Zinc finger protein transcription factors for suppressing NAV1.7 expression

Abstract

This disclosure provides a zinc finger fusion protein that inhibits the expression of Nav1.7 in the nervous system, and a method for using the protein to treat pain disorders.

Description

Cross - reference to related applications

[0001] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 496,621, entitled "Zinc Finger Protein Transcription Factors For Repressing NAV1.7 Expression", filed on April 17, 2023, the content of which is incorporated herein by reference in its entirety.

Technical Field

[0002] The present disclosure generally relates to zinc finger fusion proteins capable of suppressing NAV1.7 expression.

Background Art

[0003] Peripheral neuropathy is caused by severe damage to peripheral sensory neurons or motor neurons. The damage to neurons is permanent, and thus peripheral neuropathy is often chronic and painful. In particular, neuropathic pain is defined as pain caused by damage or changes in peripheral sensory neurons. There are various heterogeneous pathologies that can affect peripheral sensory neurons, such as surgical trauma, spinal cord injury, complex regional pain syndrome, nerve compression, vascular diseases (e.g., stroke), neurological diseases (e.g., multiple sclerosis, syringomyelia), infectious diseases (e.g., HIV, leprosy, herpes zoster), metabolic syndromes (e.g., diabetes, sarcoidosis, alcohol dependence), drugs (e.g., chemotherapeutic agents), and genetic syndromes (e.g., Fabry disease, erythromelalgia, channelopathy). Neuropathic pain is widely recognized as one of the most difficult - to - manage pain syndromes, and the outcomes are often unsatisfactory. Considering the high unmet medical needs worldwide and the lack of effective treatment for chronic painful neuropathy, there is an urgent need to develop therapeutic agents for the treatment of chronic neuropathic pain.

[0004] Voltage-gated sodium (Nav) channels are major molecular regulators of neuronal excitability in the central and peripheral nervous systems. To date, nine Nav channel isoforms (Nav1.1 to Nav1.9) with diverse roles throughout the body have been identified. Of the nine known Nav channels in humans, Nav1.7 (SCN9A gene), Nav1.8 (SCN10A gene), and Nav1.9 (SCN11A gene) are involved in pain transmission and enhancement of hyperexcitability in nociceptive neurons (small-diameter fiber neurons involved in transmitting pain signals to the brain) (Nau and Leipold, Neuroforum (2017) 23(3):A123-A130).

[0005] Of these three Nav channels, Nav1.7 has recently attracted considerable attention due to its role in various hereditary human pain disorders. Loss-of-function mutations in the SCN9A gene are associated with a clinical condition called congenital insensitivity to pain (CIP), in which patients exhibit complete loss of pain sensation (Cox et al., Nature (2006) 444:894-8). On the other hand, gain-of-function mutations in the SCN9A gene are associated with excessive pain in hereditary erythromelalgia (IEM) and paroxysmal severe pain disorder (PEPD), in which patients present with recurrent severe burning pain, erythema, warmth, and often extremity swelling (Yong et al., J Med Genet. (2004) 41:171-4, Ferreman et al., Neuron (2006) 52:767-74). All of these support the central role of Nav1.7 in the conversion and transmission of pain signals.

[0006] Nav1.7 functions as a molecular switch that modulates the transmission of pain signals to the brain following a noxious stimulus by setting the threshold for action potentials and amplifying small depolarization inputs at nociceptors in the dorsal root ganglia (DRG) (Drenth and Waxman, J Clin Invest. (2007) 117:3603-9). Due to its role in pain perception, Nav1.7 has emerged as a promising target for analgesic development. However, the development of selective Nav1.7 small or large molecule inhibitors has been challenging, partly due to the high amino acid and structural similarities between Nav channels.

[0007] Given the crucial role of Nav1.7 in pain development and the significant unmet medical need for effective treatment of painful neuropathy, there is an urgent need to develop therapeutic agents targeting this protein for the treatment of chronic neuropathic pain. [Overview of the Initiative]

[0008] This disclosure provides a fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain binds to a target sequence of the human SCN9A gene, and the target sequence comprises one of sequence numbers 16-24. In some embodiments, the target sequence is located within 1.5 kb of the transcription start site (TSS) of the SCN9A gene, for example, within 1000 bp upstream of the TSS and / or within 500 bp downstream of the TSS of the SCN9A gene.

[0009] In some embodiments, the ZFP domain comprises one or more (e.g., two, three, four, five, six, or more) zinc fingers, and / or the fusion protein represses the expression of the SCN9A gene by at least about 40, 75, 90, 9%, or 99%, and this repression is optionally detectable by well-known methods, with no or minimal detectable off-target binding or activity (e.g., binding to genes other than the SCN9A gene). Non-limiting examples of DNA-binding recognition helix amino acid sequences of the ZFPs of the present invention are shown in Table 1 or Table 3. In some embodiments, the ZFP domain of the fusion protein comprises some or all of the DNA-binding recognition helix sequences shown in a single row in Table 1 or Table 3.

[0010] In some embodiments, the transcriptional repressor domain of the fusion protein of the present invention includes a KRAB domain, which is optionally derived from human KOX1 protein. In some embodiments, a ZFP domain is linked to the transcriptional repressor domain via a peptide linker. In certain embodiments, the fusion protein contains the amino acid sequence shown in Table 2 or Table 4.

[0011] In some embodiments, the ZFP domain of the fusion protein comprises five or six DNA-binding recognition helix sequences shown in a single row in Table 1 or Table 3, bound to a target sequence shown in Table 1 or Table 3, comprising DNA-binding recognition helix sequences of a ZFP transcription factor shown in Table 1 or Table 3, comprising ligated DNA-binding recognition helix sequences as shown in Table 1, Table 2, Table 3, or Table 4, and / or comprising an amino acid sequence selected from SEQ ID NOs. 25-92.

[0012] In some embodiments, the ZFP domain of the fusion protein includes a recognition helix sequence and / or amino acid sequence shown in a single row in Tables 1-4, for each of the ZFP IDs 97028, 96986, or 96980.

[0013] In another embodiment, the Disclosure provides a nucleic acid construct comprising a coding sequence for a fusion protein of the present invention, wherein the coding sequence is operably linked to a transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is a mammalian promoter that is constitutively active or inducible in a neuron. In some embodiments, the construct is a recombinant viral construct, such as an adeno-associated virus (AAV) construct, an adenovirus construct, or a lentivirus construct.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a nucleic acid construct or a recombinant viral construct of the present invention and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides a host cell comprising a nucleic acid construct or a recombinant viral construct of the present invention. The host cell may be a human cell such as a nerve cell or a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell (iPSC)).

[0016] In another embodiment, the Disclosure provides a method for inhibiting, reducing, or repressing the expression of Nav1.7 in cells, preferably human cells, by targeting a target site / sequence within or near the SCN9A gene using a zinc finger, TALE, or CRISPR repressor, thereby inhibiting, reducing, or repressing gene expression. In some embodiments, the Disclosure provides a method for inhibiting, reducing, or repressing the expression of Nav1.7 in cells, preferably human cells, comprising expressing a zinc finger, TALE, or CRISPR repressor in the cells, wherein the zinc finger, TALE, or CRISPR repressor targets a target site / sequence within or near the SCN9A gene in the cells, thereby inhibiting, reducing, or repressing the expression of the SCN9A gene. In some embodiments, the target sequence is within 1.5 kb of the transcription start site (TSS) of the SCN9A gene. In some embodiments, the target sequence is located within 1000 bp upstream of the TSS of the SCN9A gene and / or within 500 bp downstream of the TSS. In some embodiments, the target sequence includes at least 8 consecutive bp from any of SEQ ID NOs. 16-24. In some embodiments, the target sequence includes at least 8 discontinuous bp from any of SEQ ID NOs. 16-24. In some embodiments, the target sequence includes one of SEQ ID NOs. 16-24. In some embodiments, the target sequence is targeted by a zinc finger repressor (ZFR) as described herein (e.g., Tables 1-4).

[0017] In another embodiment, the Disclosure provides a method for inhibiting the expression of Nav1.7 in human neurons, which optionally involves introducing the fusion protein of the present invention into a neuron (e.g., a nociceptive neuron in a dorsal root ganglion (DRG)) through the introduction of a nucleic acid construct or recombinant virus (e.g., AAV) encoding the fusion protein, thereby inhibiting the expression of Nav1.7 in the neuron. In some embodiments, the human neurons are located in the body of a human patient.

[0018] In one embodiment, the present disclosure provides a method for treating a pain disorder in a patient in need thereof, the method comprising administering to the patient a recombinant AAV or nucleic acid construct as herein. In some embodiments, the recombinant AAV or nucleic acid construct is introduced to the patient via an intravenous, subarachnoid, intracerebral, intraventricular, intracisional, epidural, oral, or intranasal route.

[0019] In the treatment methods described herein, patients have or are at risk of developing peripheral neuropathic pain disorders, such as hereditary erythromelopathy, paroxysmal dysarthria, small fiber neuropathy (e.g., idiopathic small fiber neuropathy, small fiber neuropathy + prediabetes, or diabetic small fiber neuropathy), large fiber neuropathy, trigeminal neuralgia, postherpetic neuralgia, and painful diabetic neuropathy.

[0020] In the therapeutic methods described herein, recombinant AAV may be, for example, AAV9 or a pseudo-AAV derived from AAV9.

[0021] Fusion proteins, nucleic acid constructs, recombinant viruses, or pharmaceutical compositions for use in the therapeutic methods described herein, and the use of fusion proteins, nucleic acid constructs, or recombinant viruses for the manufacture of agents for use in the therapeutic methods are also provided herein.

[0022] Other features, purposes, and advantages of the present invention will become apparent in the detailed description below. However, while embodiments and aspects of the present invention are shown, it should be understood that the detailed description is provided for illustrative purposes only and is not limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawing]

[0023] [Figure 1]Figure 1 illustrates the specific targeting of the SCN9A gene by engineered zinc finger protein transcription factors (ZFP-TFs), such as zinc finger protein repressors (ZF-R or ZFR). Binding of ZFP-TFs to the gene leads to a reduction in SCN9A transcription, which in turn leads to a reduction in SCN9A mRNA and Nav1.7 protein levels. [Figure 2] Figure 2 is a panel of graphs showing the mouse Scn9a repressive activity of 10 ZFP-TFs selected from a library of 288 ZFP-TFs designed to specifically target the mouse Scn9a gene. The y-axis of each graph represents mouse Scn9a mRNA expression normalized by the geometric mean of two housekeeping genes (Atp5b and Eif4a2), assessed 20 hours after transfection with mRNA encoding different ZFP-TFs in Neuro 2A (N2A) mouse neurons. mRNA doses increase from left to right (30, 100, 300 ng). Bars represent the mean of four technical replications, and error bars represent the standard deviation. The numbers below the graphs are the internal reference numbers of the ZFP-TFs. An enlarged version of the titration scale is shown in the lower right of the figure. The blue bars represent the untreated control for each panel. [Figure 3]Figure 3 is a panel of graphs representing the top nine ZFP-TFs selected for target binding assays in mice after several rounds of optimization. For each ZFP-TF panel, the volcano plot / scatter plot of Affymetrix / microarray data on the left shows transcriptome changes in primary mouse cortical neurons 7 days after transduction with ZFP-TF using the AAV6 serotype. Cells were transduced at 3E3 MOI. The numbers shown in red and green indicate the number of downregulated and upregulated off-target genes, respectively, with red circles representing downregulated off-target genes and green circles representing upregulated off-target genes. Data were derived from at least two independent experiments and six biological replications per experiment. Primary mouse cortical neurons do not express the Nav1.7 protein, so there is no repression of the Scn9a gene (represented in the volcano plot). The panel of graphs on the right shows the mouse Scn9a repressive activity of ZFP-TF in Neuro 2A (N2A) mouse neuronal cell lines. The dark blue bars in the bottom set of graphs represent untreated cells. [Figure 4] Figure 4 is a panel of graphs showing ZFP-TF and Scn9a mRNA expression levels after subarachnoid-lumbar (IT-L) delivery of AAV9 encoding the top nine ZFP-TFs in the lumbar, cervical, and thoracic DRGs of C57BL / 6 mice. The y-axis of each graph represents the absolute or normalized mRNA expression level of either ZFP-TF or mouse Scn9a, respectively. Data were collected by combining three pairs of DRGs from each level four weeks after treatment with AAV9 (dose - 2E11vg / mouse). ZFP-TFs are encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. The ZFP-TFs used are shown on the x-axis. Bars represent the mean of values ​​from eight mice, and error bars represent the standard deviation. [Figure 5]Figure 5 is a panel of graphs showing the mRNA expression levels of the neuronal marker NeuN and the neuroinflammatory markers (Iba1 and Gfap) after intrathecal-lumbar (IT-L) delivery of AAV9 encoding the top 9 ZFP-TFs of the lumbar DRG of C57BL / 6 mice. The y-axis of each graph is the normalized mRNA expression level of the marker. Data were collected by combining 3 pairs of DRGs from the lumbar region 4 weeks after treatment with AAV9 (dose - 2E11 vg / mouse). The ZFP-TFs are encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. The ZFP-TFs used are shown on the x-axis. Bars represent the mean of values from 8 mice, and error bars represent the standard deviation. [Figure 6] Figure 6A is a schematic diagram showing the Spared nerve injury (SNI) model. The SNI model is surgically created by damaging 2 of the 3 terminal branches of the sciatic nerve (tibial and common peroneal nerves) and leaving the sural nerve intact. Figure 6B is an overview and schedule table of the pain efficacy test using the SNI mouse model. WT mice were measured for mechanical and cold allodynia before undergoing SNI surgery. Seven days after SNI surgery (day 0), mechanical and cold allodynia were measured again, and mice were injected with AAV9-hSyn1-ZFP-TF at 8E11 vg / mouse. Four weeks later, the pain response was measured again, and the mice were sacrificed, and DRGs were harvested for gene expression and pathological analysis. [Figure 7]Figures 7A-7B are panels of graphs showing (Figure 7A) Scn9a and (Figure 7B) ZFP-TF mRNA expression levels after intrathecal-lumbar (IT-L) delivery of AAV9 encoding four representative ZFP-TFs used in the neuropathic evaluation pain test. ZFP-TF and Scn9a mRNA expression levels were evaluated in DRGs collected from lumbar, cervical, and thoracic levels on day 29 of the experiment from both male and female mice. The y-axis of each graph is the absolute or normalized mRNA expression level of ZFP-TF or mouse Scn9a, respectively. Data were collected by combining three pairs of DRGs from each level after treatment with AAV9-ZFP-TF. ZFP-TF is encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. The ZFP-TFs used are shown on the x-axis. Bars represent the mean of values from six mice, and error bars represent the standard deviation. [Figure 8] Figure 8 shows a microscopic image of the entire lumbar DRG after performing RNAscope in situ hybridization combined with immunohistochemistry to evaluate Scn9a mRNA levels at the single cell level 4 weeks after injection after treatment with AAV9-ZFP-TF (95676). A peripherin-specific antibody was used to identify nociceptors (green), and in addition, RNA probes for Scn9a (white) and ZFP-TF (red) were used in combination. [Figure 9] Figure 9 is a panel of graphs showing the responses of male and female mice to mechanical allodynia at different time points after different treatments in the SNI pain model. Mechanical evoked pain responses were measured on day -7 (healthy mice) and then on day 0 (7 days after SNI surgery). Twenty-eight days after IT-L injection of AAV9-ZFP-TF (94091, 95664, 95676, and 95807), mechanical allodynia was measured again to evaluate efficacy. Gabapentin (GBP) was injected 1 hour before pain measurement on day 28. Dots represent individual mice, n = 7-8, and error bars are SEM [Figure 10]Figure 10 is a panel of graphs showing the responses of male and female mice to cold-induced allodynia at different time points after different treatments in an SNI pain model. Cold-induced pain response was measured on day -7 (healthy mice) and then on day 0 (7 days after SNI surgery). Cold allodynia was measured again 28 days after IT-L injection of AAV9-ZFP-TF (94091, 95664, 95676, and 95807) to evaluate efficacy. Gabapentin (GBP) was injected 1 hour before pain measurement on day 28. Dots represent individual mice, n=7-8, and error bars are SEM. [Figure 11] Figure 11 is a panel of graphs showing the human SCN9A inhibitory activity of 12 ZFP-TFs selected from a library of 384 ZFP-TFs designed to specifically target the human / NHP SCN9A gene in neuronal cell lines. The y-axis of each graph represents human SCN9A mRNA expression normalized by the geometric mean of two housekeeping genes (A tp5b and Eif4a2), assessed 20 hours after transfection with mRNA encoding different ZFP-TFs in SK-N-MC human neurons. mRNA doses increase from left to right (3, 10, 30, 100, 300 ng). Bars represent the mean of four technical replications, and error bars represent the standard deviation. The numbers below the graphs are the internal reference numbers of the ZFP-TFs. An enlarged version of the titration scale is shown in the lower right of the figure. The orange graphs represent the untreated controls in each panel. [Figure 12]Figure 12 is a panel of graphs showing the inhibitory activity of representative ZFP-TFs on human SCN9A in human iPSC-derived GABAergic neurons. The graph on the right of each panel represents the RT-qPCR analysis of SCN9A expression after ZFP-TF treatment. The y-axis represents SCN9A mRNA expression normalized by the geometric mean of two housekeeping genes (A tp5b and Eif4A2), evaluated 32 days after transduction with AAV6 for different ZFP-TFs in human iPSC-derived GABAergic neurons. The amount of AAV6 used is shown in the legend in the lower right, with the AAV6 dose increasing from left to right (MOI of 3E3, 1E4, 3E4, 1E5, and 3E5). Bars represent the mean of four technical replications, and error bars represent the standard deviation. Blue represents the untreated control. The volcano plot / scatter plot of Affymetrix / microarray data on the left shows transcriptome changes in human iPSC-derived GABAergic neurons 32 days after transduction with ZFP-TF using the AAV6 serotype. Cells were transduced at 1E5 MOI. The numbers shown in red and green indicate the number of downregulated and upregulated off-target genes, respectively, with red circles representing downregulated off-target genes and green circles representing upregulated off-target genes. Data were derived from at least two independent experiments and six biological copies per experiment. [Figure 13]Figure 13 is a panel of graphs showing the inhibitory activity of representative ZFP-TFs in human sensory neuron progenitor cells. Expression levels of different SCN genes were measured (SCN1A (Nav1.1), SCN2A (Nav1.2), SCN3A (Nav1.3), SCN8A (Nav1.6), SCN9A (Nav1.7), SCN10a (Nav1.8), and SCN11a (Nav1.9)). The y-axis represents SCNx mRNA expression normalized by the geometric mean of two housekeeping genes (A tp5b and Eif4a2), evaluated for different ZFP-TFs in human sensory neuron progenitor cells 7 days after transduction with AAV6. The amount of AAV6 used is shown in the legend in the lower right, and the dose of AAV6 increases from left to right (MOI of 3E4, 1E5, and 3E5). The bars represent the average of four technical copies, and the error bars represent the standard deviation. [Figure 14] Figure 14 is a panel of graphs showing ZFP-TF mRNA expression levels after subarachnoid-lumbar (IT-L) delivery of AAV9 encoding the top 10 ZFP-TFs targeting the human SCN9A gene in the lumbar, cervical, and thoracic DRGs of C57BL / 6 mice. The y-axis of each graph represents the mRNA expression level of each ZFP-TF. Data were collected by combining three pairs of DRGs from each level four weeks after treatment with AAV9 (dose - 2E11vg / mouse). ZFP-TFs are encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. The ZFP-TFs used are shown on the x-axis. Bars represent the mean of values ​​from five mice, and error bars represent the standard deviation. [Figure 15]Figure 15 is a panel of graphs showing the mRNA expression levels of the neuronal marker NeuN and neuroinflammatory markers (Iba1 and Gfap) after subarachnoid-lumbar (IT-L) delivery of AAV9 encoding the top 10 ZFP-TFs in the lumbar, cervical, and thoracic DRGs of C57BL / 6 mice. The y-axis of each graph represents the normalized mRNA expression level of the marker. Data were collected by combining three pairs of DRGs from each level four weeks after treatment with AAV9 (dose - 2E11VG per mouse). ZFP-TFs are encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. The ZFP-TFs used are shown on the x-axis. Bars represent the mean of values ​​from five mice, and error bars represent the standard deviation. [Figure 16] Figure 16 shows an overview and schedule of non-human primate (NHP) trials evaluating the efficacy and safety of selected ZFP-TFs (97028, 96986, and 96980) targeting the human / NHP SCN9A gene in cynomolgus monkeys. [Figure 17] Figure 17 is a panel of graphs showing ZFP-TF mRNA expression levels after intra-arachnoid-lumbar (IT-L) delivery of AAV9 constructs encoding ZFP-TFs (97028, 96986, and 96980) targeting the human / NHP SCN9A gene. ZFP-TF mRNA expression levels were assessed using NHP DRGs collected from lumbar, cervical, and thoracic levels on day 29 of the experiment. ZFP-TFs are encoded under the human synapsin 1 promoter. The vehicle treatment group was used as a negative control. The ZFP-TFs used are shown on the x-axis. Mean copies / ng RNA are shown, combining DRGs collected from different levels for each region. Mean ± sem [Figure 18]Figure 18 is a panel of graphs showing SCN9A mRNA expression levels after intra-arachnoid-lumbar (IT-L) delivery of AAV9 constructs encoding ZFP-TFs (97028, 96986, and 96980) targeting the human / NHP SCN9A gene. SCN9A mRNA expression levels were assessed using NHP DRGs collected from lumbar, cervical, and thoracic levels on day 29 of the experiment. ZFP-TFs are encoded under the human synapsin 1 promoter. The vehicle treatment group was used as a negative control. The ZFP-TFs used are shown on the x-axis. Mean copies / ng RNA are shown, combining DRGs collected from different levels for each region. Mean ± sem [Figure 19] The panel in Figure 19 represents the total number of histopathological findings at all DRG levels (lumbar, thoracic, and cervical) after intraarachnoid-lumbar (IT-L) delivery of AAV9 constructs encoding ZFP-TFs targeting the human / NHP SCN9A gene (97028, 96986, and 96980). Histopathological findings are classified based on the severity of the incident (minimal, mild, moderate, significant, and severe) and the dose at which they occurred (low dose - 1E11, medium dose - 1E13, and high dose - 9E13). [Figure 20] The panel in Figure 20 represents the histopathological incidents for each DRG level after intra-arachnoid-lumbar (IT-L) delivery of AAV9 constructs encoding ZFP-TFs (97028, 96986, and 96980) targeting the human / NHP SCN9A gene. Histopathological findings are classified based on the severity of the incident (minimal, mild, moderate, significant, and severe) and the dose at which they occurred (low dose - 1E11, medium dose - 1E13, and high dose - 9E13). In addition, data are presented based on the type of histopathological finding, mononuclear cell infiltration (MN), and neurodegeneration. [Modes for carrying out the invention]

[0024] This disclosure provides ZFP domains that target (i.e., bind to DNA sequences) within or near the human SCN9A gene. The ZFP domains described herein may be conjugated or fused to other functional molecules or domains. For example, the ZFP domains of this disclosure may be fused to transcription factors to form zinc finger protein transcription factors (ZFP-TFs). Such ZFP-TF transcription factors may specifically target the human SCN9A gene and repress its transcription to RNA. Reducing Nav1.7 levels by introducing ZFP-TFs into a patient's DRG is expected to inhibit (e.g., reduce or halt) the transmission of pain signals. Therefore, the ZFP-TFs of the present invention may be used for the prevention and / or treatment of pain disorders such as peripheral neuropathy.

[0025] The ZFP-TF approach of the present invention for Nav1.7 inhibition offers several advantages over conventional approaches currently being tested by others, which include (i) administration of antisense oligonucleotides (ASOs) that bind to Nav1.7 mRNA and inhibit its translation, and (ii) administration of immunotherapeutic anti-Nav1.7 antibodies. ZFP-TF may require only a single administration (by introducing a ZFP-TF expression construct into the patient), while ASOs require repeated administrations. In addition, the ZFP-TF approach requires involvement of only two alleles of the SCN9A gene in the genome of each cell. In contrast, ASOs require involvement of multiple copies of SCN9A mRNA in each cell. Furthermore, while the distribution and tropism of ASOs are fixed, the ZFP-TF approach can target different cell types and nervous system regions by modifying the promoter, serotype, and administration route.

[0026] The ZFP-TF approach of the present invention is also advantageous over the antibody approach. The development of selective Nav1.7 antibody inhibitors has been difficult due to the high amino acid and structural similarities between Nav channels. To date, nine Nav channel isoforms (Nav1.1 to Nav1.9) with various roles throughout their bodies have been identified, and they all share very close sequence homology at the protein level, making it difficult to design a specific therapeutic antibody against any one of them.

[0027] I. Targeting of ZFP Domains The ZFP domain of the fusion protein of the present invention specifically binds to a target region within or near the human SCN9A gene. Figure 1 shows the binding of the ZFP domain to the DNA sequence in the SCN9A gene.

[0028] The human SCN9A gene, also known as PN1, Nav1.7, Nav1.7, voltage-gated sodium channel alpha subunit 9, and voltage-gated sodium channel subunit alpha NaV1.7, is approximately 114 kb long and maps to chr2q24.3:166,375,987~166,195,185 (GRCh38.p14). Its nucleotide sequence is available under GenBank accession number ENSG00000169432. The SCN9A gene contains 27 exons. The standard full-length isoform 1 of the human Nav1.7 protein has the following amino acid sequence (UniProt. number Q15858-1):

[0029] (Sequence ID 1)

[0030] The DNA-binding ZFP domain of the ZFP-TF directs the fusion protein to the target region of the SCN9A gene, guiding the transcriptional repression domain of the fusion protein to the target region. The repression domain recruits the transcriptional corepressor complex to modify chromatin into an unacceptable state for transcription by RNA polymerase II. The target region of the ZFP-TF can be any suitable site within or near the SCN9A gene that enables the repression of gene expression. For example, the target region may include or be adjacent to the SCN9A transcription start site (TSS) or SCN9A transcriptional regulatory elements (e.g., promoter, enhancer, RNA polymerase pause site, etc.) (either downstream or upstream). In some embodiments, the transcriptional regulatory element includes a mammalian promoter.

[0031] In some embodiments, the genomic target region is at least 8 bp long. For example, the target region may be 8 bp to 40 bp long, e.g., 12, 15, 16, 17, 18, 19, 20, 21, 24, 27, 30, 33, or 36 bp long. The target sequence may be on the sense strand of a gene or on the antisense strand of a gene. To ensure targeting accuracy by ZFP-TF and reduce off-target binding or activity, the sequence of the selected SCN9A target region preferably has less than 75% homology (e.g., less than 70%, less than 65%, less than 60%, or less than 50%) to the sequence of another gene. In certain embodiments, the target region of the ZFP-TF of the present invention is 12 to 20 bp long (e.g., 12 to 18, 15 to 19, 15, 18, or 19) bp and is located within 1500 bp upstream to 1000 bp downstream of the TSS (e.g., -1000 bp to +1000 bp, +750, or +500 bp). In some embodiments, the target region is within 1000 bp upstream of the TSS. In some embodiments, the target region is within 500 bp downstream of the TSS.

[0032] In some embodiments, the engineered ZFPs of the present invention bind to target sites (i.e., target sequences) shown in a single row in Table 1 or Table 3, and include continuous or discontinuous sequences within these target sites, preferably with little or no detectable off-target binding or activity. In some embodiments, the target sequences include and / or are within the range of any one of SEQ ID NOs: 16-24. In some embodiments, the target sequences include at least eight continuous bp of any one of SEQ ID NOs: 16-24 (i.e., target sequences shown in Table 1 or Table 3). In some embodiments, the target sequences include at least eight discontinuous bp of any one of SEQ ID NOs: 16-24 (i.e., target sequences shown in Table 1 or Table 3). In some embodiments, the target sequences include any one of SEQ ID NOs: 16-24 (i.e., target sequences shown in Table 1 or Table 3).

[0033] In some embodiments, the binding of an engineered ZFP to any of the target sequences disclosed herein results in the suppression of SCN9A gene expression by at least about 40%, about 75%, about 90%, about 95%, or about 99%.

[0034] Other criteria for further evaluating target segments include the existing availability of ZFPs that bind to such segments or related segments, the ease of designing new ZFPs that bind to a given target segment, and / or the risk of off-target binding.

[0035] II. Zinc finger protein domain A "zinc finger protein" or "ZFP" refers to a protein that has a DNA-binding domain stabilized by zinc. ZFPs bind to DNA in a sequence-specific manner. The individual DNA-binding units of a ZFP are called "zinc fingers." Each finger typically consists of seven amino acid residues and contains a DNA-binding "recognition helix" that determines the DNA-binding specificity. A ZFP domain has at least one finger, and each finger binds to 2-4 base pairs of nucleotides, typically 3-4 base pairs of DNA (continuous or discontinuous). Each zinc finger typically contains about 28-30 amino acids and chelated zinc. Engineered ZFPs may have novel binding specificities compared to natural ZFPs. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet sequence is associated with the amino acid sequence of one or more zinc fingers that bind to that particular triplet or quadruplet sequence. For example, U.S. Patents 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,140,081, 6,200,759, 6,453,242, 6,534,261, 6,979,539, and 8,586,526, as well as International Patent Publication WO See the ZFP design methods described in detail in publications 95 / 19431, 96 / 06166, 98 / 53057, 98 / 53058, 98 / 53059, International Publication 98 / 53060, International Publication 98 / 54311, International Publication 00 / 27878, International Publication 01 / 60970, International Publication 01 / 88197, International Publication 02 / 016536, International Publication 02 / 099084, and International Publication 03 / 016496. The ZFP domains described herein may be conjugated or fused to other molecules (e.g., domains), such as proteins.These ZFP fusions may include domains that enable gene activation (e.g., activation domain), gene repression (e.g., repression domain), ligand binding (e.g., ligand binding domain), high-throughput screening (e.g., ligand binding domain), local hypermutation (e.g., activation-induced cytidine deaminase domain), chromatin modification (e.g., histone deacetylase domain), recombination (e.g., recombinase domain), targeted incorporation (e.g., integrase domain), DNA modification (e.g., DNA methyl-transferase domain), base editing (e.g., base editor domain), or targeted DNA cleavage (e.g., nuclease domain). In certain embodiments, the ZFP fusions described herein include transcription factor domains. Examples of engineered ZFP domains are shown in Tables 1 to 4 below.

[0036] Table 1 shows exemplary ZFPs of this disclosure targeting human SCN9A. Capitalized letters indicate the genomic target sequences (i.e., bound sequences) of the DNA-binding recognition helix sequences, shown in a single row for each of the five or six finger ZFPs shown (i.e., F1-F5, F1-F6, or F1 and F3-F6). Exemplary peptide linker sequences shown in Table 5 are also shown between the zinc fingers for each ZFP and between the ZFP domain and the repressor domain (i.e., L1, L2, L3, L4, L5, or L6). Numbers in parentheses indicate the sequence numbers.

[0037] Table 2 shows exemplary polypeptide sequences of ZFP-TFs shown in Table 1. DNA-binding recognition helix sequences are in bold. Zinc finger linkers are underlined, while interdomain linkers are double-underlined.

[0038] Table 3 shows exemplary R→Q (Arg->Gln) variants of representative ZFP-TFs targeting the human SCN9A gene. Capitalized letters indicate the genomic target sequence (i.e., the bound sequence) of the DNA-binding recognition helix sequence, shown in a single row for each of the five or six finger ZFPs shown (i.e., F1-F5 or F1-F6). Table 3 also shows the exemplary peptide linker sequences shown in Table 5 for each ZFP shown (i.e., L1, L2, L3, L4, L5, or L6), between the zinc fingers and between the ZFP domain and the repressor domain. The symbol "^" indicates that the arginine (R) residue at the fourth position upstream of the first amino acid of the shown finger has been changed to glutamine (Q). The numbers in parentheses indicate the sequence number.

[0039] Table 4 shows the exemplary complete protein sequences of the R→Q (Arg->Gln) variants of ZFP-TF shown in Table 3. DNA-binding recognition helix sequences are in bold. Zinc finger linkers are underlined, while interdomain linkers are double-underlined.

[0040] [Table 1]

[0041] [Table 2] JPEG2026517682000004.jpg217159

[0042] [Table 3]

[0043] [Table 4]

[0044] The ZFP domain of the engineered ZFP fusion of the present invention may contain at least one zinc finger (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or more). A ZFP domain with one finger typically recognizes a target site containing three or four nucleotides. A ZFP domain with two fingers typically recognizes a target site containing six or eight nucleotides. A ZFP domain with three fingers typically recognizes a target site containing nine or twelve nucleotides. A ZFP domain with four fingers typically recognizes a target site containing twelve to fifteen nucleotides. A ZFP domain with five fingers typically recognizes a target site containing fifteen to eighteen nucleotides. A ZFP domain with six fingers can recognize a target site containing eighteen to twenty-one nucleotides.

[0045] In some embodiments, the engineered ZFP of the present invention comprises a DNA-binding recognition helix sequence having at least four amino acids from any recognition helix shown in Table 1 or Table 3. In other embodiments, the engineered ZFP of the present invention comprises a DNA-binding recognition helix sequence shown in Table 1 or Table 3. For example, the engineered ZFP may comprise the sequences F1, F2, F3, F4, F5, or F6 shown in Table 1 or Table 3.

[0046] In some embodiments, the engineered ZFP of the present invention comprises two adjacent DNA-binding recognition helix sequences shown in a single row in Table 1 or Table 3. For example, the engineered ZFP may comprise the sequences F1-F2, F2-F3, F3-F4, F4-F5, or F5-F6 shown in a single row in Table 1 or Table 3. In some embodiments, the engineered ZFP may comprise at least four amino acids of F1 and at least four amino acids of F2, at least four amino acids of F2 and at least four amino acids of F3, at least four amino acids of F3 and at least four amino acids of F4 and at least four amino acids of F5, or at least four amino acids of F5 and at least four amino acids of F6.

[0047] In some embodiments, the engineered ZFP of the present invention comprises a DNA-binding recognition helix sequence shown in a single row in Table 1 or Table 3. For example, the engineered ZFP may comprise the sequences F1, F2, F3, F4, F5, and F6 (e.g., F1-F4, F1-F5, or F1-F6) shown in a single row in Table 1 or Table 3. In some embodiments, the engineered ZFP comprises at least four amino acids from each of F1-F4, at least four amino acids from each of F1-F5, or at least four amino acids from each of F1-F6 shown in a single row in Table 1 or Table 3.

[0048] In some embodiments, the engineered ZFPs described herein include the recognition helix and backbone portions of the sequences shown in a single row in Table 2 or Table 4 as the assumed sequences after post-translational modification. For example, post-translational modification may remove an initiating methionine residue from the sequences shown in Table 2 or Table 4.

[0049] The target specificity of a ZFP domain can be improved by mutations in the ZFP backbone sequence, for example, as described in U.S. Patent No. 10,975,393. Backbone mutations include mutations made to residues on the DNA backbone that can interact nonspecifically with phosphates but are not involved in nucleotide target specificity. In some embodiments, these mutations include changing cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, these mutations include changing polar amino acid residues to neutral or nonpolar amino acid residues. In further embodiments, mutations are made at positions (-4), (-5), (-9), and / or (-14) relative to the DNA binding helix. In some embodiments, a zinc finger may contain one or more mutations at positions (-4), (-5), (-9), and / or (-14). In further embodiments, one or more zinc fingers within a multifinger ZFP domain may contain mutations at positions (-4), (-5), (-9), and / or (-14). In some embodiments, the amino acids at positions (-4), (-5), (-9), and / or (-14) (e.g., arginine (R) or lysine (K)) are mutated to alanine (A), leucine (L), serine (S), aspartic acid (D), glutamic acid (E), tyrosine (Y), and / or glutamine (Q). In some embodiments, the R residue at position (-5) is mutated to Q. The symbol "^" in Table 3 indicates that the arginine (R) residue at the fourth position upstream of the first amino acid in the indicated recognition helix is ​​changed to glutamine (Q). In each recognition helix sequence, the positions of the seven DNA-binding amino acids are numbered -1, +1, +2, +3, +4, +5, and +6. Thus, the position of the R-to-Q substitution is numbered (-5).

[0050] In some embodiments, the engineered ZFP of the present invention includes the DNA binding recognition helix sequence and associated backbone mutation shown in Table 3.

[0051] In some embodiments, the engineered ZFPs described herein include the recognition helix and backbone portions of the sequences shown in a single row in Table 4. In some embodiments, the engineered ZFPs described herein include the recognition helix and backbone portions of the sequences shown in a single row in Table 4 as the assumed sequences after post-translational modification. For example, post-translational modification may remove an initiating methionine residue from the sequences shown in Table 4.

[0052] In some embodiments, the ZFP protein of the present invention comprises a recognition helix sequence and / or amino acid sequence shown in a single row in Tables 1-4 for each of the ZFP IDs 93598, 93600, 93591, 93590, 93599, 93662, 93752, 93548, 93845, 93587, 93563, 93582, 96963, 96973, 97028, 96967, 96968, 96980, or 96986.

[0053] III. Zinc Finger Protein Transcription Factors The ZFP domains described herein may be fused to transcription factors. In some embodiments, the fusion protein of the present invention contains a DNA-binding zinc finger protein (ZFP) domain and a transcription factor domain (i.e., ZFP-TF). In some embodiments, the transcription factor may be a transcriptional repressor domain, and the ZFP domain and the repressor domain may be linked to each other by direct peptidyl linkage, a peptide linker, or by dimerization (e.g., via a leucine zipper, a STAT protein N-terminal domain, or an FK506-binding protein). As used herein, “fusion protein” refers to a complex of polypeptides having a covalent domain and polypeptides linked to each other via non-covalent bonds. The transcriptional repressor domain may be linked to the ZFP domain at any suitable position, including the C-terminus or N-terminus of the ZFP domain.

[0054] In some embodiments, the ZFP-TF of the present invention has a K content of less than about 25 nM. D They bind to these targets and repress the transcription of the human SCN9A gene by 20% or more (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more). In some embodiments, two or more of the ZFP-TFs of the present invention are expressed intracellularly to synergistically regulate intracellular SCN9A expression (see, for example, U.S. Patent Application Publications 2020 / 0101133 and 2020 / 0109406). Such synergistic ZFP-TFs may be linked by a 2A linker peptide, e.g., T2A GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 110). Thus, two or more of the ZFP-TFs of the present invention may be used simultaneously in a patient, and the ZFP-TFs bind to different target regions of the SCN9A gene to achieve optimal repression of SCN9A expression.

[0055] In some embodiments, the ZFP-TF of the present invention comprises one or more zinc finger domains. The domains may be linked together via an extensible flexible linker, for example, one domain comprising one or more (e.g., four, five, or six) zinc fingers and another domain comprising an additional one or more (e.g., four, five, or six) zinc fingers. In some embodiments, the linker is a standard interfinger linker, such that the finger array comprises one DNA-binding domain comprising eight, nine, ten, eleven, or twelve or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. For example, two ZFP domains may be linked to a transcriptional repressor TF in a configuration (N-terminus to C-terminus) of ZFP-ZFP-TF, TF-ZFP-ZFP, ZFP-TF-ZFP, or ZFP-TF-ZFP-TF (two ZFP-TF fusion proteins are fused together via a linker).

[0056] In some embodiments, ZFP-TFs are "two-handed," meaning they contain two zinc finger clusters (two ZFP domains) separated by intervening amino acids, so that the two ZFP domains bind to two discontinuous target sites. An example of a two-handed zinc finger-binding protein is SIP1, where a cluster of four zinc fingers is located at the amino terminus of the protein, and a cluster of three fingers is located at the carboxyl terminus (see Remacle et al., EMBO J. (1999) 18(18):5073-84). Each zinc finger cluster in these proteins can bind to a specific target sequence, and the space between the two target sequences can contain many nucleotides.

[0057] In some embodiments, the engineered ZFP-TFs described herein bind to target sites shown in a single row in Table 1 or Table 3, and preferably have no or little detectable off-target binding or activity. Off-target binding can be determined, for example, by measuring the activity of the ZFP-TF in an off-target gene. In some embodiments, the engineered ZFP-TFs described herein include DNA binding recognition helix sequences shown in Table 1 or Table 3. In some embodiments, the engineered ZFP-TFs described herein include two adjacent DNA binding recognition helix sequences shown in a single row in Table 1 or Table 3. In some embodiments, the engineered ZFP-TFs described herein include DNA binding recognition helix sequences shown in a single row in Table 1 or Table 3.

[0058] A. Transcriptional repressor domain The ZFP-TF of the present invention comprises an engineered ZFP domain as described herein and one or more transcriptional repressor domains that attenuate the transcriptional activity of the SCN9A gene. The one or more engineered ZFP domains and the one or more transcriptional repressor domains may be linked by a flexible linker. Non-exclusive examples of transcriptional repressor domains include the KRAB domain of KOX1 or ZIM3 (or any other KRAB domain containing a protein, see, e.g., Alerasool et al., Nature Methods (2020) 17:1093-6), KAP-1, MAD, FKHR, EGR-1, ERD, SID, TGF-beta-inducible early gene (TIEG), v-ERB-A, MBD2, MBD3, TRa, histone methyltransferase, histone deacetylase (HDAC), nuclear hormone receptors (e.g., estrogen receptor or thyroid hormone receptor), members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2. See, for example, Bird et al., Cell (1999) 99:451-4; Tyler et al., Cell (1999) 99:443-6; Knoepfler et al., Cell (1999) 99:447-50; and Robertson et al., Nature Genet. (2000) 25:338-42. Additional exemplary repressive domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al., Plant Cell (1996) 8:305-21; and Wu et al., Plant J. (2000) 22:19-27.

[0059] In some embodiments, the transcriptional repressor domain includes a Kruppel-associated box (KRAB) domain derived from the KOX1 protein. In some embodiments, the KOX1 protein is human. In some embodiments, the transcriptional repressor domain includes a sequence from the Kruppel-associated box (KRAB) domain of human zinc finger protein 10 / KOX1 (ZNF10 / KOX1) (e.g., GenBank number NM_015394.4). An exemplary KRAB domain sequence is as follows:

[0060] DAKSLTAWSR TLVTFKDVFV DFTREEWKLL DTAQQIVYRN VMLENYKNLV SLGYQLTKPD VILRLEKGEE PWLVEREIHQ ETHPDSETAF EIKSSV (Sequence 2)

[0061] This KRAB sequence variant can also be used, as long as it has the same or similar transcriptional repressor function.

[0062] B. Peptide Linker The ZFP-TFZFP domain and transcriptional repressor domain of the present invention, and / or zinc fingers within the ZFP domain, may be linked via a peptide linker, for example, a non-cleavable peptide linker of about 5 to 50 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids). Preferred linkers are typically flexible amino acid subsequences synthesized as recombinant fusion proteins. In some embodiments, the zinc fingers are linked so that there are no gaps between the linked module target subsites in the target nucleic acid molecule. In other embodiments, the zinc fingers are linked by a linker designed to allow the linked modules to bind to the target site with a gap of 1, 2, or 3 base pairs between the linked module target subsites in the target nucleic acid molecule. See, for example, U.S. Patent No. 8,772,453.

[0063] In some embodiments, the peptide linker is 3 to 20 amino acid residues long and is rich in G and / or S. Non-limiting examples of such linkers include G4S type linkers, i.e., linkers containing one or more (e.g., two, three, or four) GGGGS (SEQ ID NO: 15) motifs, or variants of that motif (such as those having one, two, or three amino acid insertions, deletions, and substitutions to that motif).

[0064] Linker design methods and exemplary linkers that can be used to ligate ZFP domains and the transcriptional repressor domains of the ZFP-TF of the present invention, and / or zinc fingers within the ZFP domain, are described in U.S. Patents 6,479,626, 7,851,216, 8,772,453, 9,394,531, 9,567,609, and 10,724,020, and PCT Publications WO1999 / 045132, WO2001 / 053480, WO2009 / 154686, WO2011 / 139349, WO2015 / 031619, and WO2017 / 136049. The proteins described herein may include any combination of appropriate linkers.

[0065] Non-restrictive examples of linkers include DGGGS (SEQ ID NO: 3), TGEKP (SEQ ID NO: 4), LRQKDGERP (SEQ ID NO: 5), GGRR (SEQ ID NO: 6), GGRRGGGS (SEQ ID NO: 7), LRKRDGERP (SEQ ID NO: 8), LRQKDGGGSERP (SEQ ID NO: 9), LRQKD(G3S)2ERP (SEQ ID NO: 10), TGSQKP (SEQ ID NO: 11), LRQKDAARGS (SEQ ID NO: 13), and LRQKDAARGSGG (SEQ ID NO: 14). Additional exemplary linkers for linking zinc fingers and / or domains are listed in Table 5. The inter-finger linkers listed in Table 5 include a portion of the backbone sequence, e.g., FQ or FA.

[0066] Table 5 shows exemplary alternative peptide linkers that can be used to link zinc finger amino acid sequences and / or ZFP sequences and functional domain sequences shown in Table 1 or Table 3.

[0067] [Table 5]

[0068] In some embodiments, for example, an engineered ZFP, such as those described herein, includes two adjacent DNA-binding recognition helix sequences linked together as shown in a single row in Table 1 or Table 3. For example, an engineered ZFP may include sequences F1-F2, F2-F3, F3-F4, F4-F5, or F5-F6 as shown in a single row in Table 1 or Table 3. In other embodiments, one or more different linkers may be used from the same linker category.

[0069] In some embodiments, for example, an engineered ZFP, as described herein, includes a ligated DNA-binding recognition helix sequence as shown in a single row in Table 1 or Table 3. For example, an engineered ZFP may include the ligated sequences F1-F4, F1-F5, or F1-F6 as shown in a single row in Table 1 or Table 3. In other embodiments, one or more different linkers may be used from the same linker category.

[0070] In some embodiments, for example, an engineered ZFP as described herein includes a recognition helix and linker portion of the sequence shown in a single row in Table 1 or Table 3. In other embodiments, one or more different linkers may be used from the same linker category. In some embodiments, an engineered ZFP as described herein includes a recognition helix, backbone, and linker portion of the sequence shown in a single row in Table 2 or Table 4. In other embodiments, one or more different linkers may be used from the same linker category. In some embodiments, an engineered ZFP-TF as described herein includes an amino acid sequence shown in a single row in Table 2 or Table 4. In some embodiments, an engineered ZFP or ZFP-TF as described herein includes a recognition helix, backbone, and linker portion of the sequence shown in a single row in Table 2 or Table 4 as the assumed sequence after post-translational modification. In some embodiments, an engineered ZFP or ZFP-TF as described herein includes an amino acid sequence such as the one shown in a single row in Table 2 or Table 4 as the assumed sequence after post-translational modification. For example, post-translational modifications may remove the initiating methionine residue from the sequences shown in Table 2 or Table 4.

[0071] IV. Expression of ZFP-TF The ZFP-TFs of this disclosure may be introduced into a patient via a nucleic acid molecule encoding them. The nucleic acid molecule may be an RNA molecule or a cDNA molecule. The nucleic acid may be introduced into the patient's brain, spinal cord, or cerebrospinal fluid (CSF) through injection of a composition comprising a lipid:nucleic acid complex (e.g., liposomes). Alternatively, the ZFP-TFs may be introduced into a patient via a nucleic acid expression vector containing a sequence encoding the ZFP-TFs. The expression vector may contain expression regulatory sequences such as promoters, enhancers, transcription signal sequences, and transcription termination sequences that enable the expression of the coding sequence for ZFP-TFs in cells of the nervous system. In some embodiments, the expression vector is retained in the cell as a stable episome. In other embodiments, the expression vector is integrated into the cell's genome.

[0072] In some embodiments, the promoter expressing the ZFP-TF described herein includes a constitutive promoter. In some embodiments, the constitutive promoter is constitutively active in neurons. In some embodiments, the promoter includes an inducible promoter. In some embodiments, the inducible promoter is inducible in neurons.

[0073] In some embodiments, the promoter on the vector for directing ZFP-TF expression in the DRG is either a constitutively active promoter or an inducible promoter. Suitable promoters include, but are not limited to, the Roussarcoma virus (RSV) long-terminal repeat (LTR) promoter (optionally containing an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing a CMV enhancer), the CMV early promoter, the Simian virus 40 (SV40) promoter, the dihydrofolate reductase (DHFR) promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the EFLα promoter, and the Moloney's mouse leukemia virus (MoMLV). This includes LTR, creatine kinase system (CK6) promoter, transthyretin promoter (TTR), thymidine kinase (TK) promoter, tetracycline-responsive promoter (TRE), hepatitis B virus (HBV) promoter, human α1-antitrypsin (hAAT) promoter, chimeric liver-specific promoter (LSP), factor E2 (E2F) promoter, human telomerase reverse transcriptase (hTERT) promoter, CMV enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene (1991) 108(2):193-9), and RU-486-responsive promoter. Neuron-specific promoters may also be used, such as the synapsin I promoter, calcium / calmodulin-dependent protein kinase II (CamKII) promoter, methyl CpG-binding protein 2 (MeCP2) promoter, choline acetyltransferase (ChAT) promoter, carbidine (Calb) promoter, CAMKII promoter, PrP promoter, GFAP promoter, or engineered or innate promoters that restrict expression in neurons and glial cells. In addition, promoters may include one or more autoregulatory elements so that ZFP-TFs can bind to their own expression levels and suppress their expression levels to a predetermined threshold. See U.S. Patent No. 9,624,498.

[0074] Any method for introducing the nucleotide sequence into cells may be used, but is not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, liposomes combined with nuclear localization signals, innate liposomes (e.g., exosomes), or viral transduction.

[0075] Viral transduction may be used for in vivo delivery of expression vectors. Various viral vectors known in the art, such as vaccinia vectors, adenovirus vectors, lentiviral vectors, poxyvirus vectors, adeno-associated virus (AAV) vectors, retroviral vectors, herpes simplex virus (HSV) vectors, and hybrid viral vectors, can be adapted by those skilled in the art for use in this disclosure. In some embodiments, the viral vector used herein is a recombinant AAV (rAAV) vector. AAV vectors are particularly suitable for neural gene delivery because they infect both divided and non-divided cells, exist as stable episomal structures for long-term expression, and have very low immunogenicity (Hadaczek et al., Mol Ther. (2010) 18:1458-61, Zais, et al., Gene Ther. (2008) 15:808-16). Any suitable AAV serotype may be used. For example, AAV may be a novel or pseudotype such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAV.PHP.B, AAV.PHP.eB, or AAVrh10, or AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9, or a serotype that is a variant or derivative of one of the AAV serotypes listed herein (i.e., AAV derived from multiple serotypes, e.g., rAAV may have an AAV2 inverted terminal repeat (ITR) and AAV in its genome). The expression vector comprises capsids 8, 5, 6, or 9. In some embodiments, the expression vector is an AAV viral vector introduced into target human cells by recombinant AAV virions containing the construct in their genome, the virion genome having ITR sequences at both ends, thereby enabling the production of AAV virions in production systems such as insect cell / baculovirus production systems or mammalian cell production systems. The AAV may be engineered so that its capsid protein has reduced immunogenicity or enhanced transduction ability in humans or non-human primates. In some embodiments, AAV9 is used.The viral vectors described herein may be produced using methods known in the art. Any suitable permissible cell or packaging cell type can be used to produce viral particles. For example, mammalian (e.g., 293) or insect (e.g., sf9) cells may be used as packaging cell lines.

[0076] V. Pharmaceutical Uses The ZFP-TF of the present invention can be used to treat peripheral neuropathic pain disorders such as hereditary erythromelalgia (IEM), paroxysmal severe pain disorder (PEPD), small fiber neuropathy (idiopathic small fiber neuropathy, small fiber neuropathy + prediabetes, diabetic small fiber neuropathy), large fiber neuropathy, trigeminal neuralgia, postherpetic neuralgia, and painful diabetic neuropathy. This disclosure provides a method for treating neurological disorders (e.g., pain disorders such as hereditary or chronic pain disorders) in subjects such as human patients in need, the method comprising introducing a therapeutically effective amount of ZFP-TF (e.g., an rAAV vector expressing it) (e.g., an amount that allows for sufficient suppression of SCN9A expression to treat the neurological disorder) into the nervous system of the subject. The term “treatment” includes symptom relief, prevention of symptom onset, slowing of disease progression, improvement of quality of life, and extension of survival.

[0077] This disclosure provides a pharmaceutical composition comprising a viral vector such as rAAV in which a recombinant genome contains an expression cassette for ZFP-TF. The pharmaceutical composition (e.g., artificial cerebrospinal fluid or CSF) may further comprise a pharmaceutically acceptable carrier such as water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin. In addition, the composition may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, stabilizers, or other reagents that enhance the efficacy of the pharmaceutical composition. The pharmaceutical composition may also comprise a delivery vehicle such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles.

[0078] Because the ZFP-TFs of this disclosure are regulated by neuron-specific promoters (e.g., synapsin promoters), ZFP-TFs will be expressed in any neuronal subtype of the central or peripheral nervous system. Nav1.7 is expressed along the length of DRG neurons, which extend peripherally and cross the blood-brain barrier (BBB) ​​centrally to form synapses with spinal secondary neurons. The role of Nav1.7 in these primary nociceptive neurons is established as essential and non-redundant in the conversion and transmission of pain signals following noxious stimuli. The target cells of this therapy are nociceptor neurons in the DRG. The ZFP-TFs of this disclosure suppress the expression of Nav1.7 in nociceptor neurons in the DRG, which prevents or reduces the transmission of pain signals to the brain, thereby stopping the brain from perceiving pain.

[0079] The DRG can be reached via intrahippocampal injection, intracerebral injection, intracisional (ICM) injection, or more commonly, intraparenchymal injection, intraventricular (ICV) injection, intra-articular (IT) injection, or intravenous (IV) injection. Other routes of administration include, but are not limited to, intraventricular, intranasal, oral, or intraocular administration. In some embodiments, the viral vector can reach the DRG after direct administration into the cerebrospinal fluid (CSF), for example, via intra-articular and / or intracerebral injection, or intracisional or intraventricular injection. In other embodiments, the viral vector is delivered directly to the target region via epidural administration.

[0080] For example, the pharmaceutical composition may be administered to the patient via intraventricular administration into the ventricular region of the patient's forebrain, such as the right ventricle, left ventricle, third ventricle, or fourth ventricle. The pharmaceutical composition may also be administered to the patient via intra-articular (IT) administration, such as by injection into the subarachnoid space of the spinal cord. The pharmaceutical composition may also be administered to the patient via intracisterna magnum (ICM) administration, such as by injection into the subarachnoid space filled with cerebrospinal fluid (CSF) between the cerebellum and the dorsal medulla oblongata.

[0081] Delivery of rAAV to the target can be achieved, for example, by intravenous administration. In certain cases, rAAV (e.g., 10 10 ~10 15 In some cases, it is desirable to deliver Vg) locally to the DRG, spinal cord, cerebrospinal fluid (CSF), nerve cells, glial cells, meninges, astrocytes, oligodendrocytes, microglia, interstitial space, etc. AAV can be delivered by needle, catheter, or related device using neurosurgical techniques known in the art, such as stereotactic injection (see, for example, Stein et al., J Vir. (1999) 73:3424-9, Davidson et al., PNAS (2000) 97:3428-32, Davidson et al., Nat Genet. (1993) 3:219-223, and Alisky and Davidson, Hum Gene Ther. (2000) 11:2315-29).

[0082] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the implementation or testing of this disclosure. In case of any conflict, including definitions, this specification shall prevail. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Throughout this specification and its embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” shall be understood to include the integer or group of integers described, but not to exclude any other integer or group of integers. All publications and other references referenced herein are incorporated by reference as if each individual reference were specifically and individually indicated to be incorporated by reference as if it were incorporated by reference as if it were incorporated by reference as if it were specifically and individually indicated. Numerous documents are referenced herein, but this reference does not constitute an endorsement that any of these documents constitutes common general knowledge in the art. As used herein, the terms “approximately” or “about” applied to one or more reference values ​​refer to values ​​similar to the given reference values. In certain embodiments, unless otherwise stated or evident from the context, the terms refer to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (greater than or less than) the given reference value.

[0083] According to this disclosure, any back reference in a dependent claim is intended as a brief statement that any combination of the claims indicated by such reference is directly and clearly disclosed. Any molecule or vector disclosed herein may be used in any of the therapeutic methods herein, and the individual being treated is as defined anywhere herein. Furthermore, the headers herein are provided for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0084] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Examples]

[0085] Example 1: Screening of anti-SCN9A ZFP-TF To identify human / NHP and mouse-specific ZFP-TFs that suppress the expression of the human SCN9A gene or its mouse homologous gene Scn9a, we designed libraries of ZFP-TFs targeting the human SCN9A or mouse Scn9a gene across the upstream 1000 bp to downstream 500 bp of the TSS and screened them for SCN9A repressive activity. In this study, the KRAB domain sequence (SEQ ID NO: 2) was used as a transcriptional repressor and fused to the C-terminus of the ZFP domain.

[0086] Human ZFP-TF screening was performed using the SK-N-MC human neuroepithelial cell line, while mouse ZFP-TF screening was performed using the mouse Neuro2A (N2A) neural crest-derived cell line. Both SK-N-MC and N2A cells express the SCN9A gene at high levels, making them suitable for testing ZFP-TFs that reduce SCN9A expression. Each cell line was cultured in a tissue culture flask until confluence. Cells were seeded onto 96-well plates and resuspended in Amaxa® SF solution. Cells were then mixed with ZFP-TF mRNA (three doses for mouse: 30, 100, 300 ng, and five doses for human: 3, 10, 30, 100, 300 ng) and transferred to Amaxa® shuttle plate wells. Cells were transfected using the Amaxa® Nucleofector® device (Lonza, program CM-137). Eagle MEM cell medium was added to each well of the plate. Cells were transferred to 96-well tissue culture plates and incubated at 37°C for 20 hours. Cells were then lysed and reverse transcription was performed using the C2CT kit according to the manufacturer's instructions. SCN9A expression levels were measured using TaqMan quantitative polymerase chain reaction (qPCR) and normalized to the geometric mean of the expression levels of the housekeeping genes ATP5B and EIF4A2. Mock transfection and transfection with ZFP-TFs known not to target SCN9A were used as negative controls. The maximum suppression achieved was 100%, but ZFP-TFs that suppressed SCN9A to a lower degree (e.g., approximately 90%, 75%, or 50% at the highest dose, or in some cases no suppression) were also identified. Figure 2 shows mouse screening data, and Figure 11 shows human ZFP-TF screening data.

[0087] Example 2: AAV production of exemplary ZFP-TF Recombinant adeno-associated virus (rAAV) vectors were generated by triple transfection. Briefly, HEK293 cells were seeded in a 10-layer CellSTACK® chamber (Corning, Acton, Massachusetts) and grown to 80% density for 3 days. Three plasmids, (i) an AAV helper plasmid containing the Rep and Cap genes, (ii) an adenovirus helper plasmid containing the adenovirus helper gene, and (iii) a transgene plasmid containing a sequence packaged adjacent to the AAV2 inverted terminal repeat, were transfected into the cells using calcium phosphate. After 3 days, the cells were harvested. The cells were then lysed by 3 rounds of freeze / thaw cycles, and cell residue was removed by centrifugation. rAAV was precipitated using polyethylene glycol. After resuspending, the virus was purified by overnight ultracentrifugation on a cesium chloride density gradient. The virus was formulated by dialysis and then filtered. After adjusting the titer (viral genome / ml) of all AAV batches by dilution with PBS + 0.001% Pluronic F-68, the AAV was dispensed into single-use doses and stored at -80°C until use. Refreezing was not performed after thawing.

[0088] Example 3: Human iPSC-derived neuron culture and ZFP-TF AAV transduction Human iPSC-derived GABAergic neurons were purchased from Cellular Dynamics International and seeded at a density of 40,000 cells per well on poly-L-ornithine and laminin-coated 96-well plates, maintained according to the manufacturer's instructions. Cells were infected with AAV expressing the desired ZFP-TF at the indicated MOI 48 hours after seeding and maintained for up to 32 days (50-75% medium change every 3-5 days). Cells were harvested at the end of the experimental period, RNA was isolated, and RT-qPCR was performed for gene expression analysis. Figure 12 shows the Nav1.7 expression levels in iPSC-derived GABAergic neuron cultures after ZFP-TF AAV injection. For microarray analysis, cells were transfected with 1E5 VG / cell 48 hours after seeding and harvested 32 days after viral transfection.

[0089] Example 4: Culture of human iPSC-derived sensory neuron progenitor cells and ZFP-TF AAV infection Human iPSC-derived sensory neuron progenitor cells were purchased from Axol Biosciences. The cells were cultured and differentiated on poly-D-lysine (PDL) coated plates according to the manufacturer's protocol. The cells were differentiated for 10 days and then infected with AAV expressing the desired ZFP-TF at the indicated MOI. Cells were harvested on day 18, RNA was isolated, and RT-qPCR was performed for gene expression analysis. Figure 13 shows the expression levels of SCN9A and other Nav channels (SCN1A, SCN2A, SCN3A, SCN8A, SCN10A, and SCN11A) in progenitor sensory neurons after infection with AAV6-ZFP-TF.

[0090] Example 5: Primary mouse neuron culture and ZFP-TF AAV infection Primary mouse cortical neurons (MCNs) were purchased from Gibco. Cells were seeded at 50,000 or 200,000 cells / well on poly-D-lysine (PDL) coated 96-well or 24-well plates, respectively, and maintained in Gibco Neurobasal medium containing GlutaMAX® I supplement, B27 supplement, and penicillin / streptomycin, according to the manufacturer's specifications. 48 hours after seeding (in DIV2), 50,000 cells / well in 96-well plates were infected with AAV-ZFP at the indicated MOI and harvested 7 days later (in DIV9; with 50% medium changes every 3-4 days), followed by RNA isolation and gene expression analysis by RT-qPCR. Alternatively, 200,000 neurons / well in 24-well plates were treated with 1E5 VG / cell to ensure 100% transduction and similarly processed for microarray analysis in DIV9.

[0091] Example 6: Off-target activity of anti-SCN9A ZFP-TF To evaluate the off-target effects of ZFP-TF on global gene expression, microarray (Clariom S Array) experiments were performed on total RNA isolated from human iPSC-derived neurons and primary mouse cortical neurons treated with AAV encoding representative ZFP-TFs. Microarray analysis was performed according to the manufacturer's protocol (Thermo Fisher Scientific), and assay results were analyzed using TAC4 software. Apparent off-target effects with FDR-corrected p-values ​​≤ 0.05 were further investigated using RT-qPCR analysis. Figure 3 shows the microarray results for ZFP-TF targeting the mouse Scn9a gene in primary mouse cortical neurons. Figure 12 shows the microarray results for ZFP-TF targeting the human SCN9A gene in human iPSC-derived GABAergic neurons transduced with ZFP-TF.

[0092] Example 7: In vivo proof-of-concept study in wild-type mice The pharmacological activity of AAV9-delivered ZFP-TF targeting the mouse Scn9a gene was evaluated in C57BL / 6 (WT) mouse DRG neurons after intra-arthritis-Lung (IT-L) injection. Transgene expression (ZFP-TF) and Scn9a gene expression levels were evaluated in lumbar, cervical, and thoracic DRGs (Figure 4).

[0093] The expression of neuroinflammatory markers (Gfap, Iba1) and neuronal loss (NeuN) markers was also evaluated in the lumbar DRG (highest transgene expression and highest Scn9a suppression) (Figure 5). To collect tissue for subsequent analysis, mice were perfused with PBS, DRG was extracted, and dissected on ice. The tissue was then rapidly frozen in liquid nitrogen and maintained at -80°C until analysis. Reverse transcription was performed using the High-Capacity RT Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Expression levels of ZFP-TF, Scn9a, Gfap, Iba1, and NeuN were measured using TaqMan quantitative polymerase chain reaction (qPCR). Gene expression levels were normalized by the geometric mean of the expression levels of the housekeeping genes Atp5b and Eif4a2. Vehicle treatment (VEH) was used as a negative control. Overall, no neuronal loss or increase in neuroinflammatory markers was observed.

[0094] The results are consistent with in vitro Scn9a suppression data obtained from mouse neuron cultures (N2A cells). Scn9a mRNA levels were significantly reduced in the lumbar DRG after treatment with any of the ZFP-TFs. While all test substances resulted in minimal suppression of Scn9a mRNA in the cervical DRG (30% suppression compared to the vehicle group), Scn9a mRNA expression levels remained unchanged in the thoracic DRG (compared to the vehicle group) for any of the test substances. This was expected based on the absolute measurements of ZFP-TF mRNA levels in each DRG level. The lumbar DRG contained approximately 100 times more ZFP-TF than the cervical and thoracic DRGs.

[0095] Neuroinflammation and transcriptional levels of neuronal markers were also evaluated as secondary endpoints. No significant upregulation of neuroinflammation markers (Iba1 and Gfap) was observed, but minimal downregulation of the neuronal marker (NeuN) was observed after intraarachnoid injection of ZFP-TF at a dose of 2E+11vg / mouse.

[0096] Example 8: Spared Nerve Injury (SNI) Neuropathic Pain Model Spared Nerve Injury (SNI) is the most widely validated mouse model of neuropathic pain and is considered the gold standard for evaluating neuropathic pain in rodents. This model is surgically generated by severing the tibial and peroneal nerves while preserving the sural nerve (Figure 6A). Damage to the tibial and peroneal nerves results in marked hypersensitivity in the lateral region of the foot innervated by the preserved sural nerve. The advantages of the SNI model are the robustness of the response and the fact that it does not require expert microsurgical skills.

[0097] Example 9: In vivo evaluation of pain behavior effectiveness in a mouse model of neuropathic pain. The overall objective of this study was to evaluate the pharmacological activity and behavioral efficacy of Scn9a-targeting AAV9-delivered ZFP-TFs administered via IT-L in male and female C57BL / 6(WT) DRG mice using an SNI model. The purpose of this study was to evaluate the pharmacology and efficacy of four representative ZFP-TFs that suppress the Scn9a gene and reduce pain in a mouse neuropathic pain model. The main points of evaluation were von Frey filament (mechanical allodynia) and cold (acetone) stimulation (cold allodynia). All mice underwent SNI surgery on day 7. Seven days after SNI surgery, the response of mice to mechanical stimulation (von Frey filament) and cold stimulation was evaluated, and mice that responded were assigned to their respective groups, as illustrated in the schematic diagram in Figure 6B. This day was designated as day 1 of the study, and a single bolus injection (10 μL) was administered to the mice on this day. Intra-arachnoid (IT) administration was performed without surgical intervention, directly by injection into the lumbar (L) region L3 / L4 or L4 / L5. The groups were assigned as follows: Group 1 (sham surgery, no procedure), Group 2 (vehicle control), Group 3 (gabapentin [GBP], 50 mg / kg, a common antineuropathic agent), Group 4 (ZFP-TF 94091, 6.81 E+11 vg / mouse), Group 5 (ZFP-TF 95664, 8.12 E+11 vg / mouse), Group 6 (ZFP-TF 95676, 7.99 E+11 vg / mouse), and Group 7 (ZFP-TF 95807, 3.36 E+11 vg / mouse). The sham surgery group underwent surgery, but no nerves were severed (only skin opening and closing), and neither the test substance nor a vehicle was administered. GBP was administered intraperitoneally (IP) approximately one hour before each evaluation trial. The dose of ZFP-TF was based on the maximum achievable concentration, given manufacturing capacity and dose volume constraints.

[0098] Scn9a mRNA expression levels were assessed using DRGs collected from the lumbar, cervical, and thoracic levels of both male and female mice on day 29 of the experiment (Figure 7A). Data were collected by combining three pairs of DRGs from each level after treatment with AAV9-ZFP-TF. ZFP-TF was encoded under the human synapsin 1 promoter. Vehicle treatment was used as a negative control. Significant reductions in Scn9a were observed at different DRG levels in both male and female mice.

[0099] Several DRGs from the lumbar region of mice treated with ZFP-TF 95676 were paraffin-embedded and processed for single-cell analysis to evaluate ZFP-TF expression and single-cell level Scn9a suppression in nociceptors. Scn9a mRNA levels were assessed using RNAscope in situ hybridization combined with immunohistochemistry, and nociceptors were identified using peripheralin-specific antibodies. High levels of Scn9a were observed in peripheralin-positive cells of lumbar DRGs isolated from the vehicle group. A significant decrease in Scn9a mRNA transcripts was observed in peripheralin-positive / ZFP-TF-positive cells from 95676-treated mice, indicating that AAV9-95676 significantly reduces Scn9a transcript expression in nociceptors (Figure 8).

[0100] The response of mice to mechanical allodynia was evaluated 28 days after injection of a representative ZFP-TF following SNI surgery (Figure 9). By day 28 post-administration, the increase in mechanical threshold (MT) was at the same level as GBP for both ZFP-TF 95664 and ZFP-TF 95676 and was statistically significant compared to the control group. In females treated with ZFP-TF 94091, there was no significant difference in MT compared to the control group. Although administered at approximately half the dose compared to other ZFP-TFs, 95807 resulted in higher MT values ​​than the control. The MT response in males was similar to that of females for ZFP-TF 94091, 95664, and 95676. However, unlike females, males treated with ZFP-TF 95807, which received half the dose, showed an increase in MT at day 28, similar to GBP, and these differences were statistically significant compared to the control group.

[0101] The response of mice to cold allodynia was evaluated 28 days after representative ZFP-TF injection following SNI surgery (Figure 10). Female mice treated with ZFP-TF also responded to cold allodynia, measured in seconds by paw withdrawal latency (PWL). Similar to mechanical allodynia, females treated with ZFP-TF 95664 and 95676 showed the highest PWL response by day 28, which was statistically significant compared to the control group and at the same level as GBP. The response of males to cold allodynia appeared to vary greatly across all groups, including the GBP and control groups. However, the trend of response appeared similar to that of mechanical allodynia, with the highest PWL response observed in males treated with ZFP-TF 95664, followed by ZFP-TF 95676 and 95807.

[0102] Example 10: In vivo evaluation of potential endogenous neurotoxicity in wild-type mice The endogenous toxicity of AAV9-delivered ZFP-TF targeting the human SCN9A gene was evaluated in C57BL / 6 (WT) mouse DRG neurons after intra-articular (IT) injection. Transgene expression (ZFP-TF) was assessed in lumbar, cervical, and thoracic DRGs. Expression of neuroinflammatory markers (Gfap, Iba1) and neuropathy (NeuN) markers was also evaluated in lumbar, cervical, and thoracic DRGs. Figure 15 shows that no significant upregulation of neuroinflammatory markers (Iba1 and Gfap) or downregulation of neuronal markers (NeuN) was observed after intra-articular injection of ZFP-TF at a dose of 2E+11vg / mouse.

[0103] Example 11: In vivo evaluation of the target involvement and safety of a ZFP-TF targeting human SCN9A in non-human primates (NHPs). Because ZFP-TF can bind to homologous sequences within the NHP SCN9A TSS, the pharmacology and target specificity of AAV9-delivered ZFP-TF were evaluated in cynomolgus monkeys in pharmacological and toxicity studies for a 4-week dose-ranging (DRF) after a single IT-L administration at three dose levels: 1E12 (low dose), 1E13 (medium dose), and 9E13 (high dose) vg / mouse (Figure 16). DRGs were collected from the lumbar (L5 and L1), thoracic (T7, T6, and T1), and cervical (C6 and C5) levels. The expression levels of ZFP-TF and SCN9A were assessed using RT-qPCR. AAV9-delivered ZFP-TF was expressed in a dose-dependent manner in all DRG regions analyzed (Figure 17). ZFP-TF96986 and 96980 inhibited SCN9A transcription in each region by 40–60% at a total tissue level compared to the vehicle group, across a 100-fold dose range, while ZFP-TF97028 showed a lower level of inhibition (Figure 18). Overall, data collected from NHP confirmed the pharmacology and specificity of AAV9-ZFR-F in this preclinical species.

[0104] All NHPs survived until scheduled necropsy. There were no abnormal clinical signs or behaviors, body weight, clinicopathology (hematology, chemistry, coagulation), or necropsy findings that could be attributed to treatment with ZFP-TF. The liver panel was included in the clinicopathological evaluation and showed no abnormalities. Histopathological diagnoses were performed on the adrenal glands, brain, DRG, epididymis, heart, kidneys, small and large intestines, liver, lungs, ovaries, pancreas, sciatic nerve, skeletal muscle, spinal cord (lumbar, thoracic, cervical), spleen, stomach, testes, thymus, and trigeminal ganglia. In all three NHPs, minimal to mild abnormalities were observed only in the DRG at all levels. Findings included mononuclear cell infiltration (MN) and neurodegeneration (ND), with most findings being minimal, and only three mild events observed in the lumbar DRG in the high-dose group (Figure 19). Most findings were in NHPs treated with ZFP-TF 96980. Minimal MN associated with AAV9-96980 was observed in the cervical DRG in the low-dose (67%), medium-dose (33%), and high-dose (67%) groups. Minimal MN was observed in the thoracic DRG in the low-dose (33%), medium-dose (67%), and high-dose (33%) groups, and in the lumbar DRG in the low-dose (100%) and medium-dose (33%) groups. Mild MN associated with 96980 was observed only in the lumbar DRG of all three NHPs in the high-dose group (Figure 20). For NHPs treated with AAV9-96980, minimal MN was observed in the lumbar DRG of both the lumbar and thoracic DRGs, in the low-dose (33%), medium-dose (33%), and high-dose (66%) groups. In general, with the exception of mild neuropathy (MN) findings in the lumbar DRG in the high-dose group, almost all findings were of minimal severity (Figure 20). Despite microscopic findings in various peripheral tissues, no abnormal behaviors indicating neurological dysfunction were observed in any of the animals.

Claims

1. A fusion protein comprising a zinc finger protein (ZFP) domain and a transcriptional repressor domain, wherein the ZFP domain binds to a target sequence of the human SCN9A gene, and the target sequence comprises one of sequence numbers 16 to 24.

2. The fusion protein according to claim 1, wherein the target sequence is located within 1.5 kb of the transcription start site (TSS) of the SCN9A gene.

3. The fusion protein according to claim 2, wherein the target sequence is located within 1000 bp upstream of the TSS of the SCN9A gene and / or within 500 bp downstream of the TSS.

4. The fusion protein according to any one of claims 1 to 3, wherein the fusion protein suppresses the expression of the SCN9A gene by at least about 40%, 75%, 90%, 95%, or 99%, and has no or minimal detectable off-target binding or activity.

5. The fusion protein according to any one of claims 1 to 4, wherein the transcriptional repressor domain comprises a KRAB domain, and the KRAB domain is optionally derived from human KOX1 protein.

6. The fusion protein according to any one of claims 1 to 5, wherein the ZFP domain is linked to the transcriptional repressor domain via a peptide linker.

7. The fusion protein according to any one of claims 1 to 6, wherein the ZFP domain includes a DNA-binding recognition helix sequence shown in Table 1 or Table 3.

8. The fusion protein according to any one of claims 1 to 7, wherein the ZFP domain includes the DNA binding recognition helix sequence shown in a single row of Table 1 or Table 3.

9. The ZFP domain of the fusion protein It contains five or six zinc fingers, It binds to the target sequence shown in Table 1 or Table 3, The ZFP transcription factor includes the DNA binding recognition helix sequence shown in Table 1 or Table 3, The DNA-binding recognition helix sequence is linked as shown in Table 1, Table 2, Table 3, or Table 4, and / or A fusion protein according to any one of claims 1 to 8, comprising an amino acid sequence selected from the amino acid sequences shown in Table 1 or Table 3 (i.e., SEQ ID NOs. 25 to 71).

10. A nucleic acid construct comprising a coding sequence of a fusion protein according to any one of claims 1 to 9, wherein the coding sequence is operably linked to a transcription regulatory element.

11. The nucleic acid construct according to claim 10, wherein the transcriptional regulatory element is a mammalian promoter that is constitutively active or inducible in neurons, and the construct is optionally a recombinant viral construct.

12. A recombinant virus comprising the nucleic acid construct according to claim 10 or claim 11.

13. The recombinant virus according to claim 12, wherein the recombinant virus is an adeno-associated virus vector, an adenovirus vector, or a lentivirus vector.

14. A pharmaceutical composition comprising a nucleic acid construct according to claim 10 or claim 11, or a recombinant virus according to claim 12 or claim 13, and a pharmaceutically acceptable carrier.

15. A host cell comprising a nucleic acid construct according to claim 10 or claim 11, or a recombinant virus according to claim 12 or claim 13.

16. The host cell according to claim 15, wherein the host cell is a human cell.

17. The host cell according to claim 15 or claim 16, wherein the host cell is a nerve cell or a pluripotent stem cell, and the stem cell is optionally an embryonic stem cell or an induced pluripotent stem cell (iPSC).

18. A method for inhibiting the expression of Nav1.7 in human neurons, comprising optionally introducing a fusion protein according to any one of claims 1 to 9 into the neurons through the introduction of a nucleic acid construct according to claim 10 or claim 11, a recombinant virus according to claim 12 or claim 13, or a pharmaceutical composition according to claim 14, thereby inhibiting the expression of Nav1.7 in the neurons.

19. The method according to claim 18, wherein the human neuron is a nociceptive neuron of a dorsal root ganglion (DRG).

20. The method according to claim 18 or claim 19, comprising introducing a recombinant adeno-associated virus (AAV) expressing the fusion protein into the cells.

21. The method according to any one of claims 18 to 20, wherein the human neuron is located in the body of a human patient.

22. A method for treating a pain disorder in a patient requiring such treatment, comprising administering to the patient a recombinant AAV or nucleic acid construct encoding a fusion protein according to any one of claims 1 to 9.

23. The method according to claim 22, wherein the recombinant AAV or nucleic acid construct is introduced into the patient via an intravenous, subarachnoid, intracerebral, intraventricular, intracisional, epidural, or intranasal route.

24. The aforementioned patient, voluntarily, Hereditary erythromelalgia, Paroxysmal severe pain disorder, Small fiber neuropathy, and optionally, idiopathic small fiber neuropathy, small fiber neuropathy + prediabetes, or diabetic small fiber neuropathy. Large-diameter fiber neuropathy, Trigeminal neuralgia, Postherpetic neuralgia, and The method according to any one of claims 21 to 23, wherein the person has or is at risk of developing a peripheral neuropathic pain disorder selected from painful diabetic neuropathy.

25. The method according to any one of claims 20 to 24, wherein the recombinant AAV is AAV9 or a pseudotype AAV derived from AAV9.

26. A method for inhibiting the expression of Nav1.7 in cells, preferably human cells, comprising targeting a target site / sequence within or near the SCN9A gene using a zinc finger, TALE, or CRISPR repressor, thereby inhibiting the expression of the said gene.

27. The method according to claim 26, wherein the target sequence is located within 1.5 kb of the transcription start site (TSS) of the SCN9A gene.

28. The method according to claim 26 or claim 27, wherein the target sequence is located within 1000 bp upstream of the TSS of the SCN9A gene and / or within 500 bp downstream of the TSS.

29. The method according to any one of claims 26 to 28, wherein the target sequence includes i) at least eight consecutive bp of any of sequence numbers 16 to 24, ii) at least eight discontinuous bp of any of sequence numbers 16 to 24, or iii) one of sequence numbers 16 to 24.

30. A fusion protein according to any one of claims 1 to 9, a nucleic acid construct according to claim 10 or claim 11, a recombinant virus according to claim 12 or claim 13, or a pharmaceutical composition according to claim 14, for use in the method according to any one of claims 18 to 29.

31. Use of a fusion protein according to any one of claims 1 to 9, or a nucleic acid construct according to claim 10 or claim 11, or a recombinant virus according to claim 12 or 13, for the production of a drug for use in the method according to any one of claims 18 to 29.

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