are not limited to, non-viral vectors (e.g., plasmids (e.g., expression plasmids), liposomes, and polymersomes) and viral vectors (e.g., adeno-associated viral vectors, HSV vectors, and lentiviral vectors). For example, the disclosed polynucleotides can be delivered using an adeno-associated viral (AAV) vector. The term “adeno-associated virus” (AAV) as used herein includes, but is not limited to, type 1 AAV, type 2 AAV, type 3 AAV (including type 3A and type 3B), type 4 AAV, type 5 AAV, type 6 AAV, type 7 AAV, type 8 AAV, type 9 AAV, type 10 AAV, type 11 AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. The genomic sequences of various AAVs and self-contained parvoviruses, as well as the sequences of ITRs, Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases (e.g., the GenBank database).

[0098] In certain instances, the AAV vectors are made with modified capsids that have been shown to improve gene transfer efficiency and potentially reduce immunogenicity compared to naturally occurring serotypes. See, e.g., Buning & Srivastava, Mol Ther Methods Clin Dev 12:248-265, (2019). For example, Anc80L65 (anc80) is a novel AAV capsid designed from a computer-reconstructed viral phylogenetic lineage that has been shown to have robust transduction capacity following local delivery to various tissues. See, Wang et al., PLoS One 12:e0182473, (2017); Hudry et al., Mol Ther Methods Clin Dev 10:197-209, (2018). Cells

[0099] In another aspect of the disclosure, cells comprising a polypeptide aptamer, a polynucleotide, or an infectious particle of the disclosure are provided. The cells can be mammalian cells, e.g., human cells. The cells can be cultured cells, e.g., human embryonic kidney (HEK) cells or induced pluripotent stem cells (iPSCs) or primary neurons, which can be isolated from a subject or in vivo. Cells expressing Na v 1.8

[0100] The inventors constructed cells comprising a heterologous polynucleotide encoding a human Na V 1.8 protein that express functional human NaV 1.8 protein, and suitable for electrophysiological experiments; also referred to in this article as "HEK1.8 cells"; see, for example Figure 3G And 11.

[0101] As used herein, "heteronucleotide" refers to a polynucleotide that is not normally present in cells and has been introduced into cells by means of, for example, transduction, transfection, liposome transfection, nuclear transfection, electroporation, or other methods known in the art. Heteronucleotides may be, for example, plasmids or other expression vectors.

[0102] Na V 1.8 The protein may have the sequence SEQ ID NO:20 and may be encoded by a heteropolynucleotide including SEQ ID NO:21.

[0103] The heteropolynucleotide may comprise sequences encoding NaV1.8a (amino acid sequence SEQ ID NO: 20) and NaVb2 (amino acid sequence SEQ ID NO: 24) by a single open reading frame (ORF), linked by a sequence encoding a 2A self-processed sequence derived from porcine cyclovir-1 (P2A) (e.g., SEQ ID NO: 26) and a furin cleavage site (e.g., R,X,X,R or R,X,K / R,R, where X is any amino acid). The furin cleavage site may be RKRR (SEQ ID NO: 27). The polynucleotide may comprise sequences encoding NaV1.8a (amino acid sequence SEQ ID NO: 20) and NaVb2 (amino acid sequence SEQ ID NO: 24), respectively. V 1.8a and Na V The nucleotide sequences of b2 are SEQ ID NO: 21 and 25. The polynucleotide may include SEQ ID NO: 22, namely pcDNA3.1-SCN10A-furin protease-P2A-SCN2B.

[0104] Cells can include human cells, such as human embryonic kidney (HEK) cells. Methods for testing the ability of compounds or reagents to regulate the function of voltage-gated sodium channels

[0105] Further envisioning, the disclosed polypeptide aptamers, polynucleotides, infectious particles, cells, or expression of Na... v Cells of 1.8 can be used in methods to test the ability of compounds or reagents to regulate the function of voltage-gated sodium channels.

[0106] For example, those skilled in the art can use disclosed peptide aptamers to modulate voltage-gated sodium channels (e.g., Na+). V 1.7) functions as a way to inhibit Na V1.7 Positive control of function. Thus, the method of testing a compound or agent can comprise: contacting the compound or agent with a cell; contacting at least one of the disclosed compositions (e.g., a disclosed polypeptide aptamer, polynucleotide, or infectious particle) with the cell; wherein if a similar change in a parameter measured in the cell (e.g., an electrophysiological parameter, e.g., current density) occurs, i.e., exhibits an effect within 5%, 10%, 20%, 30% of the effect induced by the disclosed composition, then the compound or agent is a modulator of voltage-gated sodium channel function.

[0107] Disclosed are cells expressing Na v 1.8 The cells of 1.8 can be used to determine whether a compound or agent is a Na V 1.8 modulator. The method comprises contacting a proposed Na v 1.8 modulating compound or agent with a cell expressing Na v 1.8, and measuring a parameter related to Na v 1.8 function, e.g., an electrophysiological parameter, e.g., current density, wherein if the compound or agent modulates the parameter related to Na v 1.8 function, then the compound or agent is a Na v 1.8 function modulator. Systems, kits, and platforms

[0108] In another aspect of the disclosure, systems, kits, and platforms are provided. The systems, kits, and platforms can comprise a disclosed polypeptide aptamer, polynucleotide, infectious particle, or cell. The systems, kits, and platforms can further comprise reagents for performing an electrophysiological experiment. The systems, kits, and platforms can further comprise instructions for use.

[0109] The disclosed systems, kits, and platforms can comprise a disclosed cell expressing a human Na V 1.8 protein 。 It is contemplated that such cells can be used to study or evaluate new agents that target the Na V 1.8 protein.

[0110] A number of definitions are used herein to describe the present invention, as described below and throughout the application. Definitions

[0111] The disclosed subject matter can be further described by the following definitions and terms used herein. The definitions and terms used herein are used solely for the purpose of describing particular embodiments and are not intended to limit the present invention.

[0112] As used in the specification and claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a" or "an" entity shall mean one or more entities, unless the context clearly dictates otherwise.

[0113] As used herein, "about," "approximately," "substantially" and "notably" are to be understood by those of ordinary skill in the art, and can vary somewhat depending on the context in which they are used. If certain uses of the term are not clear to those of ordinary skill in the art in the context in which they are used, "about" and "approximately" shall mean up to plus or minus 10% of the particular term, and "substantially" and "notably" shall mean more than plus or minus 10% of the particular term.

[0114] As used herein, the terms "comprises" and "comprising" are synonymous with the terms "includes" and "including," respectively. The terms "comprises" and "comprising" should be interpreted as "open transition" terms, allowing for the inclusion of additional components, in addition to the components listed in the claims. The terms "consisting of and "consisting essentially of should be interpreted as "closed transition" terms, disallowing the inclusion of additional components, in addition to the components listed in the claims. The term "consisting essentially of should be interpreted as partially closed, allowing for the inclusion of additional components, provided that the additional components do not materially alter the nature of the claimed subject matter.

[0115] The phrase "for example" should be interpreted as "for instance, including." Additionally, the use of any and all examples, or exemplary language herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. The use of the term "about" is intended to allow for variations, such as due to natural variations in materials, manufacturing, and other factors, as well as variations that can be introduced by the process of making up the claimed subject matter.

[0116] Further, in cases where a convention similar to "at least one of A, B, and C, etc." is used, in general such a construction is intended to encompass one or more of A, B, or C, in all of the various permutations of A, B, and C, i.e., A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc. It is further noted that any and all ranges recited herein include the endpoints and all the other intervening ranges between the recited endpoints unless otherwise indicated. Also, any and all ranges recited herein include the endpoints and all the other intervening ranges between the recited endpoints unless otherwise indicated. For example, a range of "1 to 10" is intended to include the endpoints 1 and 10, and all intervening ranges between 1 and 10, such as 1-3.5, 4.1-7.6, etc. Similarly, a range of "6" is intended to include the endpoint 6, and all intervening ranges between 1 and 6, such as 1, 2, 3, 4, or 6, and so on.

[0117] All such terms as "maximum," "minimum," "greater," "less," and the like are to be understood to encompass the listed term, and to refer to a range that can be subdivided into ranges and subranges. The ranges include the individual members. Thus, for example, a group having 1-3 members is a group having 1, 2, or 3 members. Similarly, a group having 6 members is a group having 1, 2, 3, 4, or 6 members, and so on.

[0118] The modal verb "may" refers to the preferential use or selection of one or more options or choices in the described embodiments or included features. When the options or choices are not disclosed with respect to a particular embodiment or a particular feature included therein, the modal verb "may" refers to an affirmative act with respect to how the described embodiment or the feature included therein is made or used, or an explicit decision that the described embodiment or the feature included therein is used with a particular skill. In the latter case, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can." EMBODIMENTS

[0119] The following embodiments are illustrative, and should not be construed as limiting the scope of the claimed subject matter. Example 1 - Peripheral targeted analgesia in rats by AAV-mediated sensory neuron-specific inhibition of multiple nociceptive sodium channels

[0120] This study reports the development of a Na v 1.7 protein. The intrinsically disordered region (IDR) of the protein was found to be useful in the discovery of a sodium channel inhibiting peptide aptamer (Na V iPA) for sensory neuron-specific analgesia mediated by adeno-associated virus (AAV). A prototype, termed Na V iPA1, was discovered to have multifaceted inhibition of I Na1.7 , I Na1.6 , I Na1.3 , and I Na1.1 , but no inhibition of I Na1.5 and I Na1.8 ; this prototype Na V iPA1 was derived from the intracellular loop 1 of Na v 1.7, which is conserved in TTXs Na V subtypes. Na V iPA1 was expressed in primary sensory neurons (PSNs) of dorsal root ganglion (DRG) and had significant inhibition of TTXs INa, but no significant inhibition of TTXr I Na . Injection of AAV6-encoded Na V iPA1 into DRG significantly attenuated the evoked and spontaneous pain behaviors of male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of PSNs showed that Na V iPA1 expression normalized the excitability of PSNs in TNI rats, suggesting that Na V iPA1 alleviated pain by reversing the injury-induced neuronal hypersensitivity. Immunohistochemistry showed that the effective expression of Na V iPA1 was limited to PSNs and their central and peripheral terminals, indicating that PSNs limited the biodistribution of AAV. Na VThe inhibitory effect of iPA1 on sodium channels was replicated in human iPSC-derived sensory neurons. These results summarize the inhibitory effect of iPA1 on sodium channels. V iPA1 is a promising analgesic lead agent, which, when combined with AAV-mediated PSN-specific blockade of multiple TTXs, can help alleviate these symptoms. V Combined, it has the potential to serve as a peripheral nerve-limiting analgesic therapy.

[0121] introduction

[0122] Voltage-gated sodium channel (Na V Na+ is a key regulator of neuronal excitability and pain perception (1). Mammals possess nine types of Na+. V Subtype, of which Na v 1.7 、 Na v 1.8 and Na v 1.9 These Na+ neurons are preferably expressed in the primary sensory neurons (PSN) of the dorsal root ganglion (DRG) (2). V The important role of subtypes in human pain has been verified (2). Na V 1.6, Na V 1.1 and Na V 1.3 is also expressed in PSN and has been reported as a potential target for analgesics (3, 4). Currently, Na... V 1.7 is Na V The primary target for developing analgesic therapies (5).

[0123] Over the past few decades, significant efforts have been made to develop selective and efficient Na+ technology. V 1.7 Blockers have been used to treat pain (6), but with limited success. Most tests are conducted on small molecule sodium for pain treatment. V 1.7 Blockers are inadequate in terms of target binding, lack target specificity or selective bioavailability along the pain axis, and their systemic distribution can lead to cardiotoxicity, movement disorders, and central nervous system (CNS) side effects (6). Developing a target for Na+ V Biologics at 1.7 represent another growing trend in the field of analgesia (7, 8). Na V 1.7 Neutralizing monoclonal antibodies have analgesic effects, but the results are inconsistent (6). (The text then abruptly shifts to a seemingly unrelated topic about spider peptides and their analgesics.) V 1.7 Blockers are effective analgesics, but they have poor membrane permeability and sodium... V 1.7 Insufficient selectivity and short half-life (6). It has been proposed to use Na... v 1.7-RNAi(6) and CRISPR-dCas9 or ZEN epigenetic Na v1.7 Inhibiting analgesic gene therapy (9) but these interventions at the mRNA and epigenetic levels lack the specificity of direct channel interventions, reduce safety, and allow off-target effects (6, 10), while anti-Cas9 immunity presents other challenges for CRISPR gene therapy (11).

[0124] Small peptides derived from nociceptive ion channels as functional interfering peptide aptamers (iPA) have high efficiency and selectivity to block specific nociceptive signaling (12, 13). The intrinsic disordered regions (IDRs) of ion channel proteins often involve a promiscuous set of interactions that play important roles in multiple signaling regulations and are considered as new promising drug targets (14). We hypothesized that Na v 1.7-IDR contains a short functional IDR domain that can play a key role in regulating Na v 1.7 function and can be developed as Na v 1.7 iPA (1.7iPA). In addition, the high conservation of Na V subtype sequences means that a given 1.7iPA can interact with other NaV subtypes with homologous sequences to Na V 1.7, thus enabling a multifaceted involvement of NaV subtypes. Since multiple PSN-Na V participate in nociceptive electrogenesis and pain pathogenesis, it is conceivable that this multifaceted Na V V involvement mediated by AAVs can have analgesic advantages over blocking a single Na V V subtype, which can have analgesic advantages over blocking a single Na V V subtype (15-17).

[0125] We here describe a new strategy by which we designed and developed Na V iPA based on NaV-IDR with high selectivity, non-toxic, and analgesic potential. The prototype Na V iPA1 was derived from the intracellular loop 1 of Na v 1.7, which is conserved among TTXs Na V V subtypes, and exhibited multifaceted inhibition of Na v 1.7, Na V 1.6, and Na V 1.3, and Na V 1.1 channels. Na V iPA1 expression in rat PSNs resulted in significant TTXs but not TTXr I Na inhibition. In a rat model, AAV-mediated Na ViPA1 selectively expressed in PSNs responsible for pain pathology, produced effective analgesic effects while avoiding ectopic biodistribution that causes side effects. In summary, these results suggest that AAV-mediated PSN-specific, multi-injurious Na V channel blockers have potential for future therapeutic development.

[0126] Results

[0127] In silico design of 1.7 iPA from NaV1.7-IDR

[0128] Candidate iPA were designed by a priori strategy aimed at defining short linear functional disordered peptides (12) from intrinsic disordered domains (IDDs), which were originally from NaV1.7 protein IDR, based on Na v 1.7 IDD contains functional sequences that modulate Na v 1.7 channel function. We analyzed the full-length of rat Na V 1.7 protein sequence using DisorderEd PredictIon CenTER (DEPICTER), which combines 10 popular algorithms for predicting IDRs within primary sequences based on the biophysical features of amino acids (aa) of the protein disorder ensemble (18). Results return a score between 0 and 1 for each residue, indicating the degree to which a given residue belongs to an ordered or disordered region (residues with scores > 0.5 are considered disordered). Results show a clear transition from ordered to disordered, with Na v 1.7 transmembrane (TM) domain and the intracellular portion, and scores indicate disordered nature of Na v 1.7 intracellular and terminal regions. Figures 1A-1C ). Specifically, the most extensive IDR is located in the intracellular loop (ICL), while the TM domain of the protein is highly ordered.

[0129] Disorder Enhanced Phosphorylation Predictor (DEPP) was used to identify potential phosphorylation sites in Na v 1.7 sequence (19). Results indicate that most of the potential phosphorylation residues (serine, threonine, and tyrosine with high DEPP score) are located in Na v 1.7-IDR, especially in the IDRs within ICL1 and ICL2. Figure 1D v Na v ​1.7 Key binding motifs or domains of the regulatory signal interaction group (20). These observations suggest a focus on Na v 1.7-IDR may be a key indicator for identifying effective regulation of Na+. v 1.7 Pathways of short peptides with channel function.

[0130] Na was further analyzed using SLiMPrints (http: / / bioware.ucd.ie / slimprints.html). v 1.7-IDR potential functional domains (21), SLiMPrints predict short linear motifs (SLiMs) based on highly conserved primary amino acid sequences, and then filter according to the prediction score (22). v The motifs predicted in the 1.7-IDR indicate that many potential functional peptides are "hotspots" for functional IDDs, including proteolytic cleavage sites, ligand-binding sites, post-translational modification (PTM) sites, and subcellular targeting sites. Based on IDR scores and phosphorylation sites, nine peptides were computationally designed and further tested as 1.7iPA candidate peptides. Figure 1E , 1B ).

[0131] 1.7 iPA constructs and transfection expression

[0132] AAV expression plasmids containing transgenic expression cassettes encoding multiple GFP-1.7iPA chimeras were constructed. Specifically, as we previously described (23), the sequence of the interchangeable iPA peptide was cloned together with the adapter sequence (GLRSRAQASNSAVDGTAGPGS) to form a GFP-adaptor-iPA oriented chimeric transgene, which was transcribed via a heterozygous human cytomegalovirus (CMV) enhancer / chicken β-actin (CBA) promoter. This yielded the pAAV-CBA-GFP-1.7iPAs (pAAV-1.7iPA) expression plasmid, in which an oligonucleotide encoding interchangeable 1.7iPA was inserted at the 3′ end of GFP ( Figure 1F The predicted protein structure of GFP1.7iPA1 was analyzed using the I-TASSER tool (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ) (24). The results showed that the overall structure of the linker-1.7iPA1 was in an unfolded and extended state, and highly flexible. Figure 1G This is consistent with a good exposure pattern for target binding. The I-TASSER tool has also identified similar structures in other GFP1.7iPA (Figure 11).

[0133] 1.7iPA on Na+ in HEK1.7 cellsV 1.7 Current (I) Na Inhibition of l.7)

[0134] By transfecting into stable expression of human wild-type Na V HEK293 cells (HEK1.7 cells) were then subjected to Western blotting (IB) to verify the stable expression of each construct. Representative assays for GFP adapter (GFP), 1.7iPA (1, 2, 3, 4, 6) are shown (Fig.). 1H, I). To characterize I... Na 1.7, in HEK1.7 cells transfected with plasmids encoding nine 1.7iPA (1.7iPA1-9), via I Na 1.7 Preliminary screening experiments were conducted using whole-cell voltage clamp. Figure 1F The peak I values ​​of nine different 1.7iPA receptors in HEK1.7 cells were summarized. Na 1.7 density (3 days post-transfection), where at least two replicates were merged. Results showed that 1.7iPA1, 4, and 6 resulted in peak I... Na 1.7 density reduction of approximately 68%, 59%, and 54%, while 1.7iPA2 reduces peak I Na 1.7 cell density increased by approximately 35%. Compared to sham-transfected (PEI transfection without plasmid transfection) HEK1.7 cells, transfection with plasmids expressing GFP adaptors and 1.7iPA3, 5, 7, 8, and 9 resulted in a peak Ig. Na 1.7 density had no significant effect. Therefore, these experiments determined that 1.7iPA1 and 1.7iPA4 (both derived from ICL1) and 1.7iPA6 (derived from ICL2) are effective iPAs (iPAs). Na 1.7 Inhibition rate > 50%. Next, in the new experiment, we focused on validating the effects of 1.7iPA1, 4, and 6 on I in HEK1.7 cells. Na 1.7 Inhibition and its channel dynamics. These results replicated previous screening test results and demonstrated that, in the presence of 1.7iPA1, 4, and 6, Na V The steady-state activation and rapid inactivation mechanics of the 1.7 channel were not significantly affected (Figure 2). The 1.7iPA1 peptide is a zwitterionic polymer, rich in 38.6% positively charged arginine or lysine (17 / 44), 23% serine (10 / 44), and 14% acidic residues (6 / 44); and is highly conserved between rodents and humans. Figure 2FDatabase searches revealed two serine phosphorylation sites and two lysine acetylation sites in high-throughput (proteomics discovery mass spectrometry) studies (https: / / www.phosphosite.org) (25), and SeqNLS (http: / / mleg.cse.sc.edu / seqNLS / ) (26) predicted nuclear localization signals. These analyses strongly suggest that 1.7iPA1 is a functional IDD peptide. Because 1.7iPA1 exhibits higher I... Na 1.7 inhibition, and with other TTXs Na V The subtype is highly homologous (see below for details), and we selected it as the prototype and named it NaViPA1 for further “lead compound” characterization.

[0135] Na V iPA1 occupies various voltage-gated ion channels with specificity

[0136] Na based on HEK cells V 1.8 Development of a stable expression system. To evaluate Na... V iPA1 affects the transmission of Na V 1.8 Channel conduction I Na Based on the potential of recombinant human Na+, which is stably expressed in HEK cells (HEK1.8), we developed a method to synthesize Na+. V 1.8 Heterogeneous System. After at least 10 rounds of G418 screening (400-800 µg / mL) and subsequent single-cell isolation using a BIOCHIPS single-cell isolation array (ThermFisher, Rockford, Illinois), cells were subjected to Na+ ionization. V Immunoblotting of 1.8a and Nab2 confirmed Na V Stable expression of 1.8. Na was found. V 1.8a and Nab2 are highly expressed on the cell membrane. Functional Na V 1.8 Expression of slowly inactivated inward IgE induced by a voltage step from -140 mV to +80 mV during whole-cell voltage-clamp recording. Na To identify, and in approximately 85% of HEK1.8, the average peak I Na 1.8 Density > 0.5 nA, and I Na 1.8 to Na V The 1.8 channel blocker A803467 (Alomone, Jerusalem, Israel) was sensitive and resistant to TTX (5 mM). We used this HEK1.8 cell line to inhibit Na+. V iPA1 to I Na The effects of 1.8 were initially screened. In comparison, CHO-Na V1.8 I in cells Na 1.8 The amplitude is usually less than 100 pA, which is insufficient for our experimental needs (Figure 12).

[0137] Na V iPA1 exhibits selectivity for ion channel occupancy. The stable NaV subtype cell lines used in this experiment, based on HEK cells, included HEK1.1, 1.3, 1.6, 1.5, and 1.8. Sequence alignment identified Na... V iPA1 and TTXs Na V The corresponding sequences of 1.1, 1.3, and 1.6 showed high homology, but low homology with TTXr NaV 1.5, 1.8, and 1.9. Figure 3A B). Na V iPA1 (fused with GFP) expression is significantly blocked by rapid activation and inactivation of Na+. V 1.1, Na V I conducted in 1.3 and 1.6 Na ( Figure 3C -E). In HEK1.5 and HEK1.8 cells ( Figure 3F -G) or transient transfection of Na V In ND7 / 23 cells at 1.8°C (Figure 13), Na V No effect on I was observed when iPA1 was present. Na 1.5 and I Na The effect of 1.8. Since no expression cell line was available, we did not test for Na. V iPA1 versus Na V 1.1 and Na V The role of channel 1.9; however, due to sequence homology (not shown), 1.7iPA1 is not expected to suppress I. Na 1.9, and at the same time, because Na V iPA1 and Na V There is no sequence homology between 1 and 9, and it is not expected to affect I. Na 1.9 Effects. In NG108-15 cells that naturally express potassium channels, Na+ was found to have an effect. V iPA1 for potassium current (BKI) Kv The negative effects of ) (12) were not recorded in DRG-PSN, but AAV-mediated Na V iPA1 expression is associated with high voltage activation (HVA) I Ca The effect of naturally expressed Na. V Na was also confirmed in NG108-15 neuronal cells and F11 DRG neuron-like cells with a molecular weight of 1.7. V iPA1's strong I Na 1.7 Inhibitory effect. These experiments show that NaV iPA1 affects BK potassium channels and HVA I Ca No effect (Figure 14).

[0138] AAV6-mediated Na in DRG-PSN V iPA1 expression inhibits TTXs I Na However, it does not inhibit TTXr I Na Since no heterologous system or cell line could fully mimic the in vivo conditions of sensory neurons, we further tested Na... V iPA1 in DRG-PSN for I Na Functional inhibition. Generation encoding GFP fusion Na V The iPA1 AAV6 vector was injected into the lumbar (L) 4 / 5 DRG of naïve male rats, and sensory neurons acutely isolated from the DRG were detected 4 weeks post-injection. AAV6 encoding a GFP adapter and an NP (1.7iPA3) derived from Na+ was used as a control. v The N-terminus of 1.7 (Figure 1) is shown after transfection into HEK1.7 and the effect on I. Na No effect (Figures 1 and 2). Voltage schemes confirmed successful isolation of TTXr I from isolated DRG neurons. Na (Nav1.8 sample) and TTXs I Na In (27, 28), the results are comparable to those recorded after adding TTX (1.0 mM) to the bath solution. Figure 15A B). Whole-cell voltage-clamp recording of small / medium-sized PSNs (≤35 mm) using a voltage protocol showed that AAV-mediated Na+... V iPA1 expression affects total I Na and TTXs I Na It produces significant inhibition, while TTXr I Na No significant inhibition ( Figure 4A -C).

[0139] Na V iPA1 affects TTXs I in human iPSC-derived sensory neurons. Na Inhibition. We used human induced pluripotent stem cell (iPSC) derived sensory neurons (hiPSC-SN, female, Anatomic, Minneapolis, Minnesota) (29) to test Na V iPA1's TTXs I Na Is inhibition a meaningful quantitative indicator for measuring functional orientation in human sensory neurons? This also allows for the production of HEK-Na without generating [the necessary inhibitory agents]. V Examining the potential effects of Na under cell overexpression ViPA1. hiPSC-SN differentiated into small PSN morphologies with a somatic cell diameter of approximately 20-25 mm, and formed extensive neurites after 4-7 days of in vitro (DIV) differentiation culture, indicating that these cells had effectively differentiated into neuronal lineages. We used a lentiviral vector (LV-GFP) ( Figure 16 We tested the transduction efficiency of hiPSC-SNs. We successfully expressed Na+ in differentiated hiPSC-SNs by performing LV transduction at a fold change in infection (MOI) of 5. V iPA1 and 1.7NP (control) Figure 4D E). We performed EP recording on hiPSC-SN (DIV25) with TTX (1 mM) added to the bath solution and separated TTXr / TTXs using a subtractive scheme. Na (27). To prevent the TTX effect, a voltage operation similar to that used in DRG neuron recording was employed; in addition, a method for isolating somatic cells was also used. Na The proposed scheme involves applying a brief pre-pulse near the peak voltage to inactivate the hiPSC-SN axonal peak without affecting the somatic peak (30). Results indicate that Na... V iPA1 significantly inhibited TTXs I in differentiated hiPSC-SN (DIV25). Na However, it does not inhibit TTXr I Na ( Figure 4F -G), comparable to rat DRG-PSN. In Na V In the presence of iPA1, the BK I recorded from hiPSC-SN (DIV21) Kv and HAVI Ca No impact was observed (Figure 17). The results indicate that Na, as defined in cell lines and rat DRG-PSN, is affected. V iPA1 for TTXs I Na The inhibitory effect can be converted into an inhibitory effect on human PSN.

[0140] Na V Molecular mechanism of iPA1: Initial testing

[0141] We first used primitive HEK cells expressing different Na+... V Cell lysates prepared from stable cell lines of the subtype and 50B11 rat DRG neurons were validated by Western blotting (IB) for Na+. v 1.7 Antibody specificity. The Na... v 1.7 Antibody (Alomone ASC-008) was derived from rat Na... v The antigenic peptide, consisting of 1.7 amino acid residues 446-460, is produced and interacts with other Na+ amino acids. VThe subtypes showed no significant sequence homology. The results indicate that Na... V 1.7 The antibody was detected only in full-length Na+ cells in HEK1.7 cells. V 1.7, while no other Na was detected. V Isotypes and naturally occurring non-expressing physical and functional Na V 1.7 50B11 cells (31) Figure 5A Immunohistochemistry (IHC) was performed on rat tissue sections using Na... V 1.7 Antibody detected Na V 1.7 Na expression exhibits high immunoreactivity density in small / medium-sized PSNs, and is also detected in the dorsal horn of the spinal cord (SDH), Ranvier's nodes of the sciatic nerve, and the dermal distal ends of the hind paw. V 1.7 ( Figure 5B -E), whose pattern is similar to that reported previously (32). These results confirm that Na V 1.7 Antibody detection of Na by IHC and Western blot V 1.7 Specificity of expression.

[0142] Because of Na V 1.7 is an integrated membrane protein, therefore we tested Na in HEK1.7 cells. V Does iPA1 expression interfere with Na? V Intracellular transport at 1.7. Our results do not support this mechanism because, in transfection with Na+... V In the fractionated preparations of iPA1 and the control, membrane Na v 1.7 Protein levels were not significantly reduced ( Figure 5F Studies have shown that IDR in membrane proteins interacts with the membrane (33). To test Na... V iPA1 versus Na v Is it possible that interference of 1.7 can be achieved by directly blocking Na? v 1.7 Implementation: GFP-Na was performed in HEK1.7 cells using GFP adapter (GFP) and GFP-1.7iPA2 (Figure 1) as controls. V Following iPA1 transfection, GFP affinity pull-down was performed using ChromoTek GFP-Trap (ChromoTek, Rosemount, Illinois). Cell lysates were prepared using lysis buffer containing 0.5% Nonidet p40 (a “non-denaturing” mild lysis detergent) to prevent interaction disruption and to maximize Na+ retention. V iPA1-protein interaction (34). Immunoblotting confirmed that full-length Na V 1.7 protein was captured in GFPNa V iPA1 was observed in the sample, but not in the control.Figure 5G Furthermore, nLC-MS / MS detected unique hNa. V 1.7 peptide ( Figure 5H ), confirming in GFPNa V The bands cut from the iPA1 affinity-guided sample by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel contained hNa. v 1.7. These results indicate that Na V iPA1 blocks Na v 1.7 Channel activation may be achieved through interaction with Na V 1.7 Protein binding, namely through intramolecular domain-domain interactions (intraDDI) (35). It has been reported that multibasic IDRs in transmembrane proteins preferentially bind to negatively charged lipids (36, 37). We hypothesize that Na... V iPA1 may be able to bind to inositol phosphate, and this hypothesis was verified using phosphatidylinositol phosphate (PIP) test strips (Echelon PIP test strips, Salt Lake City, Utah). GFPNa V iPA1 and GFP (control) were transfected into NG108-15 neuronal cells, and cell lysates were prepared using RIPA buffer containing 0.1% SDS, 1% Triton X100 (strong detergent), and 1% deoxycholic acid (anionic detergent) to maximize denaturation and disrupt Na+. V iPA1 PPI complex formation. Silver staining after SDS-PAGE gel electrophoresis revealed GFP and GFPNa. V iPA1 was well purified. Figure 5J The sample was then applied to PIP test strips. The results (Figure 5K) showed that GFP Na... V iPA1 can efficiently bind to a variety of anions, including PIP, PIP2, phosphatidic acid (PA), and phosphatidylserine (PS). In contrast, affinity-pull-down GFP did not show clear binding to lipid spots, as previously reported (38). This is consistent with literature reports that basic residues (typically aggregated in IDRs) can modulate membrane protein function through binding with lipids via electrostatic interactions (39, 40).

[0143] Na V iPA1 is a multibasic peptide rich in arginine / lysine and serine. Conserved multibasic domains in proteins with adjacent serine PTMs have been reported to play a significant role in protein function (41-43). We designed experiments to investigate the role of multibasic NLSs and multiple adjacent serine residues in Na+. V The role of iPA1 in functionality. Preliminary tests were conducted by generating Na. V The process involves iPA1mt1 (GFP fusion), in which Na is replaced by alanine.V iPA1 contains 10 serine residues, and Na is replaced by alanine. V The nine arginine or lysine residues in the multi-basic NLS predicted by iPA1 generate Na. V iPA1mt2 (GFP fusion) Figure 6A ICC data shows that for Na... V iPA1 and Na V iPA1mt1 observed Na V iPA1 nuclear localization (HEK1.7 cell transfection), but for Na... V iPA1mt2 This situation decreases ( Figure 6B -E). Immunoblotting ( Figure 6F The results showed that Na+ was detected in the extracted cytosol, membrane, and nuclear samples. V iPA1, and Na V Membrane binding and nuclear entry signals in iPA1mt1 are related to Na V iPA1 is comparable, but in Na V It disappears in iPA1mt2. For example... Figure 5F As shown, the total length Na V 1.7 was enriched in membrane samples, and Na was present in HEK1.7 cells. V iPA1, mt1, and mt2 do not appear to hinder Na V 1.7 Protein membrane integration. Whole-cell voltage-clamp recordings showed that in Na+... V When iPA1mt1 and mt2 exist, I Na1.7 Comparable to primordial cells and GFPNP-transfected HEK1.7 cells, this indicates that the polybasic arginine / lysine and multiple adjacent serine residues are Na+. V iPA1 versus Na V 1.7 The inhibition of the current is necessary. To further pinpoint the key serine sites, we generated additional NaViPA1mt3-mt6, in which alanine replaces two or three serine residues ( Figure 6A Whole-cell voltage-clamp recordings showed that Na+ with alanine substitution at different serine sites... V iPA1mt3 and 5 lost their resistance to I after transfection into HEK1.7 cells. Na1.7 The inhibitory effect of , while mt4 and mt6 showed significant I Na 1.7 Blocking effect ( Figure 6G As expected, Na V iPA1mt1 and 2 did not change after transfection into HEK1.8 cells. Na 1.8, similar to Na V iPA1 ( Figure 18These data indicate that Na V The conserved multibasic NLS and multiple adjacent serine residues within iPA1 synergistically inhibit INa1.7. V The iPA1 peptide binds to the plasma membrane polarly and enters the cell nucleus, while multiple adjacent serine residues are Na+. V iPA1 inhibits I Na 1.7 is required. However, the full length Na V 1.7 Membrane integration is determined by its TM domain rather than intracellular sequences, in Na+ V It is unaffected if iPA1 is present. Figure 6F ).

[0144] Further clarify Na V The properties of serine and other PTM residues in iPA1 inhibit various TTXs. Na The molecular mechanism, and Na V Whether the presence of iPA1 could disrupt TTXs' Nav channel activity by deceiving, reducing PTM in the full-length protein, and / or altering intradomain effects is a matter of concern from both pathophysiological and therapeutic perspectives. The goal of this study was to develop a strategy for peripherally targeted analgesia through AAV-mediated sensory neuron-specific inhibition of sodium channels. Therefore, in subsequent in vivo experiments, we focused on testing Na... V Can the DRG-PSN-targeted expression of iPA1 effectively alleviate neuropathic pain behavior?

[0145] Intraganglionic delivery of AAV-Na to rats following TNI V Analgesia after iPA1

[0146] We first conducted an experimental in vivo analgesia test. High-titer and high-purity AAV6-GFPNa were generated. V iPA1 (AAV6-Na) V iPA1 and control AAV6-GFPNP (AAV6-NP) were injected into the L4 / 5 dorsal root ganglion (DRG) of adult male rats. Three weeks after DRG-AAV injection, TNI was induced, followed by weekly sensory and behavioral assessments for 5 weeks. Tissues were then harvested for IHC characterization of transgene expression. Results (Figure 19) showed that AAV6-Na… V iPA1 injection reduces TNI-induced mechanosensitization and cryosensitization. IHC showed Na V iPA1 (fused with GFP) is efficiently expressed in DRG neurons and their peripheral (skin) and central (SDH) terminals. These data suggest that after TNI, Na VSelective sustained expression of iPA1 in PSNs of pathological DRGs can prevent the development of pain behaviors.

[0147] By DRG-AAV6-Na V iPA1 in the treatment of established neuropathic pain in male rats

[0148] Next, we extended the experiment to evaluate DRG-AAV6-Na V iPA1 in reversing established pain behaviors, including evoked responses as well as the sustained spontaneous pain after TNI. In the experimental design, sensitivity to mechanical and thermal cutaneous stimuli was assessed at baseline and once a week after TNI for 2 weeks until AAV injection. After that, rats were randomly assigned to receive AAV6-Na V iPA1 or control AAV6-NP injection, followed by weekly assessment of sensory behaviors for 6 weeks. As an endpoint experiment, a gabapentin (GBP, 100 mg / kg, i.p.) induced conditioned place preference (CPP) test was performed in both groups to assess spontaneous pain (12, 44). The behavioral measurements before AAV injection at day 14 after TNI were used as the treatment baseline (tBL) to evaluate the effectiveness of vector treatment (tBL - tBL) (Fig. 7C1-F1). Figure 7A , B). Tissues were harvested for IHC characterization of transgene and target gene expression, and whole-cell current clamp analysis of neuronal excitability was performed on isolated DRG neurons.

[0149] All rats developed multiple pain behavior patterns 2 weeks after TNI, including reduced withdrawal thresholds to mild mechanical stimuli (vF), more frequent hyperalgesic responses after noxious mechanical stimuli (pin prick), and hypersensitivity to thermal and acetone stimuli. These behaviors persisted throughout the 6-week observation period after injection of control AAV6-NP. In contrast, rats injected with AAV6-Na V Rats with iPA1 exhibited a gradual reversal of these changes, which persisted throughout the observation period and were expected to continue until the end of the observation period (Fig. 7C1-F1). Figure 7C -F). For our treatment of existing pain protocol, we used the measurements at day 14 after TNI and before AAV treatment (tBL) as the peak pain intensity (100%), and normalized the measurements of various sensory modalities after treatment relative to the measurements at tBL, and calculated the percentage of pain relief for various modalities at multiple time points (Fig. 7C1-F1). The average pain relief over the 6-week treatment period showed a 52%, 49%, 69%, and 67% reduction in mechanical and thermal pain behaviors for vF stimuli, pin prick stimuli, cold stimuli, and heat stimuli, respectively (Fig. 7C1-F1). Figure 7G AAV-Na was evaluated using a biased CPP paradigm (45).V iPA1 treatment on spontaneous pain. None of the animals in either group were excluded from the study due to their baseline preference / avoidance of the chambers (45). A significant GBP-induced CPP effect was observed in TNI rats injected with AAV6-NP, whereas TNI rats receiving AAV-Na V iPA1 treatment in TNI animals, there was no significant difference in the time spent in the initially non-preferred chamber at baseline versus during testing, suggesting that AAV-Na V iPA1 treatment significantly alleviated the persistent spontaneous pain ( Figure 7H ).

[0150] Histological examination (Figure 8) determined that AAV6-Na V In vivo transduction rate of iPA1. NaViPA1-positive neurons (GFP) accounted for 37 ± 13% (1283 out of 3447 total neurons profiled) were identified by the pan-neuronal marker β3-tubulin (n = 6 DRG, 3-4 sections per DRG, selected once every five sections from a serial series). Transduced DRG neurons included PSNs that also expressed Na V 1.7 and Na V 1.6, and expression showed multiple subcellular localizations, mainly in the PSN cytosol. No positive GFP signal was detected in GFAP-positive perineurial glial cells. GFP signal was also detected in ipsilateral dorsal horn, sciatic nerve, and cutaneous afferent nerve terminals.

[0151] These findings collectively demonstrate that injection of AAV6-encoded Na V iPA1-induced Na V iPA1 expression was restricted to PSNs of the injected DRG and their peripheral and central projections. This strategy resulted in a significant analgesic effect on established peripheral hypersensitivity to evoked and spontaneous pain behaviors in a rat model of peripheral injury-induced neuropathy via AAV6-mediated Na V iPA1 selective expression in sensory neurons of anatomic segmental DRGs responsible for the pathophysiology of pain had a clear analgesic effect on established peripheral hypersensitivity to evoked and spontaneous pain behaviors in a rat model of peripheral injury-induced neuropathy.

[0152] By AAV6-Na V iPA1 treatment reversed PSN hyperexcitability (male rats)

[0153] Enhanced excitability of nociceptive PSNs is a fundamental process in neuropathic pain (46). Thus, we used whole-cell current-clamp AP recordings of Figure 7B rat DRG dissociated neurons after the indicated treatment regimen to examine AAV6-Na Vwhether iPA1 treatment could reverse the increased neuronal excitability of nociceptive PSNs after TNI (12, 47). Although TNI results in DRG containing a mixture of damaged and undamaged axons, nerve injury can induce an increase in voltage-gated ion channel activity in axotomized neurons and adjacent intact neurons, resulting in similar electrophysiological (EP) changes and increased firing frequency in axotomized and adjacent intact DRG neurons (48, 49), possibly through interneuronal signaling and coupling (50). Thus, we recorded from randomly selected small and medium-sized neurons (diameter < 35 pm) (51) from isolated L4 and L5 DRG cultures. Transduced neurons were identified by GFP fluorescence, and excitability was assessed by measuring the baseline intensity and repetitive action potential (AP) firing during a 250 ms current pulse at 100 pA and 280 pA current injection. The results showed that the average baseline intensity of TNI rat neurons was significantly reduced compared to the sham control group, and the induced AP frequency in TNI rat neurons was significantly increased under step stimulation. In AAV6-Na V iPA1 treatment, these values returned to normal in transduced neurons, while NP-transduced neurons had no significant effect (Figure 9). These results suggest that Na V iPA1 reversal of sensory neuron hyperexcitability caused by nerve injury can contribute to its analgesic effect in reducing neuropathic pain behavior, i.e., selective blockade of TTXs Na V ion channels resulted in a significant decrease in neuronal excitability, thereby reducing pain behavior.

[0154] DRG-AAV6-Na V Analgesic effect of iPA1 treatment in female TNI rats.

[0155] There are gender differences in experimental and clinical pain and responsiveness to interventions (52). Therefore, we next tested DRG-AAV6-Na V whether iPA1 treatment could also effectively reduce hypersensitivity induced by TNI in female animals (Figure 7). The same batch of AAV6-Na V iPA1 and AAV6-NP formulations were injected. The results showed that female rats developed a similar hypersensitivity phenotype after TNI induction as male rats, and AAV6-Na V iPA1 treatment, both the induced mechanical / thermal hypersensitivity and the GBP-CPP response returned to normal, indicating a comparable analgesic effect as in male animals ( Figure 10A -E). AAV6-Na ViPA1 Immunohistochemistry (IHC) of DRG sections from female TNI rats 6 weeks later also found GFP-Na V iPA1 expression profiles were comparable to male rats ( Figure 9F -G), with an in vivo transduction rate of 39 ± 25% (766 of 1983 Tubb3-positive neurons profiled). However, transduction rates of DRG neurons have not been quantified. Thus, while not strictly compared, treatment effects were comparable between sexes, suggesting that the pain behavioral phenotype after TNI and the targeting of DRG-AAV6-Na V iPA1 treatment responsiveness did not appear to be significantly gender-biased (12).

[0156] Discussion

[0157] Sustained peripheral targeted analgesia without addiction risk is a global unmet medical need (53). Na V 1.7Current analgesic drugs primarily target NaV1.7. However, substantial evidence indicates that multiple sensory neuron NaVs are involved in nociceptive electrogenesis and pain pathogenesis (15, 54). Here, we report that targeting NaV-IDR promotes Na V iPA discovery. Prototype Na V iPA1 was initially derived from Na V 1.7, and was found to be highly selective for TTXs INa V 1.7, Na v 1.6, Na V 1.3, and Na V 1.1 conductance. DRG-PSNs exhibit a broad spectrum of inhibitory properties, but have no effect on Na V 1.8 and Na Na 1.5 conductance. DRG-PSNs have no effect on Na V 1.8 and Na V 1.5 conductance. DRG-PSNs have no effect on Na Na 1.8 and Na V iPA1 expression selectively inhibits TTXs INa, but not TTXr INa. DRG delivery of AAV6-encoded Na V iPA1 significantly attenuated pain behaviors in both male and female animals following established nerve injury, including evoked mechanical and thermal hypersensitivity, as well as persistent or spontaneous pain behaviors that are commonly seen in patients with multiple painful neuropathies (55). Moreover, Na V iPA1 blockade of TTXs INa was replicated in hiPSC-SNs, supporting its translational potential. Because multiple different types of Na VThe electrical activity required to trigger the nociceptor to generate action potential sequences (APtrains) (1) is considered to be blocked in DRG-PSN by certain specific Na+ receptors. V It has advantages in treating neuropathic pain.

[0158] In almost all cases, chronic pain is sustained by persistent afferent hyperactivity originating from peripheral pathology (56-58). Therefore, developing novel peripheral action strategies targeting pro-algia NaV inhibition in the PSN would be ideal for clinical pain management (2, 59). The strategies we describe here include a novel approach that extracts pro-algia NaV from NaV... V -IDR designed and developed highly selective and non-toxic Na+. V iPA1, and delivered to the pathological DRG using AAV. PSN-restricted inhibition of multiple pro-algae TTXs Na V It is expected to have advantages for DRG-targeted analgesia, as noted in recent expert commentary, "a single dose of Na..." V 1.7 The disappointing efficacy of post-inhibition analgesics may be related to the fact that neuronal excitability is influenced by several different Na+ molecules. V The channel is determined, and targeting only one channel may not be enough (60). It is known that Na… V 1.7 Human subjects and animal models with null mutation heterozygotes exhibited normal sensory phenotypes. Therefore, restricted AAV-mediated Na+ in DRG-PSN... V iPA1 expression may inhibit Na through a combined partial inhibition. V 1.7, Na V 1.6, Na V 1.3 and Na V 1.1 (may include Na) V 1.1) to induce analgesia while avoiding the adverse side effects of systemic distribution of small molecule inhibitors. Although the PSN cell bodies in DRG are anatomically isolated from each other and have no synaptic connections, most DRG-PSNs undergo transient depolarization when the axons of adjacent neurons in the same ganglion are repeatedly stimulated (61). This coupled activation occurs in neurons of various sizes, including small-diameter nociceptors and large-diameter low-threshold mechanoreceptors (50). Therefore, although AAV produces incomplete sensory neuron transduction, the transduced neurons can induce a decrease in pronociceptive ion channel activity in the transduced neurons and adjacent untransduced neurons, resulting in similar electrophysiological changes. Another possible advantage is that, unlike gene therapy strategies such as RNAi (62) and CRISPIR-dCas9 or ZEN epigenetic repression (9) which irreversibly reduce the production of target proteins (which may be problematic (63)), AAV-mediated Na VSelective expression of iPA1 in PSNs provides sustained and limiting block of multiple TTXs-Na V without abolishing the protein itself, thus providing a specific functional interference. It is not intended to completely block Na V 1.7 activity, as it can lead to a state of complete insensitivity to pain, thus causing accidental self-injury (64).

[0159] Painful PSNs become hyperexcitable after peripheral nerve injury, leading to the development of neuropathic pain. Multiple lines of preclinical and clinical evidence suggest that blocking peripheral nociceptive inputs can effectively relieve pain symptoms, including spontaneous pain (65, 66). Thus, targeting the peripheral PSNs can both avoid central nervous system side effects and be successful. In fact, a recent expert review stated that "the activity of primary afferent neurons is a 'low-hanging target' for the development of safe therapies for chronic pain patients" (56). Drug delivery to DRGs is mature and safe, for example, anesthesiologists use it for regional blocks, and pain specialists use it for diagnosis and treatment of radiculopathy (67). In preclinical models, the impact of drug injection into DRGs is minimal (68). It has also been demonstrated that unintentional intraganglionic injection is commonly associated with clinical transforaminal epidural steroid injection (67), a very common procedure with minimal risk of nerve injury. Thus, PSNs are particularly suitable for new analgesic treatments, particularly at the level of the pathological DRGs (59, 69). A recent study reported that central nervous system gene therapy with high-dose AAV by intravenous injection caused asymptomatic and self-limiting DRG inflammation and mild PSN degeneration in primates (70). Since these changes were very small compared to those induced by pain and neuropathy in the treatment with AAV injection, it is unlikely to be an obstacle to the clinical application of our method.

[0160] Direct delivery of AAV encoding analgesic biological agents to DRGs in preclinical models can relieve chronic pain with high transduction efficiency, flexibility of selective segmental localization, and minimal behavioral changes induced by surgical procedures (71). At the same time, injection techniques are constantly evolving to enable the minimally invasive delivery of biological agents for future clinical pain treatment (72, 73). Small peptides derived from the sequence of the target protein can act as decoy molecules to selectively interfere with its function by binding to the target signaling protein in advance (13). We have successfully employed this strategy in a rat model to induce analgesia by blocking T-type / Ca V 3.2 channel function (12) and by blocking the structural protein interaction of Ca V 2.2 channels with collapsin response mediator protein 2 (CRMP2) to block its membrane trafficking (13). Here, we extend the applicability of the DRG-AAV strategy to multiple PSN TTXs NaV Antinociceptive effect of blockers on neuropathic pain. These encouraging results confirm the effectiveness and tolerability of this approach, which indicates translational potential for the development of non-addictive peripheral pain therapeutics if further validated for long-term efficacy and minimal side effects. Besides pain caused by peripheral nerve injury, dysfunctional NaVs are also found in various pain conditions, such as osteoarthritis (OA) that is often highlighted as an unmet medical need. Therefore, targeting TTXs Na V may have therapeutic value (74).

[0161] While our study confirms the effectiveness of a rational analgesic peptide drug design strategy and provides encouraging results, we acknowledge some limitations of this study. Different sodium channels are transported to different subcellular locations (membrane, terminals, nodes of Ranvier, etc.) in PSNs, and the regulation of this process can provide multiple options for controlling neuronal excitability in different pathophysiological settings. In contrast to Na V Injury-induced peripheral hypersensitivity associated with dysfunction affects multiple sites of the peripheral sensory nervous system, including enhanced pain perception at peripheral terminals, enhanced nociceptive signal transduction at PSN somata and T-junctions, and increased neurotransmission at the dorsal horn of the spinal cord. At the early stage of this study, our experiments have not explored the blockade of TTXs Na V differential effects on peripheral nociceptor pathways; at the same time, the experimental results also do not rule out another possibility, i.e., the blockade of TTXs Na V by inhibiting afferent hyperexcitatory inputs (75), indirectly modulating the anti-nociceptive control circuit of the spinal cord and brain, thereby reducing pain. Another limitation is that Na V The molecular mechanisms of iPA1 function are not fully elucidated. Our study confirms the lack of multiple effects on BK and calcium channels, but we cannot rule out the possibility that the peptide interacts with other unknown targets that mediate protein binding. If the peptide binds to the membrane through a lipid mechanism, it can mediate the PM targeting of multiple proteins carrying special domains rich in positive charges. In future studies, elucidating these mechanisms is crucial for assessing therapeutic effects and potential side effects.

[0162] Although we have confirmed that the polybasic NLS and multiple adjacent serine residues are essential for Na V iPA1 function, Na V The phosphorylation-dependent binding of iPA1 to the membrane does not appear to be essential, as serine phosphorylation neutralizes the positive charge of Na VPositive charge of iPA1. It has been reported that polybasic peptides with unphosphorylatable serines exhibit strong membrane binding (76), and highly polar neutral serines with hydroxyl groups on the terminal carbons have stronger interactions with lipid bilayer membranes (77). Na V Other types of PTMs in iPA1 sequence residues can also play a role. It has been reported that serine PTMs can occur through multiple mechanisms, including phosphorylation, sulfation, acetylation, palmitoylation, myristoylation, and glycosylation (78-80). Different PTMs can change the charge and hydrophobicity (electrostatic), in turn inducing changes in the physicochemical properties, structure, and function of the peptide. Ion channel protein arginine methylation and lysine acetylation can enhance current density by increasing channel cell surface expression (81, 82). A recent paper reported that Halo-tagged human full-length Na V 1.7 in iPA regions does not change the membrane integration and channel function of Halo-Na V 1.7 after transfection (83). It is worth further testing whether the combined mutation of polybasic arginine / lysine with multiple adjacent serines and other conserved residues changes the polar binding of full-length Na V 1.7, thereby affecting channel function. In addition, by engineering the highly disordered Na V 1.7 released from the full-length Na V 1.7 protein, it is possible to test whether the highly disordered Na V iPA1 can confer different biological properties on Na V 1.7, such as possible binding to the membrane through electrostatic interactions and exhibiting nuclear transport capacity (12). One cannot ignore the possibility that nuclear Na V iPA1 functions as a transcription factor, affecting genes essential for regulating Na V 1.7 function, similar to the fragmented L-type calcium channel functioning as a transcription factor (84, 85). Na V iPA1 can also function as a decoy peptide, interrupting the interaction of Na V 1.7 with chaperones, as Na V iPA1 partially aligns with the putative Na V 1.7 dimerization sequence, possibly affecting channel function by decoupling Na V 1.7 dimerization assembly (86, 87), although experimental evidence for this mechanism remains to be confirmed. Na V The potential signaling pathways affected by iPA1 can be numerous, as Na V 1.7 PPI molecular network is involved in multiple pathways, and Na V 1.7 (and other TTXsNa VIntracellular segments are important interfaces for many regulatory signaling molecules, including protein-lipid interactions (35, 36). Changes in these molecules following nerve injury are crucial for ectopic PSN overactivity and pain. Future research will answer these questions.

[0163] Materials and methods

[0164] animal

[0165] Adult male and female SD rats weighing 100–125 g (Charles River Laboratories, Wilmington, Massachusetts) were used. Animals were housed individually in a temperature-controlled (22 ± 0.5°C) and relative humidity-controlled (60 ± 15%) room with a 12-hour alternating light-dark cycle, and had free access to water and food throughout the experiment. We minimized animal suffering; all surviving surgeries were performed under sterile conditions and a surgical microscope, with animals anesthetized with isoflurane (2–5%). For euthanasia during tissue collection, animals were deeply anesthetized with isoflurane before decapitation using a well-maintained guillotine. The estimated number of animals required was based on our previous experience with similar experiments (1), and no efficacy analysis was performed.

[0166] The number of rats used is detailed in the relevant chapters or illustrations of the experiment.

[0167] Computational (Computer) Design

[0168] The full-length rat NaV1.7aa sequence was retrieved from the UniProt KB Knowledgebase (UniProt Knowledgebase version 2018_11). Na was predicted using Phobius (https: / / www.ebi.ac.uk / Tools / pfa / phobius / ) (2). V 1.7 Protein™ domains, intracellular terminals, and loops. Full-length Na+ was analyzed using DEPICTER (Disordered Ed Prediction Cellular Analyzer, http: / / biomine.cs.vcu.edu / servers / DEPICTER / ) (3). v 1.7 Sequences to predict Na v 1.7 Protein IDR. Disorder-enhanced phosphorylation predictor (DEPP, http: / / www.pondr.com / cgi-bin / depp.cgi) (4) Identification of Na V1.7 Potential phosphorylation sites in the full amino acid sequence. Potential functional peptides within the IDR were further analyzed using SLiMPrints (http: / / bioware.ucd.ie / slimprints.html), a software that predicts short linear motifs (SLiMs) based on highly conserved short linear motifs (SLiMs) within the IDR (5). Peptide structure determination was analyzed by I-TASSER (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ) (6). MacVector ClustaIW (MacVector, Apex, NC) was used for vector design and sequence alignment.

[0169] Molecular cloning and AAV constructs

[0170] Construct pAAV-CBA-GFP-1.7iPA encodes GFP-Na downstream of a chimeric intron for enhanced transcription V 1.7iPA fusion protein driven by the hybrid CMV enhancer / chicken beta-actin (CBA) promoter and insertion of the woodchuck post-transcriptional regulatory element (WPRE) sequence to stabilize the mRNA V 1.7iPA downstream of the stop codon and upstream of the human growth hormone poly A signal. Subsequently, the plasmid was used for transfection experiments and AAV vector construction. AAV6-GFP-1.7iPA1, AAV6-GFP linker and AAV2 / 6-GFP-NP (Na V 1.7 N-terminal inert peptide) (referred to as AAV6-Na V 1.7iPA1, AAV6-GFP and AAV6-NP) for in vivo injections. AAV vectors were produced and purified by our laboratory using previously well-established methods (7). AAV6-GFP, AAV6-Na V iPA1 and AAV6-NP vectors had titers (GC / mL) of 2.45 x 1011, 3.05 x 1011, 2.64 x 1011, and 2.64 x 1011, respectively. 13 13 13 All in vivo experiments used the same batch of AAVs.

[0171] Site-directed mutagenesis. The QuikChange mutagenesis method (Stratagene) was used to substitute arginine / lysine and serine to alanine in pAAV-CBA-GFPNa V iPA1, resulting in Na V iPA1mt1-6. All constructs were verified by DNA sequencing before use.

[0172] Cell culture ​​

[0173] Cell lines. HEK293 cell lines stably expressing human wild-type NaV1.7 (HEK1.7) were provided by Dr. Theodore Cummins. HEK293 cell lines stably expressing human wild-type NaV1.6 (HEK1.6), NaV1.3 (HEK1.3), NaV1.1 (HEK1.1), and NaV1.5 (HEK1.5) were purchased from Charles River. CHO cells stably expressing human NaV1.8 (CHO1.8) were purchased from Charles River. Neuronal NG108-15 (NG105) neuron-like cells and F11 cells (hybrid cells of mouse neuroblastoma cells and embryonic rat DRG neurons) were purchased from ATCC (Manassas, VA). Rat DRG neurons 50B11 cells (50B11) were used as previously reported (8). These cells were cultured and transfected (PEI40) using standard techniques as previously described (1). V 1.6 (HEK1.6), Na V 1.3 (HEK1.3), Na V 1.1 (HEK1.1), Na V 1.5 (HEK1.5) were purchased from Charles River. Neuronal NG108-15 (NG105) neuron-like cells and F11 cells (hybrid cells of mouse neuroblastoma cells and embryonic rat DRG neurons) were purchased from ATCC (Manassas, VA). Rat DRG neurons 50B11 cells (50B11) were used as previously reported (8). These cells were cultured and transfected (PEI40) using standard techniques as previously described (1).

[0174] Human Na V 1.8 stable HEK293 cell generation. To generate Na V 1.8 stable expressing HEK293 cells (HEK1.8 cells), a pcDNA3.1(+)-SCN10A- Furin-P2A-SCN2B expression plasmid (Genscript) was constructed in which the CMV promoter transcribed human Na V 1.8a and Na b2 from a single open reading frame (ORF) expressing human SCN10A (NM_001293306.2) and SCN2B (NM_004588.5) linked by a 2A self-processing sequence derived from porcine teschovirus-1 (P2A) and a furin cleavage site, respectively (Figures 12, 13) (9). The final construct was sequence-confirmed and transfected into HEK293 cells and selected with G-418 (800 µg / mL) and then single cell colonies were established using BIOCHIPS single cell isolation chip (ThermoFisher) according to the manufacturer’s recommended protocol. Expression of Na V 1.8a and Na V b2 was determined by immunoblotting using cell lysates and extracted cytoplasm and cell membranes and prioritized according to functional sodium current amplitude using conventional whole-cell voltage-clamp (see below for details).

[0175] Isolated DRG neuron culture. Isolated DRG neuron culture for EP was performed as previously described (10) and studied 6-8 hours after harvesting in EP experiments.

[0176] Human induced pluripotent stem cell (iPSC)-derived sensory neurons (hiPSC-SNs). hiPSC-SNs were derived from female human ectodermal neural crest stem cells, Chrono™ Senso-MM complete growth medium was purchased from Anatomic (Minneapolis, MN)

[0177] (11). hiPSC-SN maturation differentiation culture was performed according to the manufacturer’s recommendations.

[0178] Na V Lentiviral vectors for iPA1 and NP

[0179] Na V iPA1 and NP (control) (Fig. 14). Na V iPA1 and pWPT-GFP NP were packaged into lentivirus (LV) using packaging plasmids pCMVdR8.74 and envelope plasmid pVSV-g, concentrated and the product was titrated to 1 x 107 8 to 2 x 107 8 transducing units / mL. Infection was performed by LV-GFP, GFP Na V iPA1 or LV-GFP NP infection of cultured hiPSC-SNs.

[0180] Electrophysiology (EP)

[0181] EP recordings were performed at room temperature (22-25 °C) (10, 12) in a blinded fashion, where the electrophysiologist was unaware of the treatment condition, as described previously with slight modifications. Patch pipettes with 0.9-2.5 MΩ resistance were made from borosilicate glass (King Precision Glass Co., Claremont, CA) and fire-polished. Recordings were made using an Axopatch 700B amplifier (Molecular Devices, Downingtown, PA). Digidata 1440A digitizer and pClamp 10 software (Molecular Devices, San Jose, CA) were used with a 5 kHz filter and sampled at 20 and 50 kHz (from the literature) (13-22). Series resistance (3-5 MΩ) was monitored before and after recordings, and data were discarded if resistance changed by >20%. After completing whole-cell recordings, appropriate compensation for capacitance (Cm) and series resistance (Rs) was performed.

[0182] Recording of sodium channel currents (I Na ) in cultured cell lines. Whole-cell voltage-clamp recordings I Na were performed in HEK1.7, HEK1.1, HEK1.3, HEK1.6, HEK1.5, HEK1.8, CHO1.8, NG108-15 cells and F11 cells using current density (I-V) and fast-inactivation voltage protocols. 2+ External solution composition included (in mM): 110 NaCl, 20 tetraethylammonium-Cl, 0.01 CaCl2, 0.1 CaCl2, 5 MgCl2, 10 HEPES and 5.56 mM glucose (pH 7.4, 310-315 mosM / L). Internal pipette solution included (in mM): 10 NaCl, 130 CsCl, 5 MgCl2, 5 EGTA, 2.5 Na max ATP and 10 HEPES (pH 7.2). After establishing a tight seal (maximal leak amplitude < 150 pA), membrane resistance and capacitance were determined. Voltage dependence of activation was assessed by applying 50 ms test pulses (test pulses) ranging from -100 mV to +50 mV in 5 mV or 10 mV increments, with 5 s intervals, by maintaining the holding potential. Current density was calculated by normalizing the maximal peak current with the cell capacitance. Voltage dependence of steady-state fast-inactivation was determined using a two-step protocol. Channels were inactivated using a 500 ms pre-pulse ranging from -100 mV to +10 mV in 5 or 10 mV increments. This pre-pulse was immediately followed by a 40 ms test pulse at 0 mV to determine the remaining fraction of available channels. The inward current measured during the test pulse at 0 mV was normalized to the maximal test pulse inward current of the cell. To determine the voltage dependence of activation, the peak current density during each voltage command step was fitted to a smooth curve using the Boltzmann equation: I = G rev (V-E 50 ) / [(1 + exp[(V-V max ) / k])], where G max is the maximal conductance. The normalized activation curve was fitted with the Boltzmann equation G / G 50 = 1 / (1 + exp(V 50 -Vm) / k), where G is calculated as follows: G = I / (Vm-Erev). The steady-state inactivation curve was fitted with I / Imax = 1 / (1 + exp-(V 50 -Vm) / k). In all equations, V maxand I max are the maximum conductance and maximum current, respectively. The current density is obtained by dividing the maximum peak current (pA) by the cell capacitance (pF). Rs x I max defined voltage error is minimized and readjusted before each voltage clamp protocol. Cells are excluded if the voltage error exceeds 5 mV.

[0183] TTXs and TTXr I Na recorded. I Na from single small / medium DRG neurons (diameter < 35 mm, 4 weeks after primary rat AAV-DRG injection) and hiPSC-SNs differentiated from bath solution containing the following components (in mM): 80 NaCl, 50 Choline-Cl, 30 TEA-Cl, 2 CaCl2, 0.2 CdCl2, 10 HEPES, and 5 glucose, pH = 7.3 with NaOH. The internal solution contains the following components (in mM): 70 CsCl, 30 NaCl, 30 TEA-Cl, 10 EGTA, 1 CaCl2, 2 MgCl2, 2 Na2ATP, 0.05 GTP, 10 HEPES, and 5 glucose, pH = 7.3 with CsOH. TTXr I Na and TTXs I Na (14, 23). Briefly, a 500 ms pre-pulse at -120 mV or -50 mV was applied, followed by a 50 ms test pulse at -100 mV to 40 mV, with a test pulse step of 5 mV or 10 mV for -50 mV to 0 mV. TTXs and TTXr I Na were evident after -120 mV pre-pulse; while only TTXr I Na was obtained after -50 mV pre-pulse. Na components were obtained by subtracting TTXr I Na from the total I Na recorded at DIV21-28. To isolate somatic I Na , a short pre-pulse to voltage (-40 mV) was applied near the peak to inactivate the hiPSC-SN axonal spikes, but not the somatic spikes, as previously described (24).

[0184] High voltage-activated (HVA) I Ca and voltage-gated potassium channel current (I Kv). For recordings, dissociated neurons of rat DRG (sham, AAV6-GFP, AAV6-NP and AAV6-Na V iPA1-transduced neurons, 4 weeks after AAV-DRG injection Ca BK I were performed in undifferentiated NG108-15 cells Kv Recordings were made as previously described (1).

[0185] Whole-cell current-clamp recordings were made on dissociated DRG neurons (male rats). Whole-cell current-clamp recordings were made on dissociated DRG neurons as previously described (10, 25, 26). Small and medium-sized DRG neurons (diameter < 40 μm) were isolated from sham animals, rats receiving TNI alone, and from rats injected with AAV6-GFP NP or AAV6-Na V DRG neurons with clear GFP expression isolated from TNI rats of iPA1 were used for recordings (n = 5 rats per group). Membrane input resistance was calculated by dividing the amplitude of the termination of the steady-state hyperpolarizing voltage deflection by the injected current (27). APs were generated by injecting a series of current pulses (180 to 280 pA in steps of 20 pA, 250 ms). The baseline potential was recorded for 20 ms before the stimulation pulse was injected into the neuron. Resting membrane potential (RMP) was defined as the average of the pre-stimulus 20 ms potential in the first trial, and AP threshold was defined as the minimum current required to evoke the first AP. Neurons with stable resting membrane potential (RMP) and less than -40 mV (more negative) and AP overshoot (from RMP to peak > 80 mV) were used for further data collection. AP frequency was determined by quantifying the number of APs evoked by a depolarizing current injection (250 ms).

[0186] Microinjection of AAV vectors into DRG

[0187] AAV vector solutions were microinjected into the right L4 and L5 DRG using techniques previously described (1, 28). Rats received L4 and L5 DRG injections of AAV6-Na V iPA1 or AAV6-NP (one vector per rat) in a volume of 2 μL, titrated so that a total of 2.0 x 1011 10 genomic viral particles were included. Sham in animals indicates that no virus was injected after the exposure surgery.

[0188] Animal pain models and behavioral tests

[0189] TNI. Animals were induced with anesthesia using 4% isoflurane and maintained with 2% isoflurane. TNI surgery was performed as we previously described (1). Sham rats underwent all the aforementioned procedures but without nerve ligation and transection.

[0190] Stimulus behavior testing. Behavioral testing was performed between 9:00 AM and 12:00 AM as we previously described (1). The experimenter was blinded to the treatment regimen throughout all data collection procedures. Stimuli were applied to the sural nerve innervated area on the lateral aspect of the foot. Sensory testing included evoked reflex behaviors induced by von Frey (vF) testing, pinprick testing, cold stimulus (acetone), and heat stimulus (Hargreaves test) and were performed as previously described (28).

[0191] Gabapentin (GBP) injection. Gabapentin (GBP, Sigma-Aldrich) was dissolved in normal saline immediately prior to injection and administered intraperitoneally (ip) in a volume of 0.5-1.0 ml (final dose = 100 mg / kg body weight).

[0192] Conditioned place preference (CPP). A three-chamber CPP apparatus (Med Associates, St. Albans, VT) was used in which two sliding doors separate the central chamber from two side chambers with different wall stripes and floor. CPP procedure: 1) On the first day, rats were acclimated to the CPP box for 30 min with access to all three chambers. On the pre-conditioning day, rats were placed in the middle gray chamber and allowed to explore both side chambers for 15 min each and the time spent on each side was recorded and the preferred and non-preferred chambers were determined. 2) On the conditioning day, drug-pairing was performed using a biased assignment procedure for place conditioning: saline was paired with the preferred chamber in the morning and GBP was paired with the non-preferred chamber in the afternoon, with a 6-hr interval (injections were never paired with the middle gray chamber). Conditioning consisted of the following consecutive steps: intraperitoneal injection and confinement of the animals in the preferred chamber (saline) or non-preferred chamber (GBP) for 45 min. We used a 45-min conditioning time because 30-60 min after intraperitoneal injection is the time of maximal gabapentin-induced reduction of mechanical hypersensitivity (1). Animals were conditioned for 2 days because 2 days of GBP have been reported to be sufficient to produce CPP in rodent models of pain (29, 30); 3) For post-conditioning testing, animals were returned to the middle gray chamber of the CPP chamber and given free access to all chambers for 15 min. The difference score for each animal was calculated by subtracting the time spent in the saline-paired chamber or GBP-paired chamber during the pre-conditioning period from the time spent in each chamber after pairing (post-conditioning), and then averaged within groups. Each rat was only tested once for CPP 6 weeks after AAV injection. An animal was defined as having CPP if it spent significantly more time in the GBP-paired chamber than in the saline-paired chamber.

[0193] Immunofluorescence staining

[0194] Procedures described previously (8). Primary antibodies: mouse GFP (1 :500, Santa Cruz Biotechnology (SCB), California, sc9996), rabbit GFP (1 :500, Cell signaling, Danvers, MA, 2555), rabbit Na V 1.7a (1 :400, Alomone, ASC008), rabbit Na V1.6a (1 :400, Alomone, ASC009), rabbit glial fibrillary acidic protein (GFAP, 1 : 1000, Dako, CA, Z0334), goat myelin basic protein (MBP, 1 :500, SCB, sc 13912), mouse neurofilament (NF200, 1 : 1000, Sigma-Aldrich, N6389), and mouse b3 tubulin (Tubb3, 1 :500, SCB, sc-80016). Fluorophore-conjugated (Alexa 488 or Alexa 594, 1 :2000) secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used to visualize the immunocomplexes. Immunostaining was examined and images were captured using a Nikon TE2000-S fluorescent microscope (El Segundo, CA) equipped with appropriate filters for selective detection of green and red fluorescence using a QuantiFire digital camera (Optronics, Ontario, NY). For measurement and quantification of immunostaining, cells with fluorescence intensity greater than the mean background fluorescence value of adjacent areas in IHC slides of the same negative control (primary antibody omitted) plus 2 standard deviations were defined as marker antibody-stained positive cells (n=10 for each different marker). Analysis was performed using NIH ImageJ software (http: / / rsbweb.nih.gov / ij / ).

[0195] Western blotting

[0196] Western blotting was performed as previously described (1). To detect Na v 1.7 For subcellular localization of HEK 1.7 cells and NaviPA1 and NaviPA1 mutants in HEK 1.7 cells, cells were pelleted, homogenized, and then fractionated to obtain plasma membrane, cytosol, and nuclear fractions, and extracted using ProteoExtract Subcellular Proteome Extraction Kit (Millipore, Billerica, MA). In some experiments, transferred PVDF membranes were cut in half along the protein size of approximately 70-100 kDa, and then incubated with appropriate antibodies overnight at 4°C. Antibodies: mouse GFP (1 : 1000), rabbit Na V 1.7a (1 : 1000), rabbit Na V 1.8a 1 : 1000, Alomone, ASC-016), rabbit NaVb2 (1 : 1000, Alomone, ASC-007), mouse Na + / K +ATPase 1a (NKA1a, 1 :600, SCB, sc514614), mouse Lamin B1 (1 :1000, Proteintech, 66095) and mouse Gapdh (1 :5000, Sigma-Aldrich, SAB1403850). Immunoreactive proteins were detected using Pierce enhanced chemiluminescence (ThermoFisher) on a ChemiDoc imaging system (Bio-Rad) after incubation with HRP-coupled secondary antibodies (1 :5000, Bio-Rad) for 1 h.

[0197] GFPNa V iPA1 affinity pull-down followed by immunoblotting, silver staining, mass spectrometry and PIP band assay

[0198] GFP affinity pull-down. This was performed using the ChromoTek GFP-Trap kit. Briefly, HEK 1.7 cells were transiently transfected to express GFPNa V iPA1 or GFP linker and / or GFP 1.7 iPA2 as control. After 48 h, cells were lysed with ice-cold non-denaturing lysis buffer containing (in mM) 10 Tris / Cl pH 7.5, 150 NaCl, 0.5 EDTA, and 0.5% Nonidet P40 substitute (a non-ionic and non-denaturing detergent) and protease inhibitor cocktail. Extracted cell lysate was diluted with 300 mL dilution buffer containing (in mM) 10 Tris / Cl pH 7.5, 150 NaCl, 0.5 EDTA. For GFP affinity purification, 25 mL GFP-Trap agarose beads were added to 1.5 mL reaction tubes (containing 500 mL dilution buffer) and allowed to equilibrate, followed by incubation of cell lysate (2.5 mg total protein each sample) with GFP-Trap agarose, tubes were rotated end-over-end at 4°C overnight, and beads were pelleted by centrifugation at 12,000 rpm.

[0199] silver staining, Na V 1.7 immunoblotting and mass spectrometry analysis. Extracted cell lysate (input) and GFP affinity pull-down beads (pull-down) were analyzed by loading on 4-20% SDS-PAGE gels, followed by Na V1.7 Antibodies were immunoblotted and silver-stained on a separate SDS-PAGE gel using a Pierce silver staining kit (ThermoFisher). The stained gel region of interest was excised and subjected to in-gel trypsin digestion as previously described (31). Extracted trypsin peptides were analyzed by nano-reverse phase liquid chromatography tandem mass spectrometry (nLC-MS / MS) using a nanoACQUITY (Waters Corporation, Milford, MA) online with an Orbitrap Velos Pro hybrid ion trap mass spectrometer (ThermoFisher). Instrument was set to automatically select precursor ions. Resulting MS / MS data were analyzed using the Mascot search engine (Matrix Science version 2.4) against the SWISSPROT human database.

[0200] PIP band assay. Transfected GFPNa V Cell lysates of iPA1 and GFP linker NG108-15 cells were prepared using denaturing RIPA buffer containing 10% Tris-HCl pH 7.5, 150% NaCl, 0.5% EDTA, and 0.1% SDS, 1% Triton X100, 1% deoxycholic acid, and protease inhibitor cocktail (units: mM). GFP affinity pulldown beads of NG108-15 cells were size separated using a 4-20% SDS-PAGE gel followed by silver staining. PIP band analysis (Echelon Biosciences, Salt Lake City, UT) was used to analyze purified GFPNa V iPA1 interaction with membrane lipids using purified GFP in NG108 as a control. Briefly, PIP (phosphatidylinositol phosphate) strips were blocked in 3% (w / v) fat-free BSA (Sigma-Aldrich) in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h. Membranes were then incubated with purified GFPNa ViPA1 or GFP (1.5 mg / mL each) were incubated in the same solution overnight at 4 °C with gentle agitation. Membranes were washed 3 times in 30 min in fatty acid-free BSA-TBST. Membranes were incubated with 1:2000 diluted HRP-conjugated anti-GFP monoclonal antibody (Proteintech, HRP66002) for 1 h at room temperature, then washed 6 times in 1 h in TBST, and proteins bound to membranes by enhanced chemiluminescence detection on ChemiDoc imaging system for phospholipid interaction. PIP2 lipid points: LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol (3) phosphate; PtdIns(4)P, phosphatidylinositol (4) phosphate; PtdIns(5)P, phosphatidylinositol (5) phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1 -phosphate; PtdIns(3,4)P2, phosphatidylinositol (3,4) bisphosphate; PtdIns(3,5)P2, phosphatidylinositol (3,5) bisphosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5) bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5) triphosphate; PA, phosphatidic acid; PS, phosphatidylserine. Example 2 - Na V Inhibition of Na v 1.7 / 1.8 channels by peptide aptamers

[0201] Multiple sensory neuron sodium channels (Na V ) are involved in the pathogenesis of pain, suggesting that developing strategies to specifically block multiple Na V (e.g., Na V 1.7 and Na V 1.8 (Na V 1.7 / 1.8)) can be more effective in inhibiting pain signals than blocking only a single Na V subtype. Na V proteins are rich in intrinsically disordered regions (IDRs) in their intracellular segments, which can serve as key hubs and novel drug targets in the Na V signaling network. We discovered a polyacidic peptide from the Na V 1.7-IDR in its intracellular loop 2, and voltage-clamp recordings on HEK cells stably expressing Na V 1.7 and Na V 1.8 (HEK1.7 and HEK1.8) showed that the peptide effectively inhibited the sodium currents (I V conducted by Na V 1.7 and Na Na). This dual Na V 1.7 / 1.8 inhibitory peptide aptamer (named 1.7 / 1.8 iPA) inhibits both Na V 1.7 and Na V 1.8, containing a conserved AnkG protein binding domain and a multi-PDZ domain class I protein binding domain (Pdzd2-I), suggesting that either the AnkG or Pdzd2-I domain or both within the 1.7 / 1.8 iPA are responsible for the dual Na V 1.7 / 1.8 inhibition.

[0202] The overall goal of this exploratory / innovative proposal is to investigate and determine if the AnkG and / or Pdzd2-I domains within the 1.7 / 1.8 iPA contribute to its dual Na V 1.7 / 1.8 inhibition, and if expression of the sequence-defined 1.7 / 1.8 iPA in primary sensory neurons (PSNs) will inhibit PSN excitability, supporting the 1.7 / 1.8 iPA as a potential analgesic agent. The experiments are designed to test: 1) the ion channel selectivity of the 1.7 / 1.8 iPA, including a) the effect of the 1.7 / 1.8 iPA on various Na V subtypes in HEK cells stably expressing Na V 1.7, b) the role of the AnkG and / or Pdzd2-I domains in the 1.7 / 1.8 iPA on the dual inhibition of Na V 1.7 / 1.8, c) the effect of the 1.7 / 1.8 iPA on K V 7.2 / 7.3 (both containing a conserved AnkG binding domain), and d) defining the critical 1.7 / 1.8 iPA sequence. 2) to validate the dual inhibition of the 1.7 / 1.8 iPA on TTXs and TTXr I Na channels, the effect on K V 7.2 / 7.3 channels, and the effect on rat PSN excitability. As an initial and conceptual phase of project development, this study will validate the hypothesis that the 1.7 / 1.8 iPA elicits dual blockade of Na V 1.7 / 1.8 through the combined action of the AnkG and Pdzd2-I domains, which will inhibit PSN action potential firing. Two specific objectives are proposed to explore whether the 1.7 / 1.8 iPA can be developed as a potential lead for AAV-mediated sensory neuron-specific analgesia in future studies.

[0203] Objective 1: In vitro (cell line) studies of the ion channel selectivity of the 1.7 / 1.8 iPA

[0204] Experiment 1A: Determine the effect of the 1.7 / 1.8 iPA on endogenous Na V 1.7 and Na V1.8 intracellular trafficking. This will test the hypothesis that due to Na V 1.7 and Na V 1.8 trafficking is impaired, 1.7 / 1.8 iPA reduces Na V 1.7 and Na V 1.8 conductance (HEK 1.7 and HEK 1.8 cells).

[0205] Experiment 1B: Transfection of 1.7 / 1.8 iPA into HEK 1.7, 1.8, 1.6, 1.5, 1.1 and 1.3 cells and NG108-15 neuronal cells that natively express the potassium channels K V 7.2 / 7.3, and study Na V channel occupancy specificity.

[0206] Experiment 1C: Determine the role of AnkG and / or Pdzd2-I domains in 1.7 / 1.8 iPA action. a) Site-directed mutagenesis of key amino acids (aa) in 1.7 / 1.8 iPA and / or deletion of AnkG or Pdzd2-I domains (or both) in combination with voltage clamp recordings. b) Define key amino acid sequences in 1.7 / 1.8 iPA that are critical for inhibiting Na V 1.7 / 1.8 channels and minimal impact on K V 7.2 / 7.3, and clone them into AAV plasmids for use in Study 2.

[0207] Experiment 1D: Confirm dual Na V 1.7 / 1.8 inhibition by defined 1.7 / 1.8 iPA to ensure reproducibility and compare to the effect on Na V 1.8 and Na V 1.9 expressed in ND7 / 23 cells.

[0208] Study 2: Determine the biological activity of defined 1.7 / 1.8 iPA expression in sensory neurons

[0209] Experiment 2A: Generate AAVs carrying sequence-defined 1.7 / 1.8 iPA (d1.7 / 1.8 iPA) and 1.7 NP (non-functional peptide from the N-terminus of Na V 1.7 as a control for AAV-mediated d1.7 / 1.8 iPA expression in rat PSNs.

[0210] Experiment 2B: Determine the effect of PSN-specific expression of d1.7 / 1.8 iPA on PSN electrical generation and toxicity compared to control groups. Assess d1.7 / 1.8 iPA effects on a) TTXs and TTXr NaV Conductance, b) K V 7.2 / 7.3 activity, c) PSN action potential (AP) firing, and d) effects on neurotoxicity.

[0211] Experiment 2C: Repeat TTXs and TTXr I Na recordings on PSNs expressing dl.7 / 1.8 iPA in Dr. Cummins' laboratory.

[0212] Experiment 2D: Assess behavior and safety. Determine the effects of PSN-specific expression of dl.7 / 1.8 iPA on a) normal sensory behavioral thresholds and b) DRG and afferent axon safety in vivo by immunohistochemical methods.

[0213] BACKGROUND

[0214] Peripheral sensory neuron Na V channels are ideal targets for pain control. Na V channels are key determinants of neuronal excitability 1-4 . Mammals possess nine Na V channel subtypes, of which Na V 1.7, Na V 1.8, and Na V 1.9 are primarily expressed in PSNs of dorsal root ganglia (DRGs). Na V channels are critical determinants of action potential generation and propagation, and thus inhibition of Na V channel function in PSNs can have a major impact on limiting neuronal excitability and pain signaling 5、6 . Na V 1.7 remains a primary target for developing therapeutic strategies for chronic pain, but the success rate for developing Na V 1.7-specific inhibitors as analgesics in the clinic has been limited V . Increasing evidence suggests that multiple PSN-Na 3、7 channel alterations in various pain pathologies are responsible for the development of pain V , suggesting that a multi-faceted blockade of several nociceptive Na 8 channels in PSNs has analgesic advantages over blocking only a single Na V channel subtype V .

[0215] Small molecules vs. biologies. Drug safety concerns arising from poor Na 9-12 channel subtype specificity and insufficient efficacy have limited the clinical translation of small molecule compounds for systemic administration V . Development of biologies targeting Na 13、14 channels has been limited by the need for large doses and the potential for immunogenicity V .1.7 biologicals are attractive for analgesia 15-17 . Na V 1.7 neutralizing monoclonal antibodies were reported to have analgesic efficacy, but this result has not been replicated 18 . Na V 1.7 blockers 17、19 have analgesic efficacy but poor membrane permeability, Na V 1.7 are not selective enough and have short half-life 13 . Na V 1.7-RNAi 13 and CRISPR-dCas9 or ZEN epigenetic Na V 1.7 inhibition 20 for analgesic gene therapy, but these interventions at the mRNA and epigenetic levels lack the specificity of direct channel intervention, reducing safety and allowing off-target effects to occur 13、21、22 while anti-dCas9 immunity presents an additional challenge to CRISPR gene therapy 23 Our previous studies showed that AAV-mediated PSN-specific expression of small analgesic peptides to inhibit nociceptive ion channel activity can effectively relieve pain in various animal pain models 24-26 High-dose intravenous injection of AAV caused mild and self-limiting DRG inflammation and mild PSN degeneration in primates 27 As these changes are very small compared to the changes caused by painful neuropathies treated by AAV-targeted DRG therapy, this is unlikely to be an obstacle to the translation of AAV-DRG analgesic methods in neuropathic pain.

[0216] Targeting intrinsic disordered regions (IDRs) facilitates the discovery of ion channel inhibitory peptides. Small peptides derived from nociceptive ion channels as functional interfering peptide aptamers (iPA) have high efficiency and selectivity, which can block specific nociceptive signaling 25、28 IDRs of ion channel proteins are usually involved in diverse interaction networks that play important roles in various signaling regulations and are considered as new promising drug targets 6、29-31 Nociceptive ion channel proteins are enriched in IDRs in their intracellular unstructured segments, which can serve as key hubs in the channel nociceptive signaling network 25 We have successfully identified disordered Ca V 3.2 iPA for adeno-associated virus (AAV)-mediated sensory neuron-targeted analgesia 24、25 In this study, by targeting IDRs, we derived from Na V1.7 Intracellular loop 2 (ICL2) revealed a disordered polyacidic repressive peptide (named 1.7 / 1.8iPA, 38mer), and voltage-clamp recordings showed that this peptide effectively inhibited the formation of Na+-dependent polyacidic peptides. V I conducted through channels 1.7 and 1.8 Na (Objective 1: Preliminary Data). Further validation of the selectivity of 1.7 / 1.8iPA for ion channel occupancy and sensory neuron excitability will help determine whether 1.7 / 1.8iPA can serve as a potential analgesic lead for the development of AAV-mediated PSN-specific analgesia.

[0217] B. Innovation: 1. The experiment detailed in Example 2 targets Na V The IDR explored a new method to discover double Na V 1.7 / 1.8 peptide antagonists. 2. Directly from Na... V 1.7 Protein-designed analgesic and therapeutic peptides are entirely new.

[0218] C. Method

[0219] Overall Plan The experiment will use HEK Na V Subtype stable cells and NG108 cells (or KV 7.2 / 7.3 stable expression cell lines) were tested in vitro for the effect of 1.7 / 1.8 iPA on intracellular transport of channel proteins I. Na and I Kv Effects of 7.2 / 7.3 (Target 1); and the effects of 1.7 / 1.8iPA expression defined in rat PSN on TTXs and TTXr Na V Effects on PSN excitability, normal sensory behavior, and histological safety (Objective 2).

[0220] Specific objective 1. In vitro (cell line) study of ion channel selectivity of 1.7 / 1.8 iPA.

[0221] Basic principle: The 1.7 / 1.8iPA (38-mer) contains conserved AnkG and Pdzd2-I binding domains, suggesting that the AnkG or Pdzd2-I domains (or both) within 1.7 / 1.8iPA may be its binding domains to Na+. V 1.7 and Na V 1.8 Mechanism of Dual Repression. We will mutagenesis of key amino acids within the AnkG or Pdzd2-I binding domain (or both) and combine this with voltage-clamp techniques to evaluate the role of these domains in Na+ inhibition. V 1.7 / 1.8 Role in dual inhibition. Furthermore, functional AnkG-binding domains exist in multiple Na+ domains. V and K V7.2 / 7.3, it is necessary to investigate the effects of 1.7 / 1.8 iPA on various pro-nociceptive Na V and anti-nociceptive K V channels in 7.2 / 7.3.

[0222] Preliminary data for Specific Aim 1

[0223] HEK cell-based Na V 1.8 stable expression system. Currently, there is no commercially available iPA for Na Na1.8 1.8. Therefore, we developed a protocol for stable and functional expression of recombinant human Na V 1.8 in HEK cells. We constructed a pcDNA3.1(+)-SCN10A- Furin-P2A-SCN2B expression plasmid in which the CMV promoter transcribes human Na V 1.8a and Na2B from a single open reading frame (ORF) linked by a 2A self-processing sequence derived from porcine teschovirus-1 (P2A) and a furin cleavage site, respectively. The final construct was transfected into HEK cells and selected with G-418 (400-800 µg / mL) followed by single cell colony establishment using BIOCHIPS single cell isolation chip. 32 Western blotting of Na V 1.8a and Na2B confirmed that Na V 1.8a / Na2B was stably expressed in HEK cells and highly integrated into the cell membrane. Functional Na V 1.8-expressing clonal cell lines were identified by slow inactivating inward I Na currents evoked by voltage steps from -140 mV to +40 mV in whole-cell voltage-clamp recordings, and the average peak I Na 1.8 density was about 1.0 nA in 85% of the cells and was sensitive to Na V channel blocker A803467 (100 nM of A803467 inhibited 85% of I Na1.8 ) 33 (FIG. 20).

[0224] Targeting Na V 1.7 with IDRs helped to discover dual Na V 1.7 / 1.8 inhibitory peptides. We found that IDRs from Na V1.7-ICL2 disordered peptide. This highly disordered polyacid peptide contains 35% negatively charged glutamate (E) and aspartate (D). Voltage-clamp recordings (Fig. 21) show that this 38-mer peptide inhibits I in HEK1.7 cells. Na1.7 And may reduce Na V 1.7 Sensitivity of voltage-gated activation; and unexpectedly, expression in HEK1.8 cells inhibited I... Na1.8 While these require further validation, we refer to them as the 1.7 / 1.8 repressive peptide aptamer (1.7 / 1.8iPA). Following expression of 1.7 / 1.8iPA in NG108 cells naturally expressing various KVs, large potassium (BK) I... Kv No change. However, the BK channel may not contain K. V 7.2 / 7.3 components 34 Further analysis of 1.7 / 1.8iPA and Na V Sequence alignment of the 1.8-ICL2 sequence revealed no significant homology; however, the two functional domains, the AnkG binding domain and the Pdzd2-I binding domain, showed similar homology. 35、36 This is conservative, suggesting that the AnkG or Pdzd2-I domain, or both, within 1.7 / 1.8iPA may be involved in Na... V 1.7 / 1.8 Dual inhibition. The AnkG binding domain is also conserved in KV7.2 / 7.3 (Fig. 22). AnkG expression is well-known to be enriched at potential-generating sites in the nervous system, such as axonal initiation segments (AIS) and nodes of Ranvier. 37、38 Pdzd2 via Na V The three Pdzd2 domains (class I, class IIx2, and class III) in ICL2 of 1.7 / 1.8 directly bind to Na. V 1.8 and Na V 1.7, and silencing Pdzd2 in PSN will reduce I. Na 1.8 35 .

[0225] Neurons will gate Na with high density of voltage V The KV channel targets its AIS and Ronfi junction to control the initiation and conduction of fast action potentials, respectively. 37、38 Given Na V The AnkG and Pdzd2 binding domains interact with the AnkG and Pdzd2 proteins, respectively, thereby regulating Na+. V Intracellular transport, functional expression, channel properties and neuronal excitability 35、39、40 K V 7.2 / 7.3 Controlling neuronal excitability and participating in nociceptive electrogeneration 41 Two Ks V7.2 / 7.3 share a conserved domain at the C-terminus for binding to AnkG protein 39、42 , resulting in K V 7.2 / K V 7.3 increased expression at AIS and Ranvier node surface. Thus, 1.7 / 1.8 iPA on I Na 1.7 / 1.8 dual inhibition can be through interrupting Na V 1.7 / 1.8 interaction with AnkG and / or Pdzd2 protein to inhibit neuronal excitability. On the other hand, if 1.7 / 1.8 iPA on K V 7.2 / 7.3 has a dominant inhibitory effect, then 1.7 / 1.8 iPA on neuronal excitability can be attenuated. It has been reported that silencing AnkG in neurons inhibits the inward Na V and outward K V to a lesser extent, and neuronal excitability is inhibited 38、43、44 , AnkG overexpression has a minor effect on Kv7.2 / 7.3 channel gating 39 , while deletion of Pdzd2 in neurons reduces I Na 1.8 35 . Thus, it is conceivable that the convergent effect of 1.7 / 1.8 iPA expression in PSN inhibits the excitability of PSN. We will directly test the effects of AnkG and Pdzd2-I domains of 1.7 / 1.8 iPA on I Na , I KV 7.2 / 7.3 and PSN action potential generation.

[0226] Experimental design and analysis plan for Aim 1

[0227] Experiment 1A: Determine the effects of 1.7 / 1.8 iPA on endogenous Na V 1.7 and Na V 1.8 intracellular trafficking: This will test the hypothesis that 1.7 / 1.8 iPA can reduce Na V 1.7 and Na V 1.8 channel conductance by reducing channel density in the membrane due to impaired Na V 1.7 and Na V 1.8 intracellular trafficking. Fractionated membrane, cytosolic and nuclear fractions, and total lysates prepared from 1.7 / 1.8 iPA transfected HEK 1.7 and HEK 1.8 cells will be run in parallel using 1.7 NP and mock transfection as controls for 1.7 / 1.8 iPA (GFP fusion, ~35 kDa) as well as endogenous Na V 1.7 and Na V1.8 (approximately 250 kDa) immunoblot. The proportion of target protein bands in total lysates and isolated membrane, cytoplasmic, and nuclear samples was quantified by density assay.

[0228] Experiment 1B: Studying Na V and K V 7.2 / 7.3 Channel Utilization (INa and I) Kv The specificity of 7.2 / 7.3). It has been reported that all Na... V Both AnkG and Pdzd2 binding domains are present in the cells. In the experimental design, 1) the effect of 1.7 / 1.8iPA on HEK Na was quantitatively analyzed using whole-cell voltage clamp. V Na from subtype stable cells (available from Yu's laboratory) V 1.7, Na V 1.8, Na V 1.3, Na V 1.1, Na V 1.5 and Na V 1.6 of I Na Impact (peak I) Na (density and gating characteristics), using 1.7NP and sham transfection as controls; 2) 1.7 / 1.8iPA on I Kv The effect of 7.2 / 7.3 (M current) will be naturally expressed in K. V Recording and quantitative analysis of NG108-15 (NG108) neurons in 7.2 / 7.3 45、46 We have successfully implemented the published plan. 41、47、48 Using K V 7.2 / 7.3 Selective inhibitor XE991 49 M currents in NG108 cells were recorded (Figure 23). Alternatively, stable expression of human K could be obtained from Charles River (catalog number CT6147). V 7.2 / K V 7.3 channel HEK cells (HEK-K) V 7.2 / 7.3); and 3) if I Kv 7.2 / 7.3 are suppressed, using K V 7.2 / 7.3 Specific antibodies and NG108 cells or HEK-K V 7.2 / 7.3 cells, immunoblotting will determine K V 7.2 / 7.3 Membrane integration, as described in Experiment 1A.

[0229] Experiment 1C: Determining the role of the AnkG and / or Pdzd2-I domains in 1.7 / 1.8iPA. Site-directed mutagenesis of key amino acids and / or deletion of the AnkG or Pdzd2-I domains or both will be performed, combined with the voltage-clamp technique described in Experiment 1B, to record Na V and K V 7.2 / 7.3 (based on the results of Experiment 1B), and immunoblotting to detect channel membrane integration (as described in Experiment 1A). The inhibition of Na+ will be defined... V 1.7 / 1.8 is crucial and for K V The key amino acid sequence of 1.7 / 1.8iPA with the least impact in 7.2 / 7.3 was cloned into an AAV shuttle plasmid to generate AAV for studies in Target 2. Glutamic acid (E) and the nearby casein kinase (CK2) phosphorylated serine residue (S) have been defined as Na V -Key sites for AnkG binding 50 .

[0230] Experiment 1D: Validation. Confirmation of the effectiveness of d1.7 / 1.8iPA in HEK1.7, HEK1.8, and ND7 / 23 Na. V 1.8 cells 51,52 The double Na produced in V 1.7 / 1.8 inhibition, and testing of d1.7 / 1.8iPA against optimized human Na+ functionally expressed in ND7 / 23 cells. V 1.9 The role of constructs.

[0231] Analysis Plan: Milestones and Criteria for Determining Success of Objective 1

[0232] Immunoblot assay and quantitative analysis to determine whether the presence of 1.7 / 1.8 iPA inhibits endogenous sodium production. V 1.7 and Na V 1.8 Intracellular transport in HEK1.7 and HEK1.8 cells.

[0233] Using HEK Na V Subtype stable cells, the effect of 1.7 / 1.8iPA on Na+ was measured by electrophysiological (EP) assay. V 1.7, Na V 1.8, Na V 1.1, Na V 1.3, Na V 1.6 and heart Na V 1.5% selectivity. The negative control for these Na... V The subtype had no effect on current suppression (≤5%). The results were validated and tested in Dr. Cummins' laboratory using Na. V 1.9.

[0234] Using NG108 or HEKK V 7.2 / 7.3 Cells, EP to determine if presence of 1.7 / 1.8 iPA affects K V 7.2 / 7.3 Conductance, if so, further determine K V 7.2 / 7.3 Intra-cellular transport, if so, determine if hindered.

[0235] Successful mapping and definition of Na V 1.7 / 1.8 inhibition (>50) and K V 7.2 / 7.3 conductance minimal effect (<20%) key 1.7 / 1.8 iPA amino acid sequence.

[0236] Statistical Analysis: Significance p<0.05, unpaired two-tailed Student's t-test; one-way ANOVA and Tukey's post-hoc test (as applicable). Sample power calculations were performed as appropriate and data analysis was performed in collaboration with the MCW Biostatistics Department 25 .

[0237] Comparison to Na V 1.9, verify dual Na V 1.7 / 1.8 inhibition.

[0238] Objective 1 will be completed within the first grant year (GY) and the corresponding timeline and milestones will be achieved.

[0239] Expected outcomes, potential problems, and alternative strategies for Objective 1. 1.) 1.7 / 1.8 iPA inhibits peak I Na 1.7 and 1.8 density, possibly with a shift in voltage-gated activation (Figure 21). However, these results need to be corrected for possible voltage-clamp errors, defined as the compensated Rs x Imax, which are larger in control cells at large I Na than in cells expressing 1.7 / 1.8 iPA at significantly inhibited I Na . To overcome this problem, we will characterize Na V 1.7 gating kinetics 53、54 in the presence of 1.7 / 1.8 iPA by reducing the bath Na+concentration to 50 mM. 2.) We predict that the AnkG binding domain in 1.7 / 1.8 iPA can be more dominant than Pdzd2-I because Na V 1.7 and Na V 1.8 share three Pdzd2 binding domains, class I in ICL2, class IIx2, and class III. Therefore, we predict that the AnkG domain sequence with CK2 phosphorylation sites 50Will be defined as Na V 1.7 / 1.8 dual blockade of 1.7 / 1.8 iPA. 3.) Multiple Na V Inhibition. Due to sequence homology, d1.7 / 1.8 iPA can also inhibit other conserved Na V , especially TTXs Na V 1.6, 1.3 and 1.1. Since our goal is to develop AAV-mediated, PSN-specific d1.7 / 1.8 iPA expression to achieve multiple nociceptive Na V inhibition, it can be advantageous to selectively deliver AAV to DRG to overflow block other Na V restricted in PSN 55、56 . 4.) A novel Na V 1.9 optimized cDNA will be used to test the effect of d1.7 / 1.8 iPA on Na V 1.9. If necessary, Na V 1.8 knock-out mice isolated PSN to test INa V 1.9 57 .

[0240] Specific goal 2. Determine the biological activity of defined 1.7 / 1.8 iPA expression in sensory neurons

[0241] 1) Does d1.7 / 1.8 iPA inhibit INa conducted by TTXs and TTXr, and K V 7.2 / 7.3 conducted I Kv in PSN? 2) More importantly, does d1.7 / 1.8 iPA expression inhibit (or enhance) PSN excitability?

[0242] Preliminary data, experimental design and analysis plan for goal 2

[0243] Experiment 2A: Preparation of high quality AAV. Prepare high quality AAV carrying d1.7 / 1.8 iPA and control NP (GFP) for AAV-mediated d1.7 / 1.8 iPA expression in rat PSN by DRG injection Figure 25 . 59 .

[0244] Experiment 2B: Determine the effect of PSN-specific d1.7 / 1.8 iPA expression on PSN electrical generation and biological activity. Inject AAV into lumbar (L) 4 / 5 DRG of adult rats by our established technique 25、59、60DRGs will be harvested 4 weeks after AAV injection, then DRG dissociated cultures will be established and neurons expressing dl.7 / 1.8iPA and NP (both GFP-fusion) will be identified under a fluorescence microscope for the following studies: a) whole-cell voltage-clamp recordings to determine the effect of dl.7 / 1.8iPA expression on TTXs and TTXr INa in PSNs 61-63 ; b) the effect of dl.7 / 1.8iPA on PSN-K V 7.2 / 7.3 channel activity compared to control (NP); c) whole-cell current-clamp recordings to analyze the effect of dl.7 / 1.8iPA on PSN AP firing 24、25 ; and d) neurotoxicity of dl.7 / 1.8iPA expression by imaging live PSN neurite growth, which can be used to assess neurotoxicity 64 .

[0245] Experiment 2C: Repeat recordings of TTXs and TTXr I Na and AP firing on PSNs expressing dl.7 / 1.8 will be performed. Virus vectors for infecting dissociated DRG neurons will also be provided.

[0246] Experiment 2D: Assess behavior and safety (MCW Laboratory). Assess the effect of PSN-specific dl.7 / 1.8iPA expression on normal sensory thresholds in rats. This experiment will be coordinated with Experiment 2B. Specifically, after completion of baseline sensory behavioral tests (mechanical stimuli: von Frey (vF) and pin prick, and thermal stimuli: Heat and Cold), AAVs (AAV-dl.7 / 1.8iPA and NP control vectors) will be injected into L4 / L5 DRGs, followed by behavioral assessments at a frequency of once per week for 4 weeks (10 rats per group) as we have previously described 25、65 ; then DRG dissociated cultures for EP studies and DRG / sciatic nerve IHC will be performed. Statistical data will be analyzed to compare sensory thresholds between AAV-dl.7 / 1.8iPA and NP control 65 .

[0247] Analysis Plan: Milestone criteria for judging success of Objective 2

[0248] AAV6-GFPdl.7 / 1.8iPA and AAV6-GFPNP for in vivo studies will be produced. a) AAV titer > 1 x 10 13 GC / ml, and b) high purity (ratio of AAV viral coat proteins Vp1 :Vp2:Vp3 is 1 : 1 : 10, and > 90% of total silver-stained protein on one-dimensional SDS-PAGE gel).

[0249] TTXs and TTXr I in PSNs Na . Determine if d1.7 / 1.8 iPA expression blocks TTXs and TTXr Na V (TTXs and TTXr I Na inhibition > 50%). NP has no significant effect on Na V s.

[0250] K V 7.2 / 7.3. Investigate the inhibitory effect of d1.7 / 1.8 iPA expression on PSN I KV 7.2 / 7.3. We predict that the inhibitory effect of d1.7 / 1.8 iPA will be < 20% compared to controls.

[0251] PSN excitability. Determine the effect of d1.7 / 1.8 iPA expression on PSN AP firing.

[0252] Repetition. Repeat the dual inhibition of TTXs and TTXr INa and APs in Dr. Cummins' lab

[0253] Neurotoxicity. Live cell imaging of PSN neurite growth in culture confirms minimal neurotoxicity of d1.7 / 1.8 iPA expressing PSNs compared to controls.

[0254] Sensory behavior. Determine the effect of AAV-mediated PSN-specific expression of d1.7 / 1.8 iPA on normal sensory thresholds in rats. We predict little change in normal sensory thresholds.

[0255] IHC to determine DRG d1.7 / 1.8 iPA expression, PSN neurotoxicity, and axonal neuropathy.

[0256] Statistical analysis: Significance p < 0.05 using unpaired two-tailed Student's t-test; one-way and two-way ANOVA followed by Tukey's post-hoc test (as appropriate). Sample power calculations as needed and data analysis in collaboration with our statistical consultant (MCW) based on FFS approach 25 .

[0257] Experiments in Objective 2 have been completed in the second GY and the corresponding milestones have been achieved.

[0258] Expected outcomes, potential problems, and alternative strategies for Objective 2. 1.) Inventors have extensive experience with AAV for DRG-targeted analgesia and EP recording various ion channels and PSN excitability in rodent models13,14,16,40-42, so they do not anticipate any technical challenges. 2.) Silencing AnkG in silent neurons reportedly reduces both the inward NaV and outward KV to a lesser extent, while suppressing neuronal excitability38,43,44. Pdzd2 ablation in PSN reduces INa1.835. Therefore, we anticipate that d1.7 / 1.8iPA expression in PSN will suppress AP firing of PSN, supporting the potential of d1.7 / 1.8iPA as an analgesic. 3.) d1.7 / 1.8iPA is expected to exert inhibitory effects on channels by blocking the interaction (PPI) between the channels and AnkG and / or pdzd2 proteins in PSN (not within the scope of this proposal due to R21 budget limitations). The proof-of-concept described in 2.) will help us to target the relevant PPI pathways in future studies. 4.) Safety concerns. Unlike knock-out strategies that eliminate production of the target protein, irreversible AnkG gene silencing can lead to pathological consequences, such as axonal pathology and neurological dysfunction66; inventors anticipate that d1.7 / 1.8iPA will be non-toxic because AAV-mediated selective expression of d1.7 / 1.8iPA in PSN should provide sustained and limited NaV1.7 / 1.8 blockade without eliminating the protein itself, leading to specific functional interference. 5.) AAV production. Our lab has maturely produced high-quality AAV for in vivo DRG delivery, so we do not anticipate any challenges. We use AAV6 serotype because we found that this virus can efficiently transfer genes to all DRG-PSN, including the pain subpopulation, with no significant glial cell transduction25,26,60,67. 6.) Although there are reports that women are more pain-sensitive than men, these proof-of-concept experiments will initially focus on men due to the budget limitations of R21. Gender differences will be fully explored in future expansion studies.

[0259] General methods

[0260] 1) Na V and KV7.2 / 7.3. Current density was calculated from cell capacitance and expressed in pA / pF. AP trains were recorded as we previously described Na and KV7.2 / 7.3. Current density was calculated from cell capacitance and expressed in pA / pF. AP trains were recorded as we previously described 24、25 .

[0261] 3) AAV production 60: AAV vectors will be produced by helper-free, triple-plasmid transfection of 293T cells. Plasmids include: a) AAV expression plasmid; b) pRep2 / Cap6, containing AAV2 replication genes (rep) and capsid genes (cap) from AAV6 (or other); and c) pHelper, which encodes adenovirus helper genes.

[0262] 4) DRG injection 59、65 : Following minimally invasive laminectomy, 2 μΐ of AAV or control sample will be injected into the DRG over 5 minutes via a pulled-tip glass micropipette connected to a microprocessor-controlled syringe. The pipette will be left in place for an additional 5 minutes to minimize extrusion of the injected volume, and then the wound will be sutured closed.

[0263] 5) Behavioral assessment 65、67 : Examinations will be performed in a blinded fashion, including a) brush stimulation, b) cold stimulation, c) von Frey (vF) testing, d) radiant heat stimulation, and e) noxious mechanical stimulation (pin prick).

[0264] 6) Statistical analysis: Data analysis will be assisted by the Department of Biostatistics Consulting Services at our MCW. These consulting services include assistance with the design and analysis of observational studies, assistance with the use of public databases, sample size and power calculations, and data analysis and interpretation.

[0265] References for Example 2: 1 Dib-Hajj, S. D., Cummins, T. R., Black, J. A. & Waxman, S. G. Sodium channels in normal and pathological pain. Annu Rev Neurosci 33, 325-347, doi:10.1146 / annurev-neuro-060909-153234 (2010). 2 Bennett, D. L., Clark, A. J., Huang, J., Waxman, S. G. & Dib-Hajj, S. D. The Role of Voltage-Gated Sodium Channels in Pain Signaling. Physiol Rev 99, 1079-1151, doi:10.1152 / physrev.00052.2017 (2019). 3Dib-Hajj, S. D., Yang, Y., Black, J. A. & Waxman, S. G. The Na(V)1.7 sodium channel: from molecule to man. Nat Rev Neurosci 14, 49-62, doi:10.1038 / nrn3404 (2013). 4Goodwin, G. & McMahon, S. B. The physiological function of different voltage-gated sodium channels in pain. Nat Rev Neurosci 22, 263-274, doi:10.1038 / s41583-021-00444-w (2021). 5Catterall, W. A. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13-25, doi:10.1016 / s0896-6273(00)81133-2 (2000). 6Ovsepian, S. V. & Waxman, S. G. Gene therapy for chronic pain: emerging opportunities in target-rich peripheral nociceptors. Nat Rev Neurosci, doi:10.1038 / s41583-022-00673-7 (2023). 7Emery, E. C., Luiz, A. P. & Wood, J. N. Na v 1.7 and other voltage-gated sodium channels as drug targets for pain relief. Expert Opin Ther Targets 20, 975-983, doi:10.1517 / 14728222.2016.1162295 (2016). 8Rush, A. M., Cummins, T. R. & Waxman, S. G. Multiple sodium channelsand their roles in electrogenesis within dorsal root ganglion neurons. JPhysiol 579, 1-14, doi:10.1113 / jphysiol.2006.121483 (2007). 9Eagles, D. A., Chow, C. Y. & King, G. F. Fifteen years of NaV 1.7channels as an analgesic target: Why has excellent in vitro pharmacology nottranslated into in vivo analgesic efficacy? Br J Pharmacol, doi:10.1111 / bph.15327 (2020). 10King, A. A different path: resh strategies and targets for chronicpain could deliver much-needed replacements for opioid-based painkillers.Nature 573, S4-S6 (2019). 11McDonnell, A., Collins, S., Ali, Z., Iavarone, L., Surujbally, R.,Kirby, S. etc. Efficacy of the Na v 1.7 blocker PF-05089771 in a randomised,placebo-controlled, double-blind clinical study in subjects with painfuldiabetic peripheral neuropathy. Pain 159, 1465-1476, doi:10.1097 / j.pain.0000000000001227 (2018). 12 Siebenga, P., van Amerongen, G., Hay, J. L., McDonnell, A., Gorman, D., Butt, R. et al. Lack of Detection of the Analgesic Properties of PF-05089771, a Selective Na(v) 1.7 Inhibitor, Using a Battery of Pain Models in Healthy Subjects. Clin Transl Sci 13, 318-324, doi:10.1111 / cts.12712 (2020). 13 Kingwell, K. Na v 1.7 withholds its pain potential. Nat Rev Drug Discov, doi:10.1038 / d41573-019-00065-0 (2019). 14 Kingwell, K. Navigating a new path to Na v 1.7 for pain. 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A., Flinspach, M., Gibbs, A., Shih, A. Y., Minassian, N. A., Liu, Y. et al. Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNa v 1.7. J Biol Chem 295, 1315-1327, doi:10.1074 / jbc.RA119.011318 (2020). 20Moreno, A. M., Aleman, F., Catroli, G. F., Hunt, M., Hu, M., Dailamy, A. et al. Long-lasting analgesia via targeted in situ repression of Na v1.7 in mice. Sci Transl Med 13, doi:10.1126 / scitranslmed.aay9056 (2021). 21 Chernikov, I. V., Vlassov, V. V. & Chernolovskaya, E. L. Current Development of siRNA Bioconjugates: From Research to the Clinic. Front Pharmacol 10, 444, doi:10.3389 / fphar.2019.00444 (2019). 22 Setten, R. L., Rossi, J. J. & Han, S. P. The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov 18, 421-446, doi:10.1038 / s41573-019-0017-4 (2019). 23 Crudele, J. M. & Chamberlain, J. S. Cas9 immunity creates challenges for CRISPR gene editing therapies. 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[0266] As will be apparent to those skilled in the art from the foregoing description, various substitutions and modifications can be made to the invention disclosed herein without departing from its scope and spirit. The invention exemplarily described herein can be suitably practiced in the absence of any one or more elements or limitations not specifically disclosed herein. The terms and expressions used herein are illustrative rather than restrictive, and their use does not exclude any equivalent features or portions thereof shown and described. It should be recognized that various modifications are likely to fall within the scope of the invention. Therefore, it should be understood that although the invention has been described through specific embodiments and optional features, those skilled in the art can modify and / or alter the concepts disclosed herein, and such modifications and alterations should be considered within the scope of the invention.

[0267] This document cites numerous patent and non-patent references. All cited references are incorporated herein by reference in their entirety. If a term's definition in this specification differs from its definition in any of the cited references, the definition in this specification shall prevail. Table 1. Candidate Na V 1.7iPAs for I Na 1.7 and hNaV 1.7 (HEK 1.7 cells) influence on gating properties.

[0268] Note: Data from preliminary screening results (Preliminary) Figure 1J ). Nd: not determined. *, ** and *** indicate p<0.05, 0.01 and 0.001 compared to sham, one-way ANOVA and Turkey post-hoc analysis. Table 2. Na v 1.7 iPA1 on I Na specificity and hNa V gating properties of subtypes (1.3, 1.5, 1.6 and 1.8) (HEK Na V 1.3, 1.5, 1.6 and 1.8 stable cell lines)

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Claims

1. A polypeptide aptamer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9, 50, and 52, or a sequence having at least 85% identity to one of SEQ ID NOs: 1-9, 50, and 52.

2. The polypeptide aptamer of claim 1, wherein, the sequence is SEQ ID NO: 1, 4, or 6, or a sequence having at least 85% identity to one of SEQ ID NOs: 1, 4, or 6.

3. The polypeptide aptamer of claim 1, wherein, the sequence is SEQ ID NO: 1, or a sequence having at least 85% identity to SEQ ID NO:

1.

4. The polypeptide aptamer of claim 3, wherein, the sequence is SEQ ID NO:

1.

5. The polypeptide aptamer of claim 1, wherein, the polypeptide aptamer further comprises a detectable marker.

6. The polypeptide aptamer of claim 5, wherein, the detectable marker comprises a fluorescent protein.

7. The polypeptide aptamer of claim 6, wherein, the fluorescent protein comprises a green fluorescent protein.

8. The polypeptide aptamer of claim 1, wherein, the polypeptide aptamer further comprises a linker.

9. The polypeptide aptamer of claim 8, wherein, the linker comprises SEQ ID NO:

10.

10. The polypeptide aptamer of claim 8 or 9, wherein, the polypeptide aptamer further comprises a detectable marker and a linker, wherein the detectable marker, linker, and sequence are a single contiguous peptide sequence, and arranged in order from N-terminus to C-terminus as detectable marker, linker, sequence.

11. The polypeptide aptamer of claim 1, wherein, The polypeptide aptamer binds human Na v 1.7 Channel proteins.

12. A pharmaceutical composition comprising the polypeptide aptamer of claim 1 and a pharmaceutically acceptable carrier or excipient.

13. A polynucleotide comprising a nucleotide sequence encoding the polypeptide aptamer of claim 1.

14. The polynucleotide of claim 13, wherein, the sequence encoding the polypeptide aptamer comprises one of SEQ ID NOs: 11-19.

15. The polynucleotide of claim 13 or 14, further comprising a promoter or enhancer, wherein the nucleotide sequence is operably linked to the promoter or enhancer.

16. The polynucleotide of claim 15, wherein, the promoter is a hybrid human cytomegalovirus (CMV) enhancer / chicken beta-actin (CBA) promoter.

17. The polynucleotide of claim 13, further comprising at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence.

18. An infectious particle comprising the polynucleotide of claim 13.

19. The infectious particle of claim 18, wherein, the infectious particle is a virus.

20. The infectious particle of claim 19, wherein, the virus is an adeno-associated virus (AAV).

21. The infectious particle of claim 20, wherein, the adeno-associated virus (AAV) is an AAV type 6 (AAV6).

22. A pharmaceutical composition comprising the infectious particle of claim 18.

23. A method comprising contacting a cell with the polypeptide aptamer of claim 1 or the infectious particle of claim 18.

24. A method of reducing or inhibiting stimulation of a neuron, the method comprising contacting a neuron with the polypeptide aptamer of claim 1 or the infectious particle of claim 18.

25. The method of any one of claims 23 or 24, wherein, The method results in a peak Na v 1.7 Reduction in current density of at least 50%.

26. The method of any one of claims 23-25, wherein, The method does not change the Na v 1.7 The steady-state inactivation properties of the channel.

27. A method comprising administering to a subject the pharmaceutical composition of claim 22.

28. A method of treating neuropathic pain in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 22 to treat neuropathic pain in the subject.

29. A method of treating pain resulting from a traumatic nerve injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 22 to treat pain resulting from a traumatic nerve injury in the subject.

30. The method of any one of claims 27-29, wherein, The administration includes local delivery of the pharmaceutical composition.

31. The method of claim 30, wherein, The local delivery of the pharmaceutical composition includes delivery to a dorsal root ganglion of the subject.

32. The method of any one of claims 27-31, wherein, The method reduces mechanical or cold sensitization in the subject.

33. The method of any one of claims 27-32, wherein, The subject is a human subject.

34. The method of any one of claims 27-33, wherein, The subject has chronic pain.

35. The method of any one of claims 27 or 29-34, wherein, The subject has neuropathic or neurogenic pain.

36. The method of any one of claims 27-35, wherein, The subject has been diagnosed with osteoarthritis.

37. A cell comprising a heterologous polynucleotide encoding human Na v 1.8 protein.

38. The cell of claim 37, wherein, The human Na v 1.8 The protein comprises the amino acid sequence of SEQ ID NO:

20.

39. The cell of claim 37 or 38, wherein, The nucleic acid sequence encoding human Na v 1.8 The heterologous polynucleotide encoding the protein comprises SEQ ID NO:

21.

40. The cell of claim 37, wherein, The heterologous polynucleotide also encodes a human Na v b2 protein.

41. The cell of claim 40, wherein, The Na v The b2 protein comprises the sequence SEQ ID NO:

24.

42. The cell of claim 37, wherein, The heterologous polynucleotide further comprises a self-cleaving peptide sequence, a furin cleavage site, or both a self-cleaving peptide sequence and a furin cleavage site.

43. The cell of claim 37, wherein, The polynucleotide encoding human Na v 1.8 The polynucleotide of the protein comprises SEQ ID NO:

22.

44. The cell of claim 37, wherein, The cell is a human cell.

45. The cell of claim 44, wherein, The cell is a human embryonic kidney cell.

46. A kit, system, or platform comprising the polypeptide aptamer of any one of claims 1-11 or the polynucleotide of any one of claims 13-17 and reagents for performing an electrophysiology experiment.

47. The kit, system, or platform of claim 46, wherein, The electrophysiology experiment comprises patch clamp electrophysiology.

48. A kit, system, or platform comprising the polypeptide aptamer of any one of claims 1-11 or the polynucleotide of any one of claims 13-17 and, optionally, instructions for use in treating a subject having neuropathic pain.

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