Deprivation of human pluripotent stem cell-derived trpv1+, mrgprx1+ and scn9a+ sensory neurons and their functional characterization

Abstract

A method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with at least one of nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition, the method comprising introducing into a population of human pluripotent stem cells (hPSCs) a composition comprising at least one site-directed nuclease targeting a site within the target gene, and at least one nucleic acid comprising a nucleotide sequence encoding at least one screenable, selectable marker that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene, wherein the target site is located downstream of the open reading frame of the target gene, and wherein the site-directed nuclease cleaves the target site of the target gene and the nucleic acid encoding the screenable, selectable marker is inserted at the target site; covering said population of hPSCs under an extracellular matrix comprising at least one neuronal differentiation driver to produce a sensory committed neural crest population; contacting said sensory committed neural crest population with at least one neuronal differentiation driver and at least one neurotrophic factor to produce a population of early sensory neurons (SNs); and contacting said population of early SNs with at least one neurotrophic factor to produce a population of mature hPSC-SNs expressing at least one of SN marker or one pan neuronal marker; isolating the cells expressing the at least one screenable, selectable marker, wherein said population of mature hPSC-SNs expressed said one target gene associated with nociceptive pain, chronic pain, pruriception a nociceptive- or pruriceptive- mediated condition, and wherein said population of mature hPSC-SNs responds to a nociceptive and pruriceptive stimulus.

Description

DEPRIVATION OF HUMAN PLURIPOTENT STEM CELL-DERIVED TRPV1+, MRGPRX1+ AND SCN9A+ SENSORY NEURONS AND THEIR FUNCTIONAL CHARACTERIZATIONCROSS-REFERENCE

[0001] This application claims priority to U.S. provisional patent application no. 63 / 599,832, filed November 16, 2023, the contents of which are incorporated herein in their entirety.BACKGROUND1. Technical Field

[0002] The present disclosure relates to nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition and method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition , a method of altering the expression or function of at least one target gene associated nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition in a sensory neuron (SN), a method for screening for a drug candidate that modulates one or more MRGPRX1, TRPV1 or SCN9A-mediated conditions, and a method for identifying a therapeutic target associated with nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive-mediated condition.2. Introduction

[0003] Most drugs on the market for pain and itch sensation have undesired side effects because their targets exist both inside and outside the pain pathways. Despite the urgent need for new painkillers without side effects, it is important to develop a new humanized model system. Here, we report the generation of purifiable populations of human transient receptor potential cation channel subfamily V member 1 (TRPV1)+, sodium voltage-gated channel alpha subunit 9 (SCN9A)+, and Mas-related G protein-coupled receptor X1 (MRGPRX1)+ neurons from hPSCs.SUMMARY

[0004] Additional features and advantages of the disclosure will be set forth in the description that follows, and in part will be understood from the description, or can be learned by practice of the herein disclosed principles. The features and advantages of the disclosure can be realized and obtained by means of the disclosed methods and combinations particularly pointed out in the appended claims. These and other features of the disclosure will become more fully apparent from the following description and appended claims, or can be learned by the practice of the principles set forth herein.

[0005] The methods and combinations as disclosed herein can include method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with at least one of nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition.

[0006] The methods and combinations as disclosed herein can include methods of altering the expression or function of at least one target gene associated with nociceptive pain, chronic pain, pruriception and a nociceptive / pruriceptive condition in a sensory neuron (SN).

[0007] The methods and combinations as disclosed herein can include methods of producing an enriched population of mature human pluripotent stem cell-derived nociceptive sensory neurons (hPSC-NSNs) expressing CD200 gene (CD200+), and methods for screening for a drug candidate that modulates one or more MRGPRX1, TRPV1 or SCN9A-mediated conditions.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1 A- ID illustrate generatinghPSC reporter lines;

[0009] FIGS. 2A-2E illustrate characterizing the transcriptional landscape of MRGPRX1::GFP, SCN9A::GFP, and TRPV1 ::GFP sensory neurons;

[0010] FIGS. 3A-3F illustrate purifiable and excitable populations of hPSC-derived MRGPRX1+ and TRPV1+ sensory neurons;

[0011] FIGS. 4A-4L illustrate FACS-purified TRPV1+, SCN9A+, and MRGPRX1+ hPSC- derived sensory neurons responding to various pain- and itch-inducing ligands;

[0012] FIGS. 5A-5S illustrate in vitro optimization and functional validation of candidate CRISPR sgRNAs to enable TRPV1 knockout, and SCN9A knockout;

[0013] FIGS. 6A-6I. illustrate single cell RNA-seq data and CD200 as a nociceptor marker;

[0014] FIGS.7A-7DD illustrate a KCNQ2 as a gene therapy target gene conferring robust pain- resilience effects in hPSC-derived sensory neurons and the KCNQ2 gene therapy efficacy in a rodent model resulting in bone remodeling;

[0015] FIGS. 8A-8R. illustrate CD200+ sensory neuron injection to intercept pain to treat localized pain and improve the bone remodeling by injecting hPSC-derived pain-sensing neurons;

[0016] FIGS. 9A-9K. illustrate in vivo survival and function of SCN9A+, TRPV1+, and MRGPRX1+ neurons.DETAILED DESCRIPTION

[0017] The following description of some embodiments of the invention is not intended to limit the invention to these embodiments, but rather to enable a person skilled in the art to make and use this invention. All references are incorporated by reference.

[0018] Most drugs on the market for pain and itch sensation have undesired side effects because their targets exist both inside and outside the pain pathways. Despite the urgent need for new painkillers without side effects, it is important to develop a new humanized model system. To address this issue, some embodiments of the current invention provide a generation of human transient receptor potential cation channel subfamily V member 1 (TRPV1)+, Mas-related G protein-coupled receptor XI (MRGPRX1)+ and sodium voltage-gated channel alpha subunit 9 (SCN9A)+ neurons from human pluripotent stem cells (hPSCs), followed by detailed functional characterization. Our functional analysis studies provide different levels of polymodality among the three human sensory neuron subtypes. Additionally, when being injected into rat host, the human sensory neurons can survive and rapidly generate extended axons.

[0019] It is estimated that over one-third of the world’s population suffers from persistent or recurrent pain / itching caused by neurological disorders, diabetes, car accident, war injuries, chemotherapy, etc. (7). Most drugs on the market for pain sensation have undesired side effects because their targets exist both inside and outside the pain pathways, which made the nociceptive and pruriceptive neurons as attractive targets for novel pharmacological strategies.

[0020] Recent studies have revealed that nociceptive and pruriceptive neurons encompass a remarkably heterogeneous population that entails various transductions of noxious stimulithrough numerous ion channels, membrane receptors, signaling molecules and neuropeptides and neurotransmitters, which hamper detailed understanding of human nociception as well as analgesic drug development. The transient receptor potential cation channel subfamily V member 1 (TRPV1) and sodium voltage-gated channel alpha subunit 9 (SCN9A, encoding Nav1.7) are promising targets of novel pain inhibitors(2-8), mainly because of its restricted expression in primary nociceptive neurons. However, the functional properties of human TRPV1 and SCN9A cannot be fully inferred from rodent analogs, owing to cross-species variations in their agonist activity and receptor function. While there have been innovations and advances in modelling and measuring pain in animals(9), it is also important to acquire TRPV1 and SCN9A expressing human nociceptive neurons.

[0021] Because persistent pain is often primed with peripheral pathological conditions, such as tissue inflammation and nerve injury, and its maintenance is also attributable to peripheral neuronal sensitization(10, 11), development of pain-specific treatments would greatly benefit from the identification of novel targets specifically expressed in pain pathways, especially those targets on nociceptive primary sensory neurons(12). One potential target is the Mas-related G protein-coupled receptor (MRGPR). MRGPRs comprise a family of orphan G protein-coupled receptors (GPCRs) and include many genes in humans and rodents(13-16), but their physiological functions are only partially known. However, the functional properties of MRGPRX1 cannot be fully inferred from MRGPRC owing to cross-species variation in MRGPR agonist activity and receptor function. Although mouse MrgprC shares sequence homology with human MRGPRX1, it is becoming clear that human MRGPRXl has binding and pharmacological profiles distinct from the binding and pharmacological profiles of rodent MRGPRC(17). For example, although the sequence of bovine adrenal medulla (BAM) peptide is conserved from rodents to humans(18) (e.g., BAM8-22 activates both MRGPRXl and MRGPRC)(15, 19), most MRGPRXl -selective agonists have weak or no agonist activity at MRGPRC and do not affect rodent pain behavior(20). Again, such species difference is a long- lasting barrier for developing a new pain-killer drug. One can consider human dorsal root ganglia (DRG) tissue can be an alternative, however, it is very difficult to isolate from donors at regular basis and the quantity of human DRG is also very low.

[0022] Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have emerged as a complementary system toanimal models, because the directed specification of hESCs / hiPSCs is especially useful for generating certain cell types; they produce large quantities of otherwise extremely rare cell populations such as human nociceptive and pruriceptive neurons(4, 21-24). Previous studies(25- 37) have shown that the sensory neuron population can be readily generated from hESCs / hiPSCs, however, the resulting sensory neurons are highly heterogeneous and the subtypes of sensory neurons were not sufficiently defined, purified yet or characterized in detail. Therefore, it is important to study the functions of human sensory neurons in genetically defined neuronal subsets. In this study, we generated hPSC-derived TRPV1::GFP+, MRGPRX1::GFP+ and SCN9A::GFP+ sensory neurons, followed by their functional characterization and in vivo transplantation studies.

[0023] Some embodiments of the invention disclosed herein build a foundation to develop novel drug candidates as well as a potential cell therapy strategy for managing acute pain, chronic pain, and itch conditions.

[0024] While various embodiments of the present invention are described below, it should be understood that they are presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the described illustrative embodiments but should instead be defined only in accordance with the following claims and their equivalents.

[0025] FIGS. 1A-1D illustrate generating hPSC reporter lines. As illustrated, to isolate molecularly defined subsets from hPSC-derived sensory neurons (hPSC-SNs), MRGPRX1 : :GFP, TRPV1::GFP, and SCN9A::GFP reporter cell lines were generated using the CRISPR-Cas9 system. FIG.l A illustrate schematic of CRISPR-mediated knock-in construct for MRGPRX1::GFP, TRPV1 ::GFP, and SCN9A::GFP cell lines. FIG. 1A illustrates a schematic of CRISPR-mediated knock-in construct for MRGPRX1 ::GFP, TRPV1 ::GFP, and SCN9A::GFP cell lines. For each locus, the stop codon was replaced with 2A-eGFP-PGK-Puro gene cassette (FIG. 1 A). Each clone was separately differentiated into sensory neurons and detected for GFP expression using fluorescence-activated cell sorting (FACS) after 40 DIVs (FIG. 1 B) using an optimized protocol (FIG. 1 C). Timeline of sensory neuron differentiation protocol is illustrated in FIG. 1 C. Representative morphology of hPSC-derived sensory neurons expressing BRN3A (general sensory neuron marker) and TUJ1 (pan neuronal marker) is illustrated in FIG. 1 D.Three clones were confirmed and kept for each line: MRGPRX1 : :GFP (2A4, 2A8, 2D10), TRPV1::GFP (3B1, 3B4, 3B17), and SCN9A::GFP (3A5, 3A8, 3A19).

[0026] FIGS. 2A-2E illustrate characterizing the transcriptional landscape of MRGPRX1 ::GFP, SCN9A::GFP, and TRPV1::GFP sensory neurons. Differentially expressed genes were independently analyzed in each lineage comparison using Gene Set Enrichment Analysis (GSEA) with gene sets from Molecular Signatures Database (MSigDB). To uncover molecular differences among the three subsets of hPSC-SNs, we performed bulk RNA-seq (NextSeq) to identify differentially expressed genes (DEGs) in SCN9A::GFP+ vs. SCN9A::GFP-, TRPV1::GFP+ vs. TRPV1::GFP-, and MRGPRX1::GFP+ vs. MRGPRX1::GFP- hPSC-SNs (70 DIVs). Using gene set enrichment analysis (GSEA), we first analyzed the differential enrichment of membrane receptors and channels (FIG. 2 A), as they serve important roles in regulating neuronal excitability and firing rate, making them promising novel therapeutic targets in the treatment of chronic pain. Several voltage-gated potassium channels (e.g., KCNQ2 and KCNG1), active transporters (e.g., SLC10A4 and ATP2B4), and GPCRs (e.g., ADCYAP1R1) are specifically enriched in TRPV1 ::GFP+ and SCN9A::GFP+ but not MRGPRX1 ::GFP+ neurons, suggesting that they play specific roles in the activity or function of nociceptors. On the other hand, some GPCRs (e.g., PRNP, DNM3, UTS2R, HTR2C, HTR1E, HTR2A, HTR1D, and NPY1R) and voltage-gated calcium channels (e.g., CACNG1, CACNA1S, CACNG8, and CACNA2D3) are specifically enriched in MRGPRX1::GFP+ but not SCN9A::GFP+ or TRPV1 ::GFP+ neurons, suggesting for their putative roles in mediating itch. GSEA also reveals differentially regulated pathways and transcription factors in SCN9A::GFP+, TRPV1 ::GFP+, and MRGPRX1 ::GFP+ hPSC-SNs (FIG. 2 B). The differential distributions of enriched membrane receptors and channels in SCN9A::GFP+, TRPV1::GFP+, and MRGPRX1 ::GFP+ hPSC-SNs highlight transcriptomic differences among the three subsets of sensory neurons (Fig. 2 C-E). MRGPRX1 ::GFP+ hPSC-SNs show the most diverse receptor expression profile, suggesting that they are comprised of a highly homogeneous population of neurons (FIG. 2E).

[0027] FIGS. 3A-3F illustrate purifiable and excitable populations ofhPSC-derived MRGPRX1+, TRPVl+sensory neurons. To test their functionality, the excitability and purity of sorted MRGPRX1::GFP+, TRPVl::GFP+cells were examined by their Ca2+ transient (Calbryte 590) in response to different pain- and itch-triggering stimuli. MRGPRX1+ neurons displayed significantly heightened response to BAM 8-22 (10μM) compared to MRGPRX1- neurons (FIG.3A-3B). Specifically, the purity of sorted MRGPRX1+ and TRPV1+ neurons was tested by quantifying the percentage of activated neurons in response to BAM 8-22 (10μM) and capsaicin (10μM) when compared with MRGPRX1- and TRPV1- populations, respectively (FIG. 3A-3F). Specifically, MRGPRX1+ neurons (FIG. 3B) displayed significantly heightened response to BAM 8-22 (10μM) compared to MRGPRX1 - neurons (FIG. 3A) and TRPV1+ neurons (FIG. 3E) displayed significantly heightened response to capsaicin (10μM) compared to TRPV1- neurons (FIG. 3D).

[0028] FIGS. 4A-4L. illustrate FACS-purified TRPV1+, SCN9A+, and MRGPRX1+ hPSC- derived sensory neurons responding to various pain- and itch-inducing ligands. We examined the polymodality of TRPV1+ (FIG. 4A-4E), SCN9A+ (FIG. 4F-4H), and MRGPRX1+ (FIG. 41- 4K) neurons. The TRPV1+ (FIG. 4A-4E), SCN9A+ (FIG. 4F-4H), and MRGPRX1+ (FIG. 41- 4K) neurons were characterized by their response profile to BAM 8-22 (10μM), capsaicin (10μM), mustard oil (100μM), menthol (100μM), histamine (50μM), and ATP (50μM). For example, many of TRPV1+ neurons are responding to mustard oil as well as capsaicin (FIG. 4E) whereas very small numbers of TRPV1+ neurons are responding to menthol (FIG. 4C). In FIG. 4L, we summarized the percentages of each neuron that is responding to each stimulus. Our hPSC-derived MRGPRXl+, TRPV1+, and SCN9A+ neurons displayed distinct response profiles with a preferential response to BAM 8-22, capsaicin, and histamine, respectively, while there is distinctive heterogeneity in terms of their polymodal responsiveness to additional stimuli.FIGS. 5A-5S. illustrate in vitro optimization and functional validation of candidate CRISPR sgRNAs to enable TRPV 1 knockout, and SCN9 A knockout. With the goal of developing gene therapy products that provide robust, DRG-specific suppression of chronic pain, we explored the use of CRISPR- induced AAV-delivered loss-of-function modulation and silencing (CALMS) to independently target three cation channels in the afferent pain pathway that are vital for the generation of action potentials and the maintenance of resting membrane potential. To establish robust pain silencing in DRG neurons, we first aimed at silencing two major pain targets TRPV1 and SCN9A. We systematically selected 200+ CRISPR sgRNA constructs to enable TRPV1 or SCN9A knockout (FIG. 5A-5Q). Using the most widely used in silico designing tools (CRISPick, CRISPOR, and Cas-Designer), we then selected top 10 candidate sgRNAs targeting early exons of TRPV1 or SCN9A (FIG. 5A, 5K). Three sgRNAs (red) targeting TRPV1 or SCN9A were picked based on three parameters, including high on-target efficiency scores, low off-target effects, and high out-of-frame scores, and were transfected into FACS-purified TRPV1::GFP+ hPSC-SNs (70 DIVs) or SCN9A::GFP+ hPSC-SNs (70 DIVs) using lipofectamine-mediated delivery. We next empiricallycompared the knockout efficiencies of TRPVI -knockout sgRNAs (TRPVl -sgRNAs) and SCN9A-knockout sgRNAs (SCN9A-sgRNAs) using qPCR (FIG. SB, 5L). The two most efficient TRPVI-sgRNAs and SCN9A-sgRNAs were packaged into AAV PHP.s (a serotype with a strong DRG neuron-tropism) using a dual-AAV delivery strategy (FIG. 5C, 5M). FACS-purified TRPVl ::GFP+ hPSC-SNs (70 DIVs) or SCN9A::GFP+ hPSC-SNs (70 DIVs) were transduced with two TRPVI-sgRNAs and two SCN9A-sgRNAs, respectively (FIG. 5D, data not shown). We then functionally evaluated the efficiencies of AAV-delivered TRPVI-sgRNAs and SCN9A-sgRNAs by examining their Ca2+ transients when exposed to capsaicin (10μM), histamine (50μM), mustard oil (100μM), ATP (50pM), BAM 8-22 (10μM), and KC1 (50mM). While E5 TRPVI-sgRNA demonstrated specific silencing effects upon the administration of capsaicin (the primary ligand of TRPVl) (FIG. 5F-5J), El 1 SCN9A -sgRNA displayed robust silencing to all five pain-mediating stimuli and KC1 (FIG. 5H-5Q). FIG. 5R illustrates schematic representation of using CRISPR-mediated HDR delivered by a dual-AAV system to install a protective c.2188A>G (p.Thr730Ala) mutation in KCNQ2. FIG. 5S illustrates schematic representation of using CRISPR-mediated base editing delivered by a dual-AAV system to install a protective c.2188A>G (p.Thr730Ala) mutation in KCNQ2.

[0029] FIGS. 6A-6I. illustrate single cell RNA-seq data and CD200 as a nociceptor marker.TRKA antibody has been used as the gold standard to isolate nociceptive neurons from heterogenous peripheral neuronal cultures despite the apparent limitation that TRKA is expressed in some non-nociceptive neurons and many nociceptors don’t express TRKA (Fang et a., 2005; Saito-Diaz et al., 2021). Discovering a novel non-genetic, antibody-based approach to purify nociceptive neurons from heterogeneous cultures is an important step towards testing the in vitro efficacy of gene therapy strategies in a more generalized context. To this end, we identified top enriched CD markers in TRPV1+ vs. TRPV1-, SCN9A+ vs. SCN9A- and MRGPRX1+ vs. MRGPRX1- cells, in which CD200 was shown to be significantly enriched in both SCN9A+ and TRPVl + (but not MRGPRX1+) populations (FIG. 6A). Published human DRG scRNA-seq data also demonstrates that CD200 is specifically enriched in small-fiber primary afferents and clusters differently from NEFH+ large diameter neurons (FIG. 6B, figure generated from raw sequencing data from Tavares-Ferreira et al., 2022). FACS analysis using hPSC-SNs derived from TRPVP.'.GFP and SCN9A:;GFP reporter lines and stained with anti-CD200 (APC / Cy7) acutely before sorting also corroborated the nociceptor-specific enrichment of CD200 (FIG. 6C, 6D). Interestingly, our transcriptomic data showed that the gene counts of CALCA, which encodes a subunit of calcitonin gene-related peptide (CGRP), is enriched in TRPV1+ and SCN9A+ neurons based on the gene counts (Table 3), implicating a therapeutic role of these hPSC-derivednociceptors (marked by CD200 positivity) in promoting tissue healing based on a recent publication (Lu et al., 2024).

[0030] The differentiation of hPSC-derived neural crest cells into sensory neuron subtypes emerges as a hierarchical process; for example, Runxl+ nociceptive neuronal precursors branch into peptidergic nociceptors and non-peptidergic nociceptors (Chen et al., 2006). Subsequently, these two populations are sub-diversified by expressing various neuropeptides, receptors, and channels, such as CGRP, RET, Trp channel family, and Mrgpr family (Chen et al., 2006; Kim et al., 2008; Luo et al., 2007). Although most of the specification mechanisms have been studied in mice, the human counterparts remain largely elusive.

[0031] To construct the transcriptomic continuum of human peripheral sensory neurons, we purified hPSC-derived SOX 10+ neural crest progenitor cells, RUNX1 + sensory progenitor cells as well as TRPV1+, SCN9A + and MRGPRX1+ sensory neurons during their developmental timing of peak expression in vitro (day 6-8 for neural crest progenitor cells, day 12-16 for early sensory neuron progenitor cells, and day 50-70 for mature sensory neurons) (FIG. 6 E). Sequencing libraries were generated following the Smart-seq2 protocol (Ramskold et al., 2012). A total of >30,000 cells passed through the final quality control for sequencing. 1,000 cells randomly sampled from each of the five groups were merged and clustered based on their transcriptomic identities for further analysis (FIG. 6 F). Eighteen transcriptomic clusters of developing and mature hPSC-derived DRG neurons were identified using Seurat and published human DRG neuron subtype markers (Hao et al., 2021; Lause et al., 2021; Nguyen et al., 2021; Tavares-Ferreira et al., 2022; Yu et al., 2023). Six clusters representing sensory neuron precursor cells were assigned the identities “SOX10+” or “RUNX1+”. Twelve sensory neuron subtypes were assigned, among which five are C-fiber nociceptors, four are A-fiber mechano nociceptors, two are of unknown identities, and one cluster represents a developing population of unmyelinated lineage (FIG. 6 G). CD200 is enriched in clusters “C-nociceptors”, “developing unmyelinated lineage”, “cluster 13”, “A-HTMR” and “GRIK-enriched C- noci / mechanoreceptors”, along with some low expression in SOX 10+ and RUNX1+ sensory neuron precursor cells (FIG. 6 H). Interestingly, RAB3A and RAB3C, two genes shown to promote the secretion of neuronal cell-derived extracellular (EV) vesicles during repetitive stimulation (Y ang et al., 2024), show high co-enrichment patterns with CD200 (FIG. 6H). Moreover, MiR-124, a miRNA shown to inhibit inflammatory responses through polarizingmacrophages to favor the M2 phenotype (Essandoh et al., 2016; Qin et al., 2016), is also co- enriched with CD200. These pieces of evidence indicate an attract potential of CD200+ neurons to exhibit heightened ability of secreting EVs during continuing stimulation and secrete anti- inflammatory factors such as miRNAs to polarize the phenotype of local macrophages. To investigate relevant pathways enriched in CD200+ neurons, we performed Reactome pathway analysis and identified signaling pathways that modulate the nociceptive pathways (e.g., GABA receptor activation, neurotransmitter release cycle, and opioid signaling) or local inflammatory environment (e.g., ADORA2B mediated anti-inflammatory cytokines production) are among the top enriched pathways of CD200+ compared to CD200- neurons (FIG. 61). Collectively, these data show that CD200 can serve as a reliable, specific nociceptor marker to purify cultures of primary sensory afferents in the absence of any genetic reporter system and exhibit attractive therapeutic potentials when applied in a cell therapy context.

[0032] FIGS. 7A-7DD. illustrate a KCNQ2 as a gene therapy target gene conferring robust pain-resilience effects in hPSC-derived sensory neurons and the KCNQ2 gene therapy efficacy in a rodent model resulting in bone remodeling. To identify such novel targets, the molecular profiles of TRPV1+, SCN9A+, and MRGPRX1+ neurons was analyzed. Given the vital roles of G-protein-coupled receptors (GPCRs) and ion channels in transducing noxious stimuli, generating and propagating action potentials and maintaining resting membrane potentials, we performed DEG analysis and GSEA on SCN9A+ vs. SCN9A1-, TRPV1+ vs. TRPV1-, and MRGPRX1+ vs. MRGPRX1- neurons using membrane receptor-related gene sets to identify novel pain targets. We identified that potassium voltage-gated channel subfamily Q member 2 (KCNQ2) is significantly enriched both in TRPV1 ::GFP+ neurons (Log2 FC = 2.81, p-value = 3.91E-22) and SCN9A::GFP+ neurons (Log2 FC = 2.05, p-value = 9.00E-12), but down- regulated in MRGPRX1::GFP+ neurons (Log2 FC = -3.29, p-value = 6.76E-29), suggesting that its role in maintaining resting membrane potential and regulating neuronal excitability is specific to nociceptive neurons (FIG. 7A).

[0033] In a separate study, Waxman’s group identified a gain-of-function mutation in KCNQ2 (p.T730A or c.2188A>G) that causes a hyperpolarizing shift in resting membrane potential by - 5mV, which conferred considerable resilience to pain in some patients with IEM, a well characterized human genetic model of chronic pain (Mis et al., 2019). Pharmacological modulators for KCNQ2 (retigabine and flupirtine) had been used in clinics but were laterwithdrawn due to significant side effects. Efforts to develop pharmacological KCNQ2 modulators have been limited, partly due to its expression in the Central Nervous System (CNS). We took an alternative approach by installing KCNQ2 (p.T730A) mutation in mature CD200- purified hPSC-SNs using CRISPR-Cas-mediated HDR, in which SpCas9 expressed under a compact promoter elongation factor 1α short (EFS) and a HDR template carrying the desired edit and a sgRNA targeting KCNQ2 tagged by mCherry are separately packaged into AAV-PHP.s. SpCas9 was selected over other Cas9 variants due to its broad PAM compatibility (3’NGG), which enabled the selection of two sgRNAs that cut 1 bp and 6 bp away from the mutation site, respectively (FIG. 7B, see Table 2 for a complete list of sgRNAs designed to enable the p.T730A single-base editing of KCNQ2). To improve editing efficiency, we tested a broad range of multiplicity of infection (MOI) and different ratios of AAV-SpCas9 / AAV-HDR-sgRNA and found out that AAV-SpCas9 / AAV-HDR-sgRNA administered at MOI = 20,000 and MOI = 40,000, respectively, yielded the best infection efficiency with >70% CD200-purified neurons labeled by mCherry (FIG. 7C). To functionally evaluate pain-resilient effects of KCNQ2 (p.T730A), we examined the Ca2+ transients of edited CD200-purified neurons when exposed to capsaicin, mustard oil, and KC1. Results demonstrate that the KCNQ2 (p.T730A) mutation significantly dampened the response magnitude and decreased the percentage of activated neurons (FIG. 7F, 7G). Notably, neurons installed with KCNQ2 (p.T730A) mutation displayed protracted response to KC1 and significantly diminished Ca2+ influx at axonal initial segment and distal axon (FIG. 7F).

[0034] Due to the generation of DNA double strand break (DSB), CRISPR HDR-based editing has been reported to introduce unwanted indels at the locus of editing (Chiruvella et al., 2013; Lieber, 2010). To improve the translational potential of KCNQ2 (p.T730A) editing, we designed two CRISPR BE-based strategies, in which SauriCas9-ABE8e, a DNA deaminase fused with D10A nCas9, catalyzes the deamination of adenosine to guanine without the need to generate DSB. Since the editing window of SauriCas9-ABE8e typically ranges from protospacer positions 3-16 (counting the PAM as positions 22-25), with optimal editing occurring between positions 5-15 (Anzalone et al., 2020), we designed the two sgRNAs to target the complementary strand so that the KCNQ2 (c.2188A>G) of the desired strand is placed at protospacer positions 12 and 6, respectively. While KCNQ2 BE1 installs the desired ACC (threonine) to GCC (alanine) editwith no bystander mutations, KCNQ2 BE2 also installs a CAC (histidine) to CGC (arginine) bystander mutation due to the presence of two A’s in the editing window (FIG. 7H).

[0035] We next performed TA cloning and Sanger sequencing to verify the successful installation of the mutation and estimate the editing efficiency. Results demonstrated that 16 out of 21 clones harbored edited KCNQ2 (c.2188A>G) mutation, yielding an estimated efficiency of 76% in CD200 purified hPSC-SNs (FIG. 71). To functionally examine the nociception-resilient effects of KCNQ2 (c.2188A>G) installed by CRISPR BE-mediated editing, we examined the Ca2+ transients of edited CD200-purified neurons when exposed to capsaicin, mustard oil, and KC1. Results demonstrated that the KCNQ2 (c.2188A>G) mutation significantly dampened the response magnitude and decreased the percentage of activated neurons (FIG. 7J-7L). Notably, morphological reconstruction and Simple Neurite Tracer (SNT) analysis (Arshadi et al., 2021) demonstrated that edited neurons showed fewer neuronal processes invaded by Ca2+ upon KC1 administration (FIG. 7M-7N), evidenced by the diminished complexity index, number of nodes and number of tips (FIG. 7O-7Q) compared to the unedited neurons.

[0036] We next examined the efficacy of KCNQ2 (c.2188A>G) mutation in alleviating osteoarthritic pain in a mouse model of osteoarthritis (OA). We performed anterior cruciate ligament transection (ACLT) surgery to induce OA in male and female wildtype mice preinstalled with the KCNQ2 (c.2188A>G) mutation via direct injection into the knee two weeks prior to the surgery and assessed for pain profiles two weeks after the surgery (FIG. 7R). Von Frey test showed a significant improvement in mechanical hypersensitivity in animals installed with KCNQ2 (c.2188A>G) 2 weeks after the surgery (FIG. 7S). Hargreaves test of the paw showed a favorable trend in the improvement of thermal hypersensitivity in OA mice 2 weeks after the surgery (FIG. 7T). Hargreaves test of the paw repeated 6 weeks after surgery showed a significant improvement of thermal hypersensitivity in OA mice (FIG. 7U). Primary knee hyperalgesia was analyzed 6 weeks after the surgery by measuring withdrawal threshold during direct knee press using a pressure application measurement (PAM) force transducer. PAM withdrawal threshold (PAMWT) of animals installed with KCNQ2 (c.2188A>G) showed significant improvement in primary knee hyperalgesia (FIG. 7V). To investigate whether the direct left knee injection of AAV could confer long-lasting analgesic effects and show efficacy at the contralateral knee, we monitored animals for 10 weeks after the ACLT surgery. Hargreaves and Von Frey test at bilateral paws showed significant improvements of OA-induced thermal andmechanical hypersensitivity in OA animals receiving the AAV, to a level comparable to that of animals receiving the sham surgery (FIG. 7X-7Y). PAMWT demonstrated significant improvements of bilateral primary knee hyperalgesia in animals receiving direct injection of KCNQ2 (c.2188A>G) at the left knee, although the right knee (contralateral to injection) showed less improvements compared to the left knee (FIG. 7 Z). To validate the bilateral long-term analgesic effects conferred by the KCNQ2 (c.2188A>G) installation at a biomolecular level, we harvested bilateral DRGs that innervate the knee joints (L3-L4 DRGs) at the 10- week endpoint. We observed the expression of ATF3 (a neuronal injury marker) was elevated in DRGs from OA mice that received the control AAV (ACLT-Ctrl), but the heightened ATF3 expression was revered in DRGs of OA mice that received the KCNQ2 (c.2188A>G) AAV (ACLT-AAV), to a level comparable to animals that received the sham surgery (sham-Ctrl) (FIGS. 7 AA-7BB). OA-associated bone loss is one of many pathological changes seen in joint tissues of individuals (El-Sherif et al., 2008, Haara et al., 2005, Simon et al., 2020). To examine whether the installation of KCNQ2 (c.2188A>G) could improve OA-induced bone loss, we performed microCT scans of bilateral knee joints harvested at 10 weeks after the ACLT surgery (FIG. 7 CC). Animals receiving KCNQ2 (c.2188A>G) showed reversed OA-induced bone loss at left and right knees 10 weeks after the ACLT surgery to a level comparable to the sham-operated group, evidenced by improved trabecular thickness (Tb.Th), trabecular bone pattern factor (Tb.Pf), trabecular separation (Tb.Sp) and trabecular bone volume (BV / TV), although the reversal of bone loss seen in the right knee was to a lesser extent than the left knee (FIG. 7 DD). Altogether, we have identified KCNQ2 as a novel pain target, designed and optimized two genetic strategies to install a protective mutation KCNQ2 (c.2188A>G) in hPSC-SNs via 1) a CRISPR- mediated HDR template approach and 2) a CRISPR-mediated base editing approach, and validated the long-lasting analgesic effects of the optimized KCNQ2 editing construct in a mouse model of OA. In addition to the bilateral long-lasting pain-resilient effects, we also observed that our gene therapy strategy provided a robust reversal of bone loss in bilateral knees in OA animals.

[0037] FIGS. 8A-8R illustrate CD200+ sensory neuron injection to intercept pain to treat localized pain and improve the bone remodeling by injecting hPSC-derived pain-sensing neurons. As purified CD200+ hPSC-SNs are enriched for genes conferring anti-inflammatory potentials to polarize local infiltrating macrophages and signaling pathways that modulate thenociceptive pathway, they present excellent opportunities for cell therapy to treat chronic pain caused by inflammation. We next explored the use of purified CD200+ hPSC-SNs in treating a form of localized inflammatory pain in OA. We injected CD200+ hPSC-SNs into the tibial tuberosity of immunocompromised NIH III nude mice after bilateral anterior cruciate ligament transection (ACLT) surgery-induced OA pain (FIG. 8A). We observed that animals receiving CD200+ cell injection displayed a mitigated mechanical pain hypersensitivity profile compared to the OA animals receiving heat-killed cells five weeks after injection (FIG. 8B). We also observed that OA-induced bone loss in the knee joint was reversed in the treated group at the five-week timepoint (FIG. 8C-8D). To validate the analgesic effects conferred by CD200+ injection at a biomolecular level, we isolated bilateral L3-L4 DRGS (the DRGs that innervate knee joints in mice) to assess the expression of cell injury and pain markers (i.e., ATF3 and CGRP) and found that animals that underwent the ACLT surgery but received heat-killed cells showed heightened expression of both ATF3 and CGRP. However, the expression of ATF3 and CGRP were decreased in ACLT-animals that received CD200+ cells, to a level indistinguishable from animals that underwent sham surgery (FIGS. 8E-8F). A substantial body of evidence indicates that OA involves additional recruitment of nerves that innervate the subchondral bone, and that this increased innervation is tightly associated with pain behavior in animals (Aso et al., 2020; Morgan et al., 2022). To examine whether the improved pain behavior in OA animals receiving CD200+ cells is associated with reversed recruitment of nerves innervating the knee joint, we harvested bilateral knee joints from animals at the five-week timepoint to quantify total fiber lengths (FIG. 8G). NeuroJ analysis using fluorescent intensity of PGP9.5 that stained for endogenous nerves demonstrated that the subchondral bone zone and tibia of ACLT-CD200+ animals showed significantly less innervation compared to ACLT-heat killed animals (FIG. 8H). Moreover, human-specific mitochondrial antibody (hMito) was showed that injected CD200+ neurons were localized in the tibial bone and subchondral bone area (FIG. 8G).

[0038] To exclude the alternative hypothesis that a general population of hPSC-SNs could also confer analgesic and bone uncoupling potentials, we next injected CD200+ hPSC-SNs into the tibial tuberosity of immunocompromised NIH III nude mice after bilateral ACLT, whereas the control animals were injected with CD200- cells (FIG. 81). At the five-week timepoint, PAMWT and Von Frey results demonstrated that ACLT-CD200+ animals showed significantly improved mechanical hypersensitivity while ACLT-CD200- animals maintained OA-induced mechanicalhypersensitivity with no significant improvements (FIG. 8J-8K). To examine whether the improved pain behavior in OA animals receiving CD200+ cells is associated with reversed recruitment of nerves innervating the knee joint, we harvested bilateral knees at the five- week timepoint to quantify total fiber lengths (FIG. 8L). NeuroJ analysis using fluorescent intensity of PGP9.5 that stained for endogenous nerves demonstrated that the subchondral bone zone and tibia of ACLT-CD200+ animals showed significantly less innervation compared to ACLT- CD200- animals (FIG. 8M). To assess the analgesic effects of CD200+ neurons over CD200- neurons in the context of OA at a biomolecular level, we harvested bilateral L3-L4 DRGs innervating knee joints and quantified the expression level of ATF3 and CGRP. Results demonstrated that DRGs from ACLT-CD200- mice expressed significantly higher levels of ATF3 and CGRP 5 weeks after the injection, an elevation reversed by CD200+ neuron injection (FIGS. 8O-8P). To assess the long-term efficacy of CD200+ neurons injection, we examine mechanical hypersensitivity of animals 10 weeks after injection. Von Frey and direct pressure at knee tests demonstrated that ACLT animals receiving CD200+ neurons displayed significantly improved mechanical hypersensitivity compared to ACLT animals receiving CD200- neurons, to a level indistinguishable from the control (FIGS. 8Q-8R). Altogether, we have demonstrated that the SNIIP strategy using purified CD200+ hPSC-SNs has potential to not only alleviate OA- induced pain, but also modulates the pathological bone coupling in OA and reversed OA-induced bone loss. We anticipate the use of CD200+ hPSC-SNs is not limited to OA and have great potentials for treating other pathological pain conditions.

[0039] FIGS. 9A-9K. illustrate in vivo survival and function of SCN9A+, TRPV1+, and MRGPRX1+ neurons. To explore in vivo survival and functional integration abilities, we transplanted FACS-purified hPSC-derived mature TRPV1::GFP+, SCN9A::GFP+, and MRGPRX1 ::GFP+ hPSC-SNs, and differentiating neural crest cells into the right L4 DRG of adult Rag2em2heraIl2rgemlhera / HblCrl rats (FIG. 9A). An expression cassette harboring TdTomato and excitatory (Gq) designer receptors activated by designer drugs (Gq-DREADDs) was packaged into AAV PHP.s and later transduced into hPSC-SNs prior to the injection to allow for chemogenic modulations (FIG. 9B-C). When maintained in vitro, transduced TRPV1 ::GFP+, SCN9A::GFP+, and MRGPRX1 ::GFP+ donor hPSC-SNs showed extended axons (up to 800 um - 1 mm) 5 days after plating with visible growth cone (FIG. 9D). Left (contralateral control group) and right (injected group) L4 DRGs were isolated from animals 5 weeks aftertransplantation surgery to examine the survival of hPSC-derived donor neurons. While GFP background was low in the control DRG (FIG. 9E), the injected DRGs and attached nerve roots harbored clusters of GFP -expressing hPSC-derived donor neurons with axonal sprouting (FIG. 4 F). To investigate whether the survived hPSC-derived donor neurons could establish functional connections with endogenous neurons, we examined the behavioral consequences of rats 4 weeks after the initial hPSC-SN transplantation (FIGS. 9G-9H). Spontaneous pain behaviors were assessed using video-observation of rats after intra-peritoneal administration of the DREADD activator CNO and PBS. Duration of right hind paw licking was significantly heighted after CNO administration compared to PBS (FIG. 91). Mechanical allodynia was examined using Von Frey tests 7 days after the initial CNO administration or acutely after CNO administration. The injected side displayed a significant decrease in threshold 7 days after the initial CNO administration and 1 hour after acute CNO administration FIGS. 9J-9K). Taken together, our data show that injected hPSC-SNs could survive in vivo and modulate endogenous neuronal activities by sensitization or direct excitation (FIGS. 9J-9K).

[0040] In conclusion, our study presents that TRPV1 ::GFP+, SCN9A::GFP+ and MRGPRX1 ::GFP+ hPSC-SNs can be generated and confirmed their functionality with multiple pain and itching stimuli. The hPSC-derived TRPV1 ::GFP+ and SCN9A::GFP+ neurons also display great potential for in vivo survival and functionality after transplantation. These data provide new insight on stem cell fate determination processes toward a specific sensory neuronal subtype, open new avenues for chronic pain therapeutics using cell therapy, and build a foundation to develop novel drug candidates for managing persistent pain / itching conditions.

[0041] The various embodiments described above are provided by way of illustration only and should not be construed to limit the scope of the disclosure. Various modifications and changes may be made to the principles described herein without following the example embodiments and applications illustrated and described herein, and without departing from the spirit and scope of the disclosure. For example, unless otherwise explicitly indicated, the steps of a process or method may be performed in an order other than the example embodiments discussed above. Likewise, unless otherwise indicated, various components may be omitted, substituted, or arranged in a configuration other than the example embodiments discussed above.

[0042] Further aspects of the present disclosure are provided by the subject matter of the following clauses.Materials and Methods hPSC maintenance:Healthy control hESC (H9, WiCell) were cultured using standard protocols. The hESC line was cultured with mouse embryonic fibroblasts (MEFs) (Gibco) pre-plated at 12,000-15,000 cells / cm2. hPSC culture medium contained DMEM / F12, 20% knockout serum replacement, 1 mM L- glutamine, 100 μM MEM non-essential amino acids, and 0.1 mM > -mercaptoethanol. 10 ng / mL of FGF-2 was added after sterile filtration, and cells were fed daily, and passaged weekly, using 6 U / mL dispase or mechanically.Generation of knock-in hPSC linesThe feeder-free hPSCs were dissociated into single cells using Accutase (Innovative Cell Technologies), and 1.5x 106H9 cells were resuspended in nucleofection solution V (Lonza) with 5 pg HDR donor plasmid, 2 pg i53 plasmid, 0.5 nmol sgRNA, and 0.5 nmol Cas9 nuclease (V3 or HiFi V3) for TRPV1 ::GFP, MRGPRX1 ::GFP and SCN9A::GFP (see Table 1). Nucleofection was performed with Nucleofector™ II according to the manufacturer's instruction (using the B-16 program, Lonza). The nucleofected cell suspension was subsequently plated on puromycin- resistant MEFs (DR4, Global Stem) in hESC medium with 10 μM Y-27632. Three days after nucleofection, the cells that had undergone homologous recombination were selected by adding 0.5 pg / ml of puromycin to hESC medium for two days.Sensory neuron differentiation from hESCs: hPSCs were dissociated into single cells using TrypLE™, and plated on a 0.1 % gelatin-coated dish for 10 minutes to remove MEFs. Non-adherent cells (mostly hPSCs) were collected, and plated on a 1% geltrex-coated dish (1 hour coating), at a density of 4.6 x 104cells per well of a 24-well plate, in the presence of filtered MEF-conditioned KSR media containing 10 ng / ml of FGF-2 (R&D systems) and 10 μM of Y-27632 (Cayman Chemical) (day -1). From the next day (day 0) (70% of confluence), to initiate differentiation, the medium was aspirated and KSR medium containing 10 μM SB-431542 and 500nM LDN-193189 was added. On day 2 the medium was changed to KSR medium containing 10 μM SB-431542 and 500nM LDN-193189. On day 4 themedium was changed to KSR / N2 (3:1) medium containing 10 gM SB-431542, 500nM LDN- 193189, and lOgM DAPT (final concentrations are for the combined KSR / N2 mixture). On day 6, the medium was changed to KSR / N2 (1:1) medium containing 10 gM SB-431542, 500nM LDN- 193189, and lOgM DAPT. On day 8, the medium was changed to KSR / N2 (1:3) medium containing 10 gM SB-431542, 500nM LDN-193189, and lOgM DAPT. On day 10, the medium was changed to N2 medium containing 10 gM SB-431542, 500nM LDN-193189, and lOgM DAPT. On day 12, neural crest cells were dissociated into single cells using TrypLE™ and plated on poly-lysine / laminin / fibronectin coated 12-well plates with a density of 2 x 104- 7 x 104cells per 5 ul media. 5 drops were added to each well 12-well plates. Cells were incubated in 5 ul droplets for 10 minutes at 37 °C to fully attach. NB media containing 10 ng / ml of FGF-2 and 10 gM of Y- 27632 was then gently added to each well through the wall. On day 14, 17, and 20, the medium was changed to NB media containing 200 gM dibutyrl cyclic AMP, 200gM sodium 1-ascorbate, 20 ng / ml NGF, 10 ng / ml BDNF, 10 ng / ml BDNF and 10 ng / ml NT-3. After day 21 of differentiation, half of the spent medium in each well was gently aspirated and supplemented with fresh NB medium containing 200 μM dibutyrl cyclic AMP, 200gM sodium 1-ascorbate, 20 ng / ml NGF, and 10 ng / ml GDNF. Media was changed every 3 days. Laminin and fibronectin were added to the media every week to maintain attachment.FACS purification of TRPV1+, SCN9A+, and MRGPRX1+ neurons:One day prior to FACS purification, mature hPSC -derived sensory neurons were fed with fresh NB media containing 200 gM dibutyrl cyclic AMP, 200gM sodium 1-ascorbate, 20 ng / ml NGF, and 10 ng / ml GDNF. On the day of FACS purification, sensory neuron-conditioned media was harvested from each well, filtered, and supplemented with 10 gM ofY-27632 to make post-sorting media (PSM). hPSC-derived sensory neurons were incubated with TrypLE™ for 30 minutes at 37 °C to dissociate into single cells. Pelleted cells were resuspended in FACS media (90% DPBS + 10% DMEM with DNasel, 10 gM of Y-27632, and 3% penicillin-streptomycin) and passed through 50 um strainers. 1 ul Sytox Red (ThermoFisher Scientific, S34859) was added per 1 ml cell suspension. Cells were sorted using SH800S Cell Sorter based on GFP fluorescence and collected in 1.5 ml Eppendorf tubes (filled with 400 ul PSM + 3% penicillin-streptomycin). Purified TRPV1+, SCN9A+, or MRGPRX1+ cells were pelleted, resuspended in PSM at a density of 1 x 104cells per 5 ul, and replated in 48-well plates pre-coated with poly-lysine / laminin / fibronectin. One 5 ul drop was placed per well. Cells were incubated in 5 ul droplets for 10 minutes at 37 °C to fully attach and added with PSM media. Note that 3% penicillin- streptomycin was added to the FACS media and PSM in the collecting tubes but not to the final replating media.Purification of CD200+ hPSC-derived neuronsOne day prior to FACS purification, mature hPSC -derived sensory neurons were fed with fresh NB media containing 200 μM dibutyrl cyclic AMP, 200μM sodium 1-ascorbate, 20 ng / ml NGF, and 10 ng / ml GDNF. On the day of FACS purification, sensory neuron-conditioned media was harvested from each well, filtered, and supplemented with 10 μM ofY-27632 to make post-sorting media (PSM). hPSC-derived sensory neurons were incubated with TrypLE™ for 30 minutes at 37 °C to dissociate into single cells. Peleted cells were resuspended in FACS media (90% DPBS + 10% DMEM with DNasel, 10 μM of Y-27632, and 3% penicillin-streptomycin) and passed through 50 um strainers. 1 ul Sytox Red (ThermoFisher Scientific, S34859) was added per 1 ml cell suspension. Mix 10 μg of APC / Cy7 Anti-human CD200 antibody *OX-104* (AAT Bioquest, 120001D0) to cells harvested from one plate suspended in 5 mL of FACS media and incubate for 10 min at 37°C. Resuspend the cells and transfer to sorting tubes (with 50 um strainer cap) using a pl 000 micropipette and store the tube on ice. Sort the cells using SH800S Cell Sorter based on GFP fluorescence (for TRPV1+ or SCN9A+ identity), Sytox red (for live cells) and APC / Cy7 fluorescence (for CD200+ identity) and collected in 1.5 ml Eppendorf tubes (filled with 400 ul PSM + 3% penicillin-streptomycin). Purified CD200+ cells were pelleted, resuspended in PSM at a density of 1 x 104cells per 5 ul, and replated in 48-well plates pre-coated with poly- lysine / laminin / fibronectin. One 5 ul drop was placed per well. Cells were incubated in 5 ul droplets for 10 minutes at 37 °C to fully attach and added with PSM media. Note that 3% penicillin- streptomycin was added to the FACS media and PSM in the collecting tubes but not to the final replating media.Animals and surgical procedureFemale and male rats (Charles River, SRG rat, strain code: 707) aged about 50 days were used in the study with approval by the JHU IACUC committee (PI: Dong). Prior to surgery, animals were anesthetized with ketamine (2 mg / lb) and kept under anesthesia for the duration of the surgery.For transplantation into the DRG, a midline incision was performed through the skin and fascia, and then the surrounding muscles were dissected and retracted laterally to expose the lower lumbar vertebral column. A hemi-laminectomy was performed to expose the spinal cord dura and the attached nerve root and L4 DRG. 0.5 ul cell suspension containing 1x 104TRPV1+ neurons, 2.7 x 104SCN9A+ neurons, and 3 x 104differentiating neural crest cells were injected into the DRG or the adjacent nerve bundles. Cells were delivered using a glass capillary to minimize physical damage. The glass capillary was left in place for 1 minute to prevent backflow. Grafted animals were maintained and monitored for 4 weeks before left and right L4 DRGs were surgically removed. Left (control) and right L4 DRGs were stained for GFP (Abeam, abl3970) and TUJ1 (ThermoFisher Scientific, MAI-118) to examine the survival of hPSC-derived sensory neurons.We purchased male NIH III nude from Charles River Laboratories (Wilmington, MA). We anesthetized mice at 8 weeks of age with xylazine (Rompun, Sedazine, AnaSed; 10 mg / kg, intraperitoneally) and ketamine (Vetalar, Ketaset, Ketalar; 100 mg / kg, intraperitoneally). Then, the anterior cruciate ligament transection (ACLT) surgery was performed to induce instability of the knee. Sham ACLT operations were performed by opening the joint capsule of the knee of independent mice. The tibial tuberosity of sham or ACLT mice was injected with 40 k cells / knee of CD200+ or CD200- hPSC-SNs 2 weeks after surgery.Behavioral testingBehavioral tests were performed 2 weeks after the cell injection and 4 weeks after surgery. All behavioral tests were performed by the same investigators, who were blinded to the allocation of groups. The von Frey hair was applied perpendicular to the plantar surface of the hind paw (avoiding the toe pads) for 2-3 s. If no response, the next higher strength of hair was used, up to the maximum level of 6 g of bending force. If a withdrawal response occurred, the paw was re- tested, starting with the next descending von Frey hair until no response occurred. Four more measurements were made after the first difference was observed. The 50% PWT was determined by using the following formula:50% PWT 1 / 4 10Xf pkd —10; 000; where Xf is the exact value (in log units) of the final test of von Frey hair, K is the tabular value for the pattern of the last six positive / negative responses, and d is the mean difference (in log units)between stimuli. The threshold force required to elicit paw withdrawal (median 50% withdrawal) was determined twice on each hind paw (and averaged) on each testing day, with sequential measurements separated by at least 10 min.Virus infection of hPSC-derived TRPV1+ neuronsFACS-purified TRPV1+ neurons were cultured for 7 days before infection using AAV PHP.S- CAG-tdTomato (MOI = 10,000, 20,000, and 100,000). Ad-NULL (MOI = 100) was used to co- infect TRPV1+ neurons for improved infection efficiency and tdTomato overexpression. Infected TRPV1+ cells were cultured for additional 5 days, washed for three times in PSM, and injected into animals.Immunohistochemical analysis:Culuture neurons were fixed with 4% PFA for 1 hours at room temperature. After 2 washes with PBS and the samples were blocked in PBS with 5% Bovine Serum Albumin (BSA) (Sigma Aldrich) and 0.2% Triton X-100 (Sigma Aldrich) for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. Primary antibodies were washed 3 times with PBS with 0.3% Triton X-100 (PBST). The samples were incubated with secondary antibodies for 1-2 hours at room temperature. After secondary incubation, slides were washed 3 times with PBST and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Fisher Vector Lab). The staining was performed using primary antibodies such as BRN3a (Abeam, ab245230), TUJ1 (ThermoFisher Scientific, MAI-118) and appropriate 488, 568, and 647-conjugated Alexa fluor secondary antibodies (Invitrogen) were utilized.Single cell transcriptomics and pathway analysisR (version 4.3.2) and Seurat (version 5) were used for the single-cell RNA-seq analysis. Sequencing reads were separately acquired for SOX10+, RUNX1+, TRPV1+, SCN9A+ and MRGPRX1+ cells and imported as separate Seurat objects. Data were normalized (NormalizeData) within each group and verified by FeatureScatter (nFeature RNA vs. nCount RNA > 0.9 for all five groups). Five Seurat objects were then merged into one (merged) with the original identity stored as metadata in each object. ElbowPlot(merged) was performed to calculate the major principle components to avoid over-clustering. To remove technical and otherbatch-to-batch variations, SCTransform was performed for normalization and variance stabilization of molecular count data from scRNA-seq experiments. Highly similar clusters without clearly distinguishable markers were merged to produce the final 18 clusters. All clusters representing subtypes of mature sensory neurons (i.e., TRPV1+, SCN9A+ and MRGPRX1+) are verified for neuronal markers SNAP25, RBFOX1 and THY1. Standard Seurat analysis pipeline was followed to identify enriched genes in each cluster. Violin plot was used to visualize top genes co-enriched with CD200. Enriched pathways in CD200+ cells were identified using the open- source, open access, manually curated and peer-reviewed Reactome pathway database (https: / / reactome.org).StatisticsAll data are shown as mean ± SEM and were subjected to statistical analysis. Significance was analyzed by 1-way ANOVA using Dunnett’s or Tukey’s multiple-comparisons test or were analyzed by 2-tailed unpaired Student’s t test. P < 0.05 was considered significant. The n values indicate the number of independent biological samples. Data were analyzed and represented with GraphPad Prism.Table 1CRISPR-Cas9 ribonucleoprotein (RNP) and homology-directed recombination (HDR) template design. The PAM sequence is labeled in blue, restriction enzyme sites are shown in yellow, and the genomic sequence is shown in green. A silent mutagenesis is designed on the right homology arm for the TRPV1 locus. The PAM sequence for all loci is designed to be located within lObp of the insertion site. The RNP complex for each reporter line was assembled immediately prior to nucleofection. 1.5x 106H9 cells were nucleofected with 5pg HDR donor plasmid, 2 pg i53 plasmid, 0.5 nmol sgRNA, and 0.5 nmol Cas9 nuclease (V3 or HiFi V3) using Amaxa Lonza Nucleofector II system. Nucleofected cells were then plated with lOμM Y-27632 in pre- warmed media onto the culture dish plated with Drug Resistance 4 Mouse embryonic fibroblast feeder cells (DR4). Three days following the nucleofection, nucleofected cells were selected for puromycin (0.5pg / ml) resistance for two days.Table 2CRISPR-mediated HDR and CRISPR-mediated base editing designs to install the protective KCNQ2 (c.2188A>G, p.Thr730Ala) mutation in primary sensory neurons as a treatment of chronic pain.Table 3.Gene counts of CALCA, CALCB, TRPV1, SCN9A. SOXIO and RUNX1 in mature hPSC-derived TRPV1+ and SCN9A+ sensory neurons and in early SOX10+ or RUNX1+ sensory neurons.REFERENCES1. E. Gascon, A. Moqrich, Heterogeneity in primary nociceptive neurons: from molecules to pathology. Arch Pharm Res 33, 1489-1507 (2010).2. M. Duitama et al., TRP Channels as Molecular Targets to Relieve Cancer Pain. Biomolecules 12, (2021).3. I. Drissi, W. A. Woods, C. G. Woods, Understanding the genetic basis of congenital insensitivity to pain. BrMed Bull 133, 55-78 (2020).4. A. S. Yekkirala, D. P. Roberson, B. P. Bean, C. J. Woolf, Breaking barriers to novel analgesic drug development. Nat Rev Drug Discov 16, 545-564 (2017).5. L. Basso, C. AItier, Transient Receptor Potential Channels in neuropathic pain. Curr Opin Pharmacol 32, 9-15 (2017).6. E. C. Emery, A. P. Luiz, J. N. Wood, Navi.7 and other voltage-gated sodium channels as drug targets for pain relief. Expert Opin Ther Targets 20, 975-983 (2016).7. J. Lotsch, G. Geisslinger, Pharmacogenetics of new analgesics. BrJ Pharmacol 163, 447- 460 (2011).8. G. Goodwin, S. B. McMahon, The physiological function of different voltage-gated sodium channels in pain. Nat Rev Neurosci 22, 263-274 (2021).9. K. E. Sadler, J. S. Mogil, C. L. Stucky, Innovations and advances in modelling and measuring pain in animals. Nat Rev Neurosci 23, 70-85 (2022).10. J. E. Pope, T. R. Deer, J. Kramer, A systematic review: current and future directions of dorsal root ganglion therapeutics to treat chronic pain. Pain Med 14, 1477-1496 (2013).11. A. I. Basbaum, D. M. Bautista, G. Scherrer, D. Julius, Cellular and molecular mechanisms of pain. Cell 139, 267-284 (2009).12. S. Q. He etal., MrgC agonism at central terminals of primary sensory neurons inhibits neuropathic pain. Pain 155, 534-544 (2014).D. E. Gloriam, R. Fredriksson, H. B. Schioth, The G protein-coupled receptor subset of the rat genome. BMC Genomics 8, 338 (2007). L. Zhang etal., Cloning and expression of MRG receptors in macaque, mouse, and human. Brain Res Mol Brain Res 133, 187-197 (2005). P. M. Lembo et al., Proenkephalin A gene products activate a new family of sensory neuron-specific GPCRs. Nat Neurosci 5, 201-209 (2002). X. Dong, S. Han, M. J. Zylka, M. I. Simon, D. J. Anderson, A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106, 619-632 (2001). R. Schmidt, J. Butterworth, D. O'Donnell, V. Santhakumar, M. Tomaszewski, Cyclic dimers of C-terminal gamma2-MSH analogs as selective antagonists of the human sensory nerve-specific receptor (SNSR-4). Adv Exp Med Biol 611, 111-112 (2009). K. Mizuno, N. Minamino, K. Kangawa, H. Matsuo, A new family of endogenous "big" Met-enkephalins from bovine adrenal medulla: purification and structure of docosa- (BAM-22P) and eicosapeptide (BAM-20P) with very potent opiate activity. Biochem Biophys Res Commun 97, 1283-1290 (1980). S. K. Han et al., Orphan G protein-coupled receptors MrgAl and MrgCll are distinctively activated by RF-amide-related peptides through the Galpha q / 11 pathway. Proc Natl Acad Sci U SA 99, 14740-14745 (2002). P. Kunapuli et al., Identification of small molecule antagonists of the human mas-related gene-Xl receptor. Anal Biochem 351, 50-61 (2006). A. Lampert et al., Human sensory neurons derived from pluripotent stem cells for disease modelling and personalized medicine. Neurobiol Pain 8, 100055 (2020). K. Mittal, K. Schrenk-Siemens, Lessons from iPSC research: Insights on peripheral nerve disease. Neurosci Lett 738, 135358 (2020). S. D. Dib-Hajj, S. G. Waxman, Sodium Channels in Human Pain Disorders: Genetics and Pharmacogenomics. Annu Rev Neurosci 42, 87-106 (2019). S. Viventi, M. Dottori, Modelling the dorsal root ganglia using human pluripotent stem cells: A platform to study peripheral neuropathies. IntJ Biochem Cell Biol 100, 61-68 (2018). M. Alsaloum et al., Contributions of NaV1.8 and NaV1.9 to excitability in human induced pluripotent stem-cell derived somatosensory neurons. Sci Rep 11, 24283 (2021). M. Zeidler et al., NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC-Derived Nociceptor Maturation. Adv Sci (Weinh) 8, e2102354 (2021). K. Saito-Diaz, J. R. Street, H. Ulrichs, N. Zeltner, Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells. Stem Cell Reports 16, 446-457 (2021). C. L. Schoepf et al., Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (IPSC)-Derived Nociceptors. Brain Sci 10, (2020). L. A. McDermott et al., Defining the Functional Role of NaV1.7 in Human Nociception. Neuron 101, 905-919 e908 (2019).J. E. Meents et al., The role of Navi.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients. Pain 160, 1327-1341 (2019). B. Namer et al., Pain relief in a neuropathy patient by lacosamide: Proof of principle of clinical translation from patient-specific iPS cell-derived nociceptors. EBioMedicine 39, 401-408 (2019). I. Jones et al., Development and validation of an in vitro model system to study peripheral sensory neuron development and injury. Sci Rep 8, 15961 (2018). B. Zimmer et al., Human iPSC-derived trigeminal neurons lack constitutive TLR3- dependent immunity that protects cortical neurons from HSV-1 infection. Proc Natl Acad Sci U SA 115, E8775-E8782 (2018). A. J. Alshawaf et al., Phenotypic and Functional Characterization of Peripheral Sensory Neurons derived from Human Embryonic Stem Cells. Sci Rep 8, 603 (2018). J. Schwartzentruber et al., Molecular and functional variation in iPSC-derived sensory neurons. Nat Genet 50, 54-61 (2018). E. Eberhardt et al., Pattern of Functional TTX-Resistant Sodium Channels Reveals a Developmental Stage of Human iPSC- and ESC-Derived Nociceptors. Stem Cell Reports 5, 305-313 (2015). S. M. Chambers et al., Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 30, 715-720 (2012). Lu, Y., Nayer, B., Singh, S. K., Alshoubaki, Y. K., Yuan, E., Park, A. J., Maruyama, K., Akira, S., & Martino, M. M. (2024). CGRP sensory neurons promote tissue healing via neutrophils and macrophages. Nature (London), 628(8008), 604-611. 10.1038 / s41586- 024-07237-y El-Sherif, H., Kamal, R., M.D., & Moawyah, O., M.D. (2008). Hand osteoarthritis and bone mineral density in postmenopausal women; clinical relevance to hand function, pain and disability. Osteoarthritis and Cartilage, 16(1), 12-17. doi:10.1016 / j.joca.2007.05.011 Haara, M. M., Arokoski, J. P. A., Kroger, H., Karkkainen, A., Manninen, P., Knekt, P., et al. (2005). Association of radiological hand osteoarthritis with bone mineral mass: A population study. Rheumatology (Oxford, England), 44(12), 1549-1554. doi :10.1093 / rheu matology / kei084 Simon, D., Tascilar, K., Unbehend, S., Bayat, S., Berlin, A., Liphardt, A., et al. (2020). Bone mass, bone microstructure and biomechanics in patients with hand osteoarthritis. Journal of Bone and Mineral Research, 35(9), 1695-1702. doi:10.1002 / jbmr.4046 Chen, C., Broom, D. C., Liu, Y., de Nooij, J. C., Li, Z., Cen, C., Samad, O. A., Jessell, T. M., Woolf, C. J., & Ma, Q. (2006). Runxl Determines Nociceptive Sensory Neuron Phenotype and Is Required for Thermal and Neuropathic Pain. Neuron (Cambridge, Mass.), 49(3), 365-377. 10.1016 / j. neuron.2005.10.036 Luo, W., Wickramasinghe, S. R., Savitt, J. M., Griffin, J. W., Dawson, T. M., & Ginty, D. D. (2007). A Hierarchical NGF Signaling Cascade Controls Ret-Dependent and Ret- Independent Events during Development of Nonpeptidergic DRG Neurons. Neuron (Cambridge, Mass.), 54(5), 739-754. 10.1016 / j.neuron.2007.04.027Kim, A. Y., Tang, Z., Liu, Q., Patel, K. N., Maag, D., Geng, Y., & Dong, X. (2008). Pint, a Phosphoinositide-Binding Protein, Functions as a Regulatory Subunit ofTRPV1. Cell, 133(3), 475-485. 10.1016 / j.cell.2008.02.053 Nguyen, M. Q., von Buchholtz, L.,J., Reker, A. N., Ryba, N. J., & Davidson, S. (2021). Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons. eLife, 1010.7554 / eLife.71752 Yu, H., Usoskin, D., Nagi, S., Hu, Y., Kupari, J., Bouchatta, 0. C., et al. (2023). Single- soma deep RNA sequencing of human DRG neurons reveals novel molecular and cellular mechanisms underlying somatosensation. bioRxiv [Preprint], doi: 10.1101 / 2023.03.17.533207 Essandoh, K., Li, Y., Huo, J., & Fan, G. (2016). MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response. Shock (Augusta, Ga.), 46(2), 122-131. doi:10.1097 / SHK.0000000000000604 Qin, Z., Wang, P., Su, D., & Liu, X. (2016). miRNA-124 in immune system and immune disorders. Frontiers in Immunology, 7, 406. doi:10.3389 / fimmu.2016.00406 Aso, K., Shahtaheri, S. M., Hill, R., Wilson, D., McWilliams, D. F., Nwosu, L. N., et al. (2020). Contribution of nerves within osteochondral channels to osteoarthritis knee pain in humans and rats. Osteoarthritis and Cartilage, 28(9), 1245-1254. doi:10.1016 / j.joca.2020.05.010 Morgan, M., Thai, J., Nazemian, V., Song, R., & Ivanusic, J. J. (2022). Changes to the activity and sensitivity of nerves innervating subchondral bone contribute to pain in late-stage osteoarthritis. Pain (Amsterdam), 163(2), 390-402. doi:10.1097 / j. pain.0000000000002355Sequence List:

Claims

AMENDED CLAIMS received by the International Bureau on 25 May 2025 (25.05.2025)We claim:

1. A method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with at least one of nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition, the method comprising: introducing into a population of human pluripotent stem cells (hPSCs) a composition comprising: at least one site-directed nuclease targeting a site within the target gene, and at least one nucleic acid comprising a nucleotide sequence encoding at least one screenable, selectable marker that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene, wherein the target site is located downstream of the open reading frame of the target gene, and wherein the site-directed nuclease cleaves the target site of the target gene and the nucleic acid encoding the screenable, selectable marker is inserted at the target site; covering said population of hPSCs under an extracellular matrix comprising at least one neuronal differentiation driver to produce a sensory committed neural crest population; contacting said sensory committed neural crest population with at least one neuronal differentiation driver and at least one neurotrophic factor to produce a population of early sensory neurons (SNs); and contacting said population of early SNs with at least one neurotrophic factor to produce apopulation of mature hPSC-SNs expressing at least one of SN marker or one pan neuronal marker; isolating the cells expressing the at least one screenable, selectable marker, wherein said population of mature hPSC-SNs expressed said one target gene associated with nociceptive pain, chronic pain, pruriception a nociceptive- or pruriceptive- mediated condition, and wherein said population of mature hPSC-SNs responds to a nociceptive and pruriceptive stimulus.

2. The method of claim 1, wherein the target gene is SCN9A, TRPV1, MRGPRX1, or KCNQ2.

3. The method of claim 2, wherein the site-directed nuclease is a CRISPR system comprising a ribonuclear protein (RNP) comprising CRISPR nuclease complexed with a guide RNA (gRNA) or a single guide RNA (sgRNA).

4. The method of claim 3, wherein CRISPR nuclease is a Type II Cas9 nuclease or a Type V Cfpl nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

5. The method according to any one of claims 1 to 4, wherein the at least one screenable, selectable marker is a fluorescent reporting protein, an antibiotic resistance protein, or a combination thereof.

6. The method according to claim 5, wherein the fluorescent reporting protein is selected from green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), lacZ, firefly Rennila protein, luciferase, red cyan protein, and yellow cyan protein.

7. The method of claim 6, wherein the fluorescent reporting protein is selected from greenfluorescent protein (GFP).

8. The method of claim 5, wherein the antibiotic resistance protein is selected from hygromycin, neomycin, zeocin, and puromycin.

9. The method of claim 8, wherein the antibiotic resistance protein is puromycin.

10. The method according to any one of claims 2 to 9, wherein the target gene is SEQ ID NO.1, the nucleic acid encoding the at least one screenable, selectable marker comprises SEQ ID NO.2, and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO.

111. The method according to any one of claims 2 to 9, wherein the target gene is SEQ ID NO.3, the nucleic acid encoding the at least one screenable, selectable marker comprises SEQ ID NO.2, and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO.

312. The method of claim 10, wherein the hPSC-SNs are enriched in voltage gated potassium channel genes, active transporter genes, GPCR receptors, or any combination thereof.

13. The method of claim 11, wherein the hPSC-SNs are enriched in voltage gated potassium channel genes, active transporter genes, GPCR receptors, or any combination thereof.

14. The method of claims 12 to 13, wherein the voltage gated potassium channel genes comprise KCNQ2 and KCNG2.

15. The method according to claim 12 to 13, wherein the active transporter genes comprise SLC10A4 and ATP2B4.

16. The method according to claim 12 to 13, wherein the GPCR receptors comprise ADCYAP1R1.

17. The method according to any one of claims 2 to 9, wherein the target gene is SEQ ID No.4, the nucleic acid encoding the at least one screenable, selectable marker comprises SEQ ID NO.2, and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO. 4.

18. The method according to claim 17, wherein hPSC-SNs are enriched in voltage gated calcium channel genes, GPCR receptors, or any combination thereof.

19. The method according to claim 18, wherein the voltage gated calcium channel genes comprise CACNG1, CACNA1S, CACNG8, and CACNA2D3.

20. The method according to claim 18, wherein the GPCR receptors comprise PRNP, DNM3, UTS2R, HTR2C, HTR1E, HTR2AHTR1D, and NPY1R.

21. The method according to any one of claims 1 to 20 wherein the composition further comprises at least one agent capable of favoring the homology-directed repair (HDR) pathway over the non-homologous end-joining (NHEJ) pathway.

22. The method according to claim 21, wherein the agent is an HDR activator or NHEJ inhibitor.

23. The method according to claim 21, wherein the HDR activator is selected from the group comprising: RAD51, RAD52, DMC1, CtIP, or any combination thereof.

24. The method according to claim 21, wherein the NHEJ inhibitor selected from E1B55K, E4orf6, 53BP1(DM), Rif, p53, or any combination thereof.

25. A population of hPSC-SNs produced by the method according to any one of claims 1-24.

26. The method of claim 1, wherein the target gene is associated with nociceptive pain.

27. The method of claim 1, wherein the target gene is associated with chronic pain.

28. A population of hPSC-SNs produced by the method according to any one of claims 26-27.

29. A method of altering the expression or function of at least one target gene associated with nociceptive pain, chronic pain, pruriception and a nociceptive / pruriceptive condition in a sensory neuron (SN), the method comprising: introducing into SN a composition comprising: at least one site-directed nuclease targeting a site within the target gene, and optionally, at least one nucleic acid comprising a nucleotide sequence encoding a gene expression altering sequence that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene, and wherein the site-directed nuclease cleaves the target site of the target gene and the nucleic acid encoding the gene expression altering sequence is inserted at the target site; wherein the expression or function of said at least one target gene is altered.

30. The method of claim 29, wherein the site-directed nuclease is a CRISPR system comprising a ribonuclear protein comprising CRISPR nuclease complexed with a guide RNA (gRNA) or a single guide RNA (sgRNA).

31. The method of claim 30, wherein CRISPR nuclease is a Type II Cas9 nuclease or a Type V Cfpl nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

32. The method of 29, wherein the composition is a vector composition comprising:(a) a polynucleotide sequence encoding a type of Cas9 protein variant or a fusion protein comprising at least one variant from of Cas9 protein;(b) a polynucleotide sequence encoding at least one guide RNA (gRNA) or a single guide RNA (sgRNA);(c) one or more promoters, each promoter operably linked to the polynucleotide sequence encoding the at least one gRNA and the polynucleotide sequence encoding the Cas9 protein or fusion protein;(d) optionally, a polynucleotide sequence encoding at least one gene expression altering sequence that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene.

33. The method of claim 32, wherein variants of Cas9 protein comprise SpCas9, SaCas9.

34. The method according to claim 32, wherein the vector is a viral vector.

35. The method according to claim 34, wherein the viral vector is an Adeno-associated virus (AAV) vector.

36. The method according to claim 35, wherein the AAV vector is an AAV8 vector, an AAV1 vector, an AAV6.2 vector, an AAVrh74 vector, an AAV9 vector, or an AAV PHP.s.

37. The method according to any one of claims 32-36 wherein the vector composition comprises a single vector comprising:(a) a polynucleotide sequence encoding a variant from the class of Cas9 proteins or a fusion protein comprising a variant from the class of Cas9 proteins;(b) a polynucleotide sequence encoding at least one guide RNA (gRNA) or a single guide RNA (sgRNA);(c) one or more promoters, each promoter operably linked to the polynucleotide sequence encoding the at least one gRNA and the polynucleotide sequence encoding the Cas9 protein or fusion protein;(d) optionally, a polynucleotide sequence encoding at least one gene expression altering sequence that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene.

38. The method according to any one of claims 32-37 wherein the vector composition comprises two or more vectors comprising(a) a polynucleotide sequence encoding a type of Cas9 variant protein n or a fusion protein comprising the Cas9 protein;(b) a polynucleotide sequence encoding at least one guide RNA (gRNA) or a single guide RNA (sgRNA);(c) one or more promoters, each promoter operably linked to the polynucleotide sequence encoding the at least one gRNA and the polynucleotide sequence encoding the Cas9 protein or fusion protein;(d) optionally, a polynucleotide sequence encoding at least one gene expression altering sequence that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene.

39. The method according to any one of claims 32-37 wherein the vector composition comprises: a first vector comprising (i) a polynucleotide sequence encoding a type of Cas9 protein variant or a fusion protein comprising the variant of Cas9 protein; and (ii) a promoter operably linked to the polynucleotide sequence encoding the variant of Cas9 protein or a fusion protein comprising the variant of Cas9 protein a second vector comprising (i) a polynucleotide sequence encoding at least one guide RNA (gRNA) or a single guide RNA (sgRNA); (ii) a promoter operably linked to the polynucleotide sequence encoding the at least one guide RNA (gRNA) or a single guide RNA (sgRNA) and, optionally, (iii) a polynucleotide sequence encoding at least one gene expression altering sequence that is flanked by (a) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (b) a nucleotide sequence homologous with a region located downstream of the target site within the target gene.

40. The method according to any one of claims 1 -39, wherein the dual AAV-mediated delivery comprises TRPV1 sgRNA and SaCas9, consisting of SEQ ID NOs. 5, 6, 7.

41. The method according to any one of claims 1 -39, wherein the dual AAV-mediated delivery comprises SCN9A sgRNA and SaCas9, consisting of SEQ ID NOs. 5, 8, 9.

42. The method according to any one of claims 29-39, wherein the expression of the target gene is inhibited.

43. The method according to any one of claims 29-40, wherein the target gene is from SCN9A, TRPV1, MRGPRX1, KCNQ2 or any combination thereof.

44. The method according to any one of claims 29-40, wherein the target gene is SCN9A and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs.: 1.

45. The method according to any one of claims 29-41, wherein the target gene is TRPV1 and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO.3.

46. The method according to any one of claims 29-41, wherein the target gene is KCNQ2, the nucleic acid encoding the gene expression altering sequence comprising SEQ ID NO: 10, and the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs. : 10.

47. The method according to claim 46 wherein KCNQ2 function is altered by introducing the mutation c.2188A>G, p.Thr730Ala set forth in SEQ ID. NO. 10.

48. The method of claims 46 to 47, wherein the composition administered to SN further comprises a second gRNA comprising a spacer sequence corresponding to a target sequence consisting of SEQ ID NO. 10.

49. The method according to claim 48, wherein KCNQ2 function is altered by introducing a CAC (histidine) to CGC (arginine) mutation.

50. The method according to any one of claims 46 to 49, wherein the composition administered to SN further comprises at least one nucleic acid comprising a nucleotide sequence encoding a base editor operably linked to a promoter, wherein the base editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain.

51. The method of claim 50, wherein the base editor further comprises one or more nuclearlocalization sequences (NLS).

52. The method of claims 50 to claim 51 , wherein the base editor is ABE8e, ABE8e(V 106W), ABE9, ABE20, ABE7.10, or a variant thereof.

53. The method according to any one of claims 50 -to 52, wherein the base editor is SaKKH- ABE8e, SauriCas9-ABE8e, CjCas9-ABE8e, Nme2Cas9-ABE8e, or SaCas9-ABE8e, or a variant thereof.

54. The method according to any one of claims 50 to 53, wherein the base editor is SauriCas9- ABE8e.

55. The method according to any one of claims 29 to 54, wherein the sensory neurons expressing mutation have diminished and protracted Ca2+response to KC1 stimulation as compared to neurons expressing wild type KCNQ2.

56. The method according to any one of claims 29-55, wherein the sensory neurons is produced according to the method of claim 1.

57. The method according to any one of claims 29-55, wherein the sensory neurons is produced according to the method of claims 29 to 30.

58. A population of sensory neurons produced by the method according to any one of claims 29-57.

59. Method of producing an enriched population of mature human pluripotent stem cell- derived nociceptive sensory neurons (hPSC-NSNs) expressing CD200 gene (CD200+), the method comprising: contacting hPSC-NSNs produced by the method of claim 1 with a CD200-specific antibody; andseparating the antibody-bound cells from the non-bound cells in order to obtain the purified cells.

60. The method according to claim 59, wherein the CD200-specific antibody is OX-104.

61. The method of claim 60, wherein the separation step is carried out by means of MACS or FACS.

62. The method according to claim 61, wherein the separation step is carried out by means of FACS.

63. The method according to any one of claims 59 to 62, wherein the enriched CD200+ hPSC- NSNs population is also expresses MiR-24 miRNA.

64. A method of treating nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition in a subject in need thereof, the method comprising administering to a subject the population of sensory neurons having the function of KCNQ2 altered according to methods of any one of claims 46 to 54 into a subject.

65. A method of treating nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition in a subject in need thereof, the method comprising administering to a subject the population of CD200+ sensory neurons of any one of claims 59 to 63.

66. The method according to claims 64 to 65, wherein the administering is by implanting the population of sensory neurons onto one or more affected locations of the subject.

67. The method according to claim 66, wherein the population of sensory neurons is implanted at the one or more sites of nociceptive pain and chronic pain.

68. The method according to any one of claims 64 to 67, wherein the population of sensory neurons is autologous to the subject.

69. The method according to any one of claims 64 to 67, wherein the population of sensory neurons is allogenetic to the subject.

70. The method according to any one of claims 64 to 69, wherein the nociceptive, chronic, and pruriceptive condition is osteoarthritis.

71. The method according to any one of claims 64 to 69, wherein the nociceptive, chronic, pruriceptive condition is derived from neuroinflammation or neurodegeneration, or a combination thereof.

72. The method according to any one of claims 1 -35, wherein the dual AAV-mediated delivery comprises KCNQ2, consisting of SEQ ID NOs. 11, 12, 13.

73. The method according to any one of claims 1 -35, wherein the dual AAV-mediated delivery comprises KCNQ2, consisting of SEQ ID NOs. 14, 15, 16.

74. A method for screening for a drug candidate that modulates one or more MRGPRX1, TRPV1 or SCN9A-mediated conditions the method comprising: contacting at least one population of hPSC-SNs expressing one of MRGPRX1, TRPV1 or SCN9 A produced according to any one of claims 1-24 with a drug candidate; and detecting a response to a nociceptive and pruriceptive stimulus in said at least one population of hPSC-SNs expressing one of MRGPRX1, TRPV1 or SCN9Ato thereby select said drug candidate for modulating said response.