Methods of treating central nervous system cancers using muscarinic receptor antagonists

Abstract

Disclosed herein are methods of treating cancers of the central nervous system (e.g. CNS tumors) in a subject involving providing to the subject a muscarinic acetylcholine receptor M1 and / or a muscarinic acetylcholine receptor M3 antagonist to the subject. Further disclosed herein are co-cultures comprising cholinergic neurons and glioma cells that may be used for therapy screening / testing, and methods of producing the same.

Description

PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196METHODS OF TREATING CENTRAL NERVOUS SYSTEM CANCERS USING MUSCARINIC RECEPTOR ANTAGONISTSSTATEMENT REGARDING RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 690,062, filed September 3, 2024, the entire contents of which are incorporated herein by reference for all purposes.TECHNICAL FIELD

[0002] Disclosed herein are methods of treating cancers of the central nervous system (e.g. CNS tumors) in a subject involving providing to the subject a muscarinic acetylcholine receptor Ml and / or a muscarinic acetylcholine receptor M3 antagonist to the subject.BACKGROUND

[0003] High-grade gliomas, including glioblastoma and diffuse midline glioma (DMG) are the most common malignant brain tumor types and leading causes of brain-tumor-related death in adults and children, respectively. DMGs, which are driven by oncogenic mutations in genes encoding histone H3 (H3K27M), originate most commonly in the pons of the brainstem and are also referred to as diffuse intrinsic pontine glioma (DIPG). H3K27M-altered DMGs also occur in the thalamus and spinal cord. Regardless of their specific location in these midline structures, DMGs arise from and closely resemble oligodendrocyte precursor cells (OPCs).

[0004] OPCs communicate extensively with neurons, both through neuron-to-OPC synapses and via paracrine factors such as brain-derived neurotrophic factor (BDNF). Within these neuron-to-OPC networks, neuronal activity promotes the proliferation of normal OPCs, oligodendrogenesis, and adaptive myelin changes that contribute to a range of functions including memory and learning in the healthy brain. In a similar pattern, neuronal activity also drives glioma proliferation and invasion. To date, research efforts have chiefly focused on the influence of glutamatergic and GABAergic neurons on glioma pathophysiology, while the effects of neuromodulatory neuron types - cholinergic, serotonergic, adrenergic andPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 dopaminergic neurons - remain unknown.SUMMARY

[0005] In some aspects, provided herein are methods of treating a central nervous system cancer in a subject, comprising providing to the subject at least one muscarinic acetylcholine receptor antagonist (MRA). In some embodiments, the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist and / or a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist. In some embodiments, the at least one MRA comprises a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist and a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the at least one MRA is an antagonist of muscarinic acetylcholine receptor Ml and muscarinic receptor antagonist M3 (e.g. is a single agent that is an antagonist at both receptors).

[0006] In some embodiments, the nervous system cancer is a glioma. For example, in some embodiments, the glioma is a diffuse midline glioma (DMG). In some embodiments, the subject is a pediatric subject. For example, in some embodiments the subject is a pediatric subject at the glioma is a DMG. In some embodiments, the glioma is a glioblastoma. In some embodiments, the subject is an adult subject. For example, in some embodiments the subject is an adult subject and the glioma is a glioblastoma.

[0007] In some embodiments, the MRA penetrates the blood-brain barrier. In some embodiments, the at least one MRA is selected from diclyomine, scopolamine, biperiden, trihexyphenidyl, clemastine, PIPE-359, Ar-[3-oxo-3-[4-(4-pyridinyl)-l-piperazinyl]propyl]-2,l,3- benzothiadiazole-4-sulfonamide (VU0255035), dicycloverine, benztropine, muscarinic toxin 7 (MT7), 4-DAMP, darifenacin, procyclidine, zamifenacin, solifenacin, fesoterodine, tramadol, hyoscyamine, diphenhydramine, cyproheptadine, mequitazine, oxybutynin, tolterodine, dicylomine, atropine, mecamylamine, olanzapine, amitriptyline, desipramine, desmethyldespiramine, dosulepin, doxepin, femoxetine, imipramine, lofepramine, nortriptyline, paroxetine, sertraline, chlorprothixene, chlorpromazine, clozapine, cymamazine, N-PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 dexmethylclozapine, flurperlapine, loxapine, mesoridazine, olanzapine, thoridazine, and zotepine.

[0008] In some aspects, provided herein is a muscarinic acetylcholine receptor antagonist (MRA) for use in a method of treating a central nervous system cancer in a subject. In some aspects, provided herein is a muscarinic acetylcholine receptor antagonist (MRA) for use in the manufacture of a medicament for the treatment of a central nervous system cancer in a subject.

[0009] In some embodiments, the MRA comprises a muscarinic acetylcholine receptor Ml antagonist and / or a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the MRA comprises muscarinic acetylcholine receptor Ml antagonist. In some embodiments, the MRA comprises a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the MRA comprises a muscarinic acetylcholine receptor Ml antagonist and a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the MRA is an antagonist of muscarinic acetylcholine receptor Ml and muscarinic acetylcholine receptor M3 (e.g. is a single agent that is an antagonist at both receptors).

[0010] In some embodiments, the nervous system cancer is a glioma. For example, in some embodiments, the glioma is a diffuse midline glioma (DMG). In some embodiments, the subject is a pediatric subject. For example, in some embodiments the subject is a pediatric subject at the glioma is a DMG. In some embodiments, the glioma is a glioblastoma. In some embodiments, the subject is an adult subject. For example, in some embodiments the subject is an adult subject and the glioma is a glioblastoma.

[0011] In some embodiments, the MRA penetrates the blood-brain barrier. In some embodiments, the MRA is selected from diclyomine, scopolamine, biperiden, trihexyphenidyl, clemastine, PIPE-359, / V-[3-oxo-3- [4- (4 -pyridinyl) - 1 -piperazinyl] ropyl] -2, 1,3- benzothiadiazole-4-sulfonamide (VU0255035), dicycloverine, benztropine, muscarinic toxin 7 (MT7), 4-DAMP, darifenacin, procyclidine, zamifenacin, solifenacin, fesoterodine, tramadol, hyoscyamine, diphenhydramine, cyproheptadine, mequitazine, oxybutynin, tolierodine, dicylomine, atropine, mecamylamine, olanzapine, amitriptyline, desipramine, desmethyldespiramine, dosulepin, doxepin, femoxetine, imipramine, lofepramine, nortriptyline, paroxetine, sertraline, chlorprothixene, chlorpromazine, clozapine, cymamazine, N- dexmethylclozapine, flurperlapine, loxapine, mesoridazine, olanzapine, thoridazine, and zotepine.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0012] In some aspects, provided herein is a co-culture comprising cholinergic neurons and glioma cells.

[0013] In some aspects, provided herein is a method of producing a cholinergic neuronglioma co-culture. In some embodiments, provided herein is a method comprising culturing cholinergic neurons and glioma cells in a media mixture comprising cholinergic neuron maintenance medium, and a tumor stem cell growth factor-free medium comprising DMEM, neurobasal-A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture. In some embodiments, the method comprises culturing the cholinergic neurons and glioma cells in the media mixture for at least 24 hours. In some embodiments, the method comprises culturing the cholinergic neurons and glioma cells in the media mixture for at least 48 hours.

[0014] In some embodiments, the cholinergic neuron maintenance medium and the tumor stem cell growth factor-free medium are present in the media mixture at a ratio of about 0.8: 1.2 to 1.2:0.8. (v / v), respectively. In some embodiments, the cholinergic neuron maintenance medium and the stem cell growth factor-free medium are present in the media mixture at a ratio of about 1:1.

[0015] In some embodiments, the cholinergic neurons are obtained by differentiating human induced pluripotent stem cells (hiPSCs) into the cholinergic neurons, wherein the differentiating comprises delivering a neurogenic transcription factor to the hiPSCs followed by culturing for 7 or more days. In some embodiments, the neurogenic transcription factor is delivered virally. In some embodiments, the cholinergic neurons are transduced with a vector configured for optogenetic manipulation to co-culturing the cholinergic neurons and the glioma cells in the media mixture.

[0016] In some embodiments, provided herein is a method comprising transducing cholinergic neurons with a vector configured for optogenetic manipulation; culturing the transduced cholinergic neurons and glioma cells in a media mixture comprising cholinergic neuron maintenance medium, and a tumor stem cell growth factor-free medium comprising DMEM, neurobasal-A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture; and performing optogenetic manipulation on the cholinergic neuron-glioma co-culture. In some embodiments, the method comprises culturing the transduced cholinergic neurons and the glioma cells in the media mixture for at least 24 hours.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0017] In some embodiments, the method comprises culturing the transduced cholinergic neurons and the glioma cells in the media mixture for at least 48 hours. In some embodiments, the cholinergic neuron maintenance medium and the tumor stem cell growth factor-free medium are present in the media mixture at a ratio of about 0.8: 1.2 to 1.2:0.8. (v / v), respectively. In some embodiments, the cholinergic neuron maintenance medium and the stem cell growth factor- free medium are present in the media mixture at a ratio of about 1:1. In some embodiments, the cholinergic neurons are obtained by differentiating human induced pluripotent stem cells (hiPSCs) into the cholinergic neurons, wherein the differentiating comprises delivering a neurogenic transcription factor to the hiPSCs followed by culturing for 7 or more days. In some embodiments, the neurogenic transcription factor is delivered virally.

[0018] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1-1H show cholinergic neuronal activity-regulated modulation of oligodendrocyte precursor cells. (FIG. 1A) Schematic of experimental paradigm for Optogenetic stimulation of cholinergic neurons in either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN). 5-week-old ChAT-IRES-Cre+ / wtx Ai230flx / wtmice (P35-38) were stimulated for 30 minutes followed by perfusion after 3 hours. (FIG. IB) Optogenetic stimulation of cholinergic neurons in LDT increases OPC proliferation (EdU+ / Pdgfra+) in thalamus which is not seen after PPN stimulation (CTL (not stimulated), PPN stimulated, and LDT stimulated, n=5 mice / group). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 1C) Optogenetic stimulation of cholinergic neurons in PPN increases OPC proliferation (EdU+ / Pdgfra+) in pons (CTL, PPN, and LDT, n=5 mice). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. ID) Confocal micrographs show thalamic OPC response after optogenetic stimulation of LDT (top image) and in pons after optogenetic stimulation of PPN (bottom image). ChRmine-oScarlet: green; Pdgfra: white; EdU: red, scale bars = lOOum. (FIG.IE) - (FIG. 1H) OPC response in (FIG. IE) prefrontal cortex, (FIG. IF) ventral tegmental area, (FIG. 1G) nucleus accumbens, and (FIG. 1H) hippocampus after optogenetic stimulation ofPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 cholinergic neurons (CTL, PPN, and LDT, n=5 mice / group). Unpaired two-tailed t-test; **p < 0.01, ns: non- significant. Data shown as mean, error bars indicate range.

[0020] FIGS. 2A-2H show interactions between cholinergic midbrain neurons and diffuse midline glioma. (FIG. 2A) Schematic of experimental paradigm for optogenetic stimulation of cholinergic neurons in either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN) in mice bearing H3K27M DMG. Four-week-old ChAT-IRES-Cre+ / wtx Ai230flx / wtmice (P28-30) were allografted with a H3K27M MADR tumour model into either the pons or thalamus, with optic ferrule placement into the LDT or PPN three weeks after allografting. Optogenetic stimulation for 30 minutes of the LDT or PPN was performed four weeks after allografting, followed by perfusion 24 hours after stimulation. (FIG. 2B) Proliferation index (EdU+ / GFP+) of thalamus allografts in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=5 mice / group). Unpaired two-tailed t-test; ****p < 0.0001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 2C) Proliferation index (EdU+ / GFP+) of pons allografts in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=5 mice / group). Unpaired two-tailed t-test; ****p < 0.0001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 2D) Confocal micrographs showing proliferating GFP+tumour cells in the right thalamus in non-stimulated (“CTL”) (upper images) and LDT- stimulated (“LDT”) (bottom images) mice. GFP: green, EdU: red, scale bars = 100pm. (FIG. 2E) Proliferation index (EdU+ / HNA+) of patient-derived pontine xenografts (SU- DIPG 17) in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=4 mice / group). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 2F) Confocal micrographs illustrate proliferating HNA+glioma cells in non-stimulated (“CTL”) (upper images) and PPN-stimulated (“PPN”) (bottom images) mice after xenografting patient-derived diffuse midline glioma line (SU- DIPG17) into pons. HNA: green, EdU: red, scale bars = 100pm. (FIG. 2G) Schematic of experimental paradigm for investigating reciprocal signalling effects of tumour cells to cholinergic midbrain neurons. Four-week-old ChAT-IRES-Cre+ / wtx Ai230flx / wtmice (P28-30) were either allografted with a H3K27 DMG or injected with buffered saline (HBSS) into pons and were perfused four weeks after surgery. Cholinergic neuronal activity of LDT and PPN were measured by cFos staining and normalized to four-week-old healthy ChAT-IRES-Cre+ / wtx Ai230flx / wtmice. (FIG. 2H) An increased neuronal activity of cholinergic neurons (cFos+inPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196ChAT+neurons) in LDT and PPN was observed in tumor-bearing mice when compared to healthy and saline-injected mice (healthy, n=5 mice; saline, PPN, and LDT, n=3 mice). Unpaired two-tailed t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

[0021] FIGS. 3A-3J show direct effects of acetylcholine on diffuse midline glioma. (FIG. 3A) Confocal micrographs showing cholinergic neurons generated from human induced pluripotent stem cells (hiPSCs) of a healthy 12-year-old male. MAP2: turquoise, ChAT: red, merged: white, scale bars = 100pm. (FIG. 3B) Representative confocal micrographs demonstrating dense neuron-to-glioma networks after co-culturing hiPSC-derived cholinergic neurons and DMG cells. MAP2: turquoise, Nestin: white, EdU: red, scale bars = 100pm. (C) Quantification of glioma cell proliferation (EdU+ / Nestin+ / MAP2‘) when co-cultured with hiPSC- derived cholinergic neurons. Unpaired two-tailed t-test; ***p < 0.001. Data shown as mean, error bars indicate range. (FIG. 3D) Confocal images of cholinergic neuron-glioma co-culture with PSD95-RFP-expressing DMG cells (SU-DIPG13FL-PSD95). Yellow boxes indicate colocalizations of synapsin (presynaptic cholinergic neuron) and PSD95 (postsynaptic glioma cell expressing PSD95-RFP). Nestin: white, MAP2: turquoise, PSD95-RFP: red, synapsin: green, scale bars = 20pm (left) and 4pm (right). (FIG. 3E) 3-dimensional rendering of the image in (D), illustrating presynaptic cholinergic neuron (MAP2, turquoise, asterisk) with presynaptic puncta (green, synapsin) co-localizing with postsynaptic puncta (PSD95-RFP, red) expressed by postsynaptic glioma cells (nestin, white, arrow). (FIG. 3F) Proliferation index (EdU+ / DAPI+) of a patient-derived DMG cell line (SU-DIPG17) after exposure to different concentrations of acetylcholine. (FIG. 3G) Representative confocal micrographs showing the proliferation of a patient-derived cell line without acetylcholine (upper images) and after exposure to 5pM acetylcholine (bottom images). DAPI: blue, EdU: red, scale bars = 100pm. (FIG. 3H) Proliferation index (EdU+ / DAPI+) of a patient-derived cell line (SU-DIPG17) after exposure to various muscarinic and nicotinic receptor agonists (nicotine and muscarine) and antagonists (mecamylamine and scopolamine). One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; **p < 0.01, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 31) 3D migration assay analysis comparing distance of glioma cell spread 72 h after seeding after exposure to acetylcholine. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; *p < 0.05, **p < 0.01. Data shown as mean, error bars indicate range. (FIG.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-1963J) Representative images showing the glioma cell migration at timepoint zero and after 72h in control- and acetylcholine-treated wells. Scale bars = 1000pm.

[0022] FIGS. 4A-4D show cholinergic receptor gene expression and receptor mechanisms in gliomas. (FIG. 4A) Heatmap of pseudo-bulk analysis of cholinergic receptors gene expression across central nervous system tumours from various studies with single-cell or single-nucleus RNA sequencing data. The red- marked boxes indicate studies with DMG samples. (FIG. 4B) Scatter plot correlating cholinergic receptor gene expression values with an OPC-like score in DMG samples. (FIG. 4C) Two-dimensional representation of the association between CHRM1 expression (red dots: centered value > 1) and the OPC-like (y axis) as well as OC-like and AC- like (x axis) scores for H3K27M DMGs. (FIG. 4D) Two-dimensional representation of the association between CHRM3 expression (red dots: centered value > 1) and the OPC-like (y axis) as well as OC-like and AC-like (x axis) scores for H3K27M DMGs.

[0023] FIGS. 5A-5D show validation of experimental paradigms. (FIG. 5A) Schematic of experimental paradigm for validation of optogenetic stimulation of cholinergic neurons in either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN). 5-week-old ChAT- IRES-Cre+ / wtx Ai230flx / wtmice (P35-38) were stimulated for 30 minutes followed by perfusion after 90 minutes to assess neuronal activity changes by cFos staining. (FIG. 5B) Quantification of activated cholinergic neurons (cFos+ / mCherry+ / ChAT+) after optogenetic stimulation of LDT (CTL, LDT, n=3 mice). Unpaired two-tailed t-test; ***p < 0.001. Data shown as mean, error bars indicate range. (FIG. 5C) Quantification of activated cholinergic neurons (cFos+ / mCherry+ / ChAT+) after optogenetic stimulation of PPN (CTL, PPN, n=3 mice). Unpaired two-tailed t-test; **p < 0.01. Data shown as mean, error bars indicate range. (FIG. 5D) Top panel shows confocal micrographs showing cFos staining in cholinergic neurons of nonstimulated mice (left image), PPN-stimulated mice (middle image), and LDT-stimulated mice (right image). ChRmine-oScarlet: red, cFos: green, scale bars = 50pm. Bottom panel shows confocal micrographs highlighting the OPC-like character (Pdgfra+) in tumour cells (GFP+) of the H3K27M MADR model. GFP: green, Pdgfra: white, scale bars = 50pm.

[0024] FIGS. 6A-6I show midbrain cholinergic neurons promote DMG growth in part through paracrine factors such as BDNF. (FIG. 6A) Schematic of experimental paradigm for xenografting with optogenetic stimulation of either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN) in immunodeficient mice. Four-week-old NSG mice (P28-30)PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 were injected with AAV-Efla-ChR2::YFP or AAV-Efla-YFP into LDT or PPN and xenografted into the pons with patient-derived H3K27-altered DIPG cells (SU-DIPG17) three weeks after viral vector delivery. After five weeks of glioma growth, optical ferrules were placed either in the LDT or PPN and optogenetic stimulation was performed one week later. Mice were perfused 24 hours after optogenetic stimulation. (FIG. 6B) Schematic of experimental paradigm for collection of conditioned media (CM) after ex vivo optogenetic stimulation of cholinergic neuronal cell bodies within the LDT or PPN in midbrain explants of 4-week-old ChAT-IRES- Cre+ / wtx Ai230flx / wtmice. (FIG. 6C) Quantification of DMG cell proliferation (EdU+ / DAPI+) when adding CM after ex vivo stimulation of LDT or PPN compared to CM from non- stimulated slices. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; **p < 0.01, ***p < 0.001, ns: non- significant. Data shown as mean, error bars indicate range. (FIG. 6D) Confocal micrographs showing EdU-labeled DMG proliferation after adding CM from nonstimulated slices (upper images) and from LDT stimulation (bottom images). DAPI: blue, EdU: red, scale bars = 100 pm. (FIG. 6E) Proliferation index (EdU+ / DAPI+) after fractionation of the CM by molecular weight using size-exclusion ultracentrifugal filters on CM from LDT and PPN explants. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; **p < 0.01, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 6F) Quantification of glioma cell proliferation (EdU+ / DAPI+) with addition of neurexin (which binds to and sequesters NLGN34) or ANA-12 (specific TrkB -inhibitor) to the CM from LDT and PPN. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; **p < 0.01, ***p < 0.001. Data shown as mean, error bars indicate range. (FIG. 6G) - (FIG. 6H) Measurement of BDNF and acetylcholine levels in CM from (G) LDT, and (H) PPN. (FIG. 61) Western blot analysis of NLGN3 in CM from non-stimulated midbrain explants (“Control”) as well as CM from optogenetically stimulated PPN explants and from LDT explants.

[0025] FIGS. 7A-7G show acetylcholine promotes DMG cell proliferation in monoculture and cortical neuron co-culture in a dose dependent manner. (FIG.7A) - (FIG. 7C) Proliferation index (EdU+ / DAPI+) of three patient-derived H3K27M DMG cell cultures (FIG. 7A: SU- DIPG92, FIG. 7B: SU-DIPG13fl, FIG. 7C: SU-DIPG19) after exposure to different concentrations of acetylcholine. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant. Data shown as mean, error bars indicate range. (FIG. 7D) Proportions of neuronal subpopulations within thePCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 cortical neuron cultures from mouse pups used for the co-culture experiment shown in E-G. (FIG. 7E) Confocal micrographs representing the proliferative effect of adding acetylcholine (IpM) to a neuron-glioma co-culture. HNA: blue, EdU: red, MAP2: green, scale bars = 100pm. (FIG. 7F) - (FIG. 7G) Proliferation index (EdU+ / HNA+) of patient-derived DMG cell cultures when co-cultured with cortical neurons from mouse pups and after exposure to different concentrations of acetylcholine. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; p < 0.05, ***p < 0.001, ****p < 0.0001. Data shown as mean, error bars indicate range.

[0026] FIGS. 8A-8C show Muscarinic targets for DMG therapy. (FIG. 8A) Heatmap illustrating intra- and intertumoral heterogeneity of acetylcholine receptor gene expression in six scRNA-seq DMG samples (Filbin dataset, Filbin, M.G., et al. (2018). Science 360, 331-335.) The data were centered across all cells per sample. (FIG. 8B) Scatter plot correlating cholinergic receptor gene expression values with an OPC-like score in medulloblastoma samples. (FIG. 8C) Correlation of cholinergic receptor gene expressions with different cell like-states (AC, Gl / S, G2 / M, MES-1, MES-2, NPC-1, NPC-2, and OPC) in all available CNS tumour entities. The radius of each circle corresponds to the -log10(p-value), and the circle filling is the correlation. Non-significant p-values are not shown. Red dashed boxes indicate studies containing DMG samples.

[0027] FIGS. 9A-9D show pharmacological inhibition of CHRM1 and CHRM3 abolishes the effects of cholinergic neuronal activity on DMG cells in vitro. (FIG. 9A) Timeline for the generation of hiPSC-derived cholinergic motor neurons and co-culturing with glioma cells.(FIG. 9B and FIG. 9C) Proliferation index (EdU+ / H3K27M+) of a patient-derived DMG cell line (SU-DIPG17) when co-cultured and treated with Ml (VU0255035) and M3 (4- DAMP) receptor antagonists, or vehicle control. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; ****p < 0.0001, ***p < 0.001, ns = nonsignificant. Data = mean ± SEM; n = five independent experiments, each with three wells per condition; each data point represents the mean of three wells per condition for a given experiment. (FIG. 9D) Co-localization of VAChT (presynaptic cholinergic neurons, indicated by NF-H / M+) and CHRM1 / 3 (post- synaptic glioma cells, indicated by H3K27M+) in the DMG cell line when co-cultured with hiPSC-derived cholinergic motor neurons and treated with Ml (VU0255035) and M3 (4-DAMP) receptor antagonists, or vehicle control. Unpaired two-tailed Welch’s t test; **p < 0.01. Data = mean +PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196SEM; n = four independent experiments, each with three wells per condition; each data point represents the mean of three wells per condition for a given experiment.

[0028] FIGS. 10A-10E show CHRM1 and CHRM3 mediate the effects of cholinergic neuronal activity in pontine and thalamic DMG. (FIG. 10A) Schematic of the experimental paradigm for local antagonism of muscarinic receptors Ml and M3 in thalamic allografts. (FIG. 10B) Proliferation index (EdU+ / GFP+ ) of thalamic allografts in mice optogenetically stimulated in LDT or mock-stimulated controls, with or without administration of Ml (VU0255035) or M3 receptors (4-DAMP) pharmacological inhibitors. “Mock,” “Vehicle,” and “M1 / M3 inhibition” n = 5 mice / group; “Ml inhibition” and “M3 inhibition” n = 4 mice / group. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; ****p < 0.0001, ns: non- significant. Data = mean ± SEM. (FIG. 10C) Fiber photometry recordings showing averaged GCaMP-labeled calcium transients in thalamic glioma cells with simultaneous LDT stimulation and either local vehicle or CHRM1 / 3 antagonist infusion (n = 4 mice / group). (FIG. 10D) Proliferation index (EdU+ / HNA+ ) of pontine xenografts in mice optogenetically stimulated in the PPN or mock-stimulated controls, with either control (Cas9- expressing) patient-derived DMG cells (DIPG17) or dual CHRM1 / 3 CRISPR-mediated knockout (DIPG17- CHRM1 / 3-K0). “DIPG17” n = 4 mice / group; “DIPG17- CHRM1 / 3-KO” n = 5 mice / group. One-way ANOVA with Tukey’s post hoc analysis; ****p < 0.0001, ns: not significant. Data = mean ± SEM. (FIG. 10E) Confocal micrographs showing proliferating HNA+ tumor cells in the pons of either control (Cas9-expressing) patient-derived DMG cells (DIPG17) or dual CHRM1 / 3 CRISPR-mediated knockout cells (DIPG17-CHRM1 / 3-KO) in PPN-stimulated mice. HNA, green; EdU, red, scale bars, 50 pm.DETAILED DESCRIPTION

[0029] The data presented herein demonstrates that the activity of cholinergic midbrain neurons modulates both healthy OPC and malignant DMG proliferation in a circuit- specific manner at sites of long-range cholinergic projections. Single-cell RNA sequencing analyses revealed prominent expression of the muscarinic receptor genes CHRM1 and CHRM3 in primary patient DMG samples, particularly enriched in the OPC-like tumor subpopulation. Acetylcholine, the neurotransmitter cholinergic neurons release, exerts a direct effect on DMG tumor cells, promoting increased proliferation and invasion through muscarinic receptors.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196Pharmacological blockade of Ml and M3 acetylcholine receptors abolished the activity -regulated increase in DMG proliferation in cholinergic neuron- glioma co-culture and in vivo. These findings demonstrate that midbrain cholinergic neuron long-range projections to midline structures promote activity-dependent DMG growth through Ml and M3 cholinergic receptors, and that inhibition of Ml and / or M3 cholinergic receptors can be used for treatment of cancers of the central nervous system, including DMG.

[0030] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0031] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.

[0032] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0033] As used herein, the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. OtherPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.

[0034] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0035] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi- specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, antibody fragments, Fab fragments, F(ab’) fragments, F(ab’)i fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290- 1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitopebinding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0036] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The polypeptides may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain.

[0037] An “effective amount” refers to an amount sufficient to elicit a desired biological response (e.g., treating a condition). As will be appreciated by those skilled in the art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, a “therapeutically effective amount” is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0038] A “subject” or “patient” broadly refers to any living organism, and more specifically to an animal including human and non-human animals. In some embodiments, the subject is a mammal. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human. The subject may be an adult subject (e.g. an adult human of 18 years of age or older). The subject may be a juvenile (e.g. pediatric) subject. The term “juvenile” or “pediatric” when used in reference to a human subject refers to a subject 17 years of younger. In some embodiments, the subject has or is suspected of having cancer.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0039] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or condition, or one or more signs or symptoms thereof. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease disorder or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. “Treating” a cancer may refer to decreasing the size of the tumor(s), decreasing the number of tumors, decreasing the risk of metastasis of the tumor(s), etc.

[0040] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of disclosed muscarinic receptor antagonists into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. The administration can be by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.

[0041] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Methods of Treating Cancer

[0042] In some aspects, provided herein are methods of treating cancer in a subject. In some embodiments, provided herein are methods of treating a central nervous system (CNS) cancer in a subject, comprising providing to the subject at least one muscarinic acetylcholine receptor antagonist (MRA). Muscarinic acetylcholine receptors (mAchRs) refers to a family of G-protein coupled receptors (GPCRs) which are broadly expressed in the central nervous system and the peripheral nervous system. Muscarinic acetylcholine receptors are divided into five subtypes, muscarinic acetylcholine receptor Ml (“Ml”), muscarinic acetylcholine receptor M2 (“M2”),PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 muscarinic acetylcholine receptor M3 (“M3”), muscarinic acetylcholine receptor M4 (“M4), and muscarinic acetylcholine receptor M5 (“M5”). Muscarinic acetylcholine receptors are stimulated by the natural agonist acetylcholine. Muscarinic acetylcholine receptors are so named due to their sensitivity to muscarine compared to nicotine. In contrast, nicotinic acetylcholine receptors (nAChrs) are sensitive to nicotine rather than muscarine.

[0043] In some embodiments, the at least one MRA is a muscarinic acetylcholine receptor Ml antagonist and / or a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist and a muscarinic acetylcholine receptor M3 antagonist. In some embodiments, the method comprises providing to the subject a single MRA, wherein the MRA is an antagonist of both Ml and M3. In some embodiments, the method comprises providing to the subject a single MRA, wherein the MRA is an antagonist of Ml. The MRA may be an antagonist at other muscarinic acetylcholine receptors in addition to Ml (e.g. one or more of M2, M3, M4, and / or M5). In some embodiments, the method comprises providing to the subject a single MRA, wherein the MRA is an antagonist of M3. The MRA may be an antagonist at other muscarinic acetylcholine receptors in addition to M3 (e.g. one or more of Ml, M2, M4, and / or M5). In some embodiments, the method comprises providing to the subject more than one MRA. For example, in some embodiments the method comprises providing to the subject a first MRA that is an antagonist of Ml and a second MRA that is an antagonist at M3. The first and second antagonist may be provided in a single composition or may be provided to the subject in separate compositions, which may be provided at separate time points or concurrently.

[0044] In some embodiments, the at least one MRA comprises a non-selective muscarinic acetylcholine receptor antagonist. The term “non-selective” indicates that the MRA is an antagonist at each muscarinic acetylcholine receptor subtype (e.g. each of Ml, M2, M3, M4, and M5). In some embodiments, a non-selective MRA has comparable activity (e.g. comparable binding affinity) at each subtype. However, the term “non-selective” does not necessarily indicate that the binding affinity is equal for each subtype, rather the non-selective MRA may have differential binding affinity for the different receptor subtypes. In some embodiments, the MRA comprises a non-selective MRA with increased affinity for Ml compared to other subtypes. In some embodiments, the MRA comprises a non-selective MRA with increasedPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 affinity for M3 compared to other subtypes. In some embodiments, the MRA comprises a non- selective MRA with increased affinity for Ml and M3 compared to other subtypes.

[0045] In some embodiments, the MRA is a selective antagonist. The term “selective” indicates that the MRA is has specificity for one or more given muscarinic acetylcholine receptor subtypes. For example, in some embodiments the MRA comprises a selective Ml antagonist. As another example, in some embodiments the MRA comprises a selective M3 antagonist. The MRA may be an antagonist at more than muscarinic acetylcholine receptor subtypes and still be considered “selective”, so long as it is not broadly antagonistic to each subtype. For example, the MRA may be a selective antagonist for Ml and between one and three additional subtypes selected from M2, M3, M4, and M5. As another example, the MRA may be a selective antagonist for M3 and between one and three additional subtypes selected from Ml, M2, M4, and M5. In some embodiments, the MRA is selective for both Ml and M3 subtypes.

[0046] The blood brain barrier is a selective semi-permeable membrane between the blood and the brain. The blood brain barrier plays an important role in regulation of transport between the blood and the central nervous system and can prevent the central nervous system from toxicity. However, the blood brain barrier can also prevent drugs or agents with a desired action in the central nervous system from effectively reaching the target tissue. In some embodiments, the MRA is able to penetrate (e.g. pass through) the blood brain barrier and exert effects centrally (e.g. within the central nervous system). For example, in some embodiments the MRA penetrates the blood brain barrier and exerts anti-tumor effects at or near the site of the central nervous system tumor(s). In some embodiments, the MRA provided to the subject along with an agent that increases blood brain barrier permeability or otherwise facilitates transport of the MRA across the blood brain barrier and into the central nervous system.

[0047] In some embodiments, the MRA is a natural product. A natural product refers to a substance that is derived or obtained from a natural source, as compared to a synthetic product which is man-made. While a majority of known MRAs are synthetic, common natural product MRAs include atropine and scopolamine, both of which are belladonna alkaloids extracted from plants (e.g. Atropa belladonna, or nightshade). In some embodiments, the MRA is a synthetic product. In some embodiments, the MRA is a small molecule (e.g. a compound), a peptide, a protein, or an antibody.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0048] In some embodiments, the muscarinic acetylcholine receptor antagonist is an antihistamine, an antidepressant, or an antipsychotic. For example, known antihistamines, antidepressants, and antipsychotics may also be muscarinic receptor antagonists. First generation antihistamines, along with antidepressants and antipsychotics (which exert their effects in the central nervous system) are commonly able to penetrate the blood brain barrier and may therefore also be useful muscarinic receptor antagonists for use in methods of treatment of central nervous system cancer (e.g. CNS tumors) as described herein.

[0049] In some embodiments, the muscarinic acetylcholine receptor antagonist is an Ml antagonist. Exemplary Ml antagonists include, but are not limited to, diclyomine, scopolamine, biperiden, trihexyphenidyl, clemastine, PIPE-359, AT-[3-oxo-3-[4-(4-pyridinyl)-l- piperazinyl]propyl]-2,l,3-benzothiadiazole-4-sulfonaimde (VU0255035), dicycloverine, benztropine, and muscarinic toxin 7 (MT7). In some embodiments, the muscarinic receptor antagonist is an M3 antagonist. Exemplary M3 antagonists include, for example, 4-DAMP, darifenacin, procyclidine, zamifenacin, solifenacin, and fesoterodine. In some embodiments, the MRA is an antagonist of both Ml and M3. Exemplary such MRAs include, but are not limited to, tramadol, hyoscyamine, diphenhydramine, cyproheptadine, mequitazine, oxybutynin, tollerodine, and dicylomine. In some embodiments, the MRA is a non-selective MRA.Exemplary non-selective MRAs include, for example, atropine, mecamylamine, and olanzapine. In some embodiments, the MRA is an antidepressant with muscarinic acetylcholine receptor antagonist activity. For example, in some embodiments the MRA is amitriptyline, desipramine, desmethyldespiramine, dosulepin, doxepin, femoxetine, imipramine, lofepramine, nortriptyline, or paroxetine. In some embodiments, the MRA is an antipsychotic with muscarinic acetylcholine receptor antagonist activity. For example, in some embodiments the MRA is chlorprothixene, chlorpromazine, clozapine, cymamazine, N-dexmethylclozapine, flurperlapine, loxapine, mesoridazine, olanzapine, thoridazine, or zotepine.

[0050] In some embodiments, the MRA is an antibody or a peptide that binds to and there by blocks or allosterically inhibits the muscarinic acetylcholine receptor. In some embodiments, the antibody or the peptide is conjugated to a moiety and / or delivered by a mechanism that increases blood brain barrier permeability, thereby facilitating transport of the antibody or the peptide across the blood brain barrier and to the site of action (e.g. to the muscarinic acetylcholine receptors in the central nervous system).PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0051] In some embodiments, the MRA is able to cross the blood brain barrier and therefore can be administered to the subject peripherally from the site of the tumor(s) (e.g. not injected directly into the brain or the spinal cord) and still reach the desired location to exert the therapeutic effect. In some embodiments, the MRA is administered to the subject orally. In some embodiments, the MRA is administered to the subject by topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose) or parenteral administration (e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrastemal injection), or by implant of a depot, for example, subcutaneously or intramuscularly.

[0052] In some embodiments, the administration is via the cerebrospinal fluid. In select embodiments, the administration comprises intrathecal, intracistemal, intranasal, or intracerebroventricular administration. Intrathecal drug administration can be accomplished by lumbar puncture or by an implanted intrathecal drug delivery device (IDDD).Intracerebroventricular administration facilitates administration of drugs into a lateral cerebral ventricle, e.g., by direct injection or via an implanted device (reservoir and catheter).

[0053] In some embodiments, the methods disclosed herein may further comprise opening or increasing the permeability of the blood-brain barrier (BBB) prior to administration, particularly for administration methods which target the agent(s) to the bloodstream. For example, mannitol has been used as an osmotic substance, for increasing BBB permeability. Convection enhanced delivery (CED) is another technique that has been explored to bypass the BBB. CED is performed by first inserting a small catheter directly into the targeted brain region and then by slowly infusing the drug directly into the tissue, thereby bypassing the BBB. Pulsed ultrasound (US) or ultrasound beams temporarily disrupts the BBB. Recently administration of gold nanoparticles having the therapeutic agent followed by laser pulses modulated the BBB permeability to successfully deliver a variety of agents to the brain.

[0054] In some embodiments, the MRA is comprised in a composition (e.g. a pharmaceutical composition) comprising a pharmaceutically acceptable carrier or excipient. Reference to providing or administering the MRA to the subject is inclusive of providing or administering a composition (e.g. a pharmaceutical composition) comprising the MRA to the subject. ThePCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 phrase “pharmaceutically acceptable,” as used in connection with compositions of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce undesirable reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. The pharmaceutically acceptable carrier should also be compatible with the active ingredient of the composition (e.g., the MRA). Any of the pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0055] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0056] The dose of the MRA provided to the subject may depend on the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration, the precise MRA used, and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount is sufficient to treat the cancer in the subject. For example, in some embodiments the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of the cancer in the subject. For example, in some embodiments the effective amount reduces the size and / or number of tumors in the subject, and / or prevents tumors from metastasizing in the subject. In some embodiments, the subject is a human.

[0057] It will be appreciated that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present disclosure. ThePCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 amount and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[0058] The MRA may be provided to the subject in be in a single dose or in multiple doses throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the exact MRA and route of administration used for therapy, the severity of the cancer, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0059] In some embodiments, a given dose is provided to the subject continuously or intermittently over the course of a suitable dosing window. For example, the dosing window may be 10 minutes to 6 hours, 20 minutes to 5 hours, 30 minutes to 4 hours, or about 1 to 3 hours. In some embodiments, the MRA is provided to the subject once per day. In some embodiments, the MRA is provided to the subject multiple times per day. In some embodiments, the MRA is provided to the subject every other day, every 3 days, every 4 days, every 5 days, ever)' 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, ever ' 6 months, annually, etc. MRA may be administered until a desired reduction of symptoms is achieved.

[0060] In some embodiments, the dose of the MRA given to the subject is about 1 pg of the MRA per kg body weight of the subject (e.g. 1 pg / kg) to about 100 mg / kg. For example, in some embodiments the dose is about 1 pg / kg to about 100 mg / kg, about 10 pg / kg to about 50 mg / kg, about 50 pg / kg to about 25 mg / kg, about 100 pg / kg to about 10 mg / kg, or about 500 pg / kg to about 1 mg / kg.

[0061] In some embodiments, the MRA is provided to the subject in combination with other anti-cancer therapies. Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This isPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0062] In some embodiments, the methods may further comprise administration with one or more additional therapies to treat the cancer or ease symptoms thereof. For example, the methods may comprise providing to the subject the MRA in combination with one or more medicines to control seizures, steroids to reduce brain swelling, agents to improve alertness, and / or agents to improve cognitive impairments. As another example, in some embodiments the methods comprise providing to the subject the MRA in combination with one or more therapies for the cancer, including surgery (e.g. surgical removal of the glioma), radiation therapy, chemotherapy, immunotherapy, tumor treating fields therapy (e.g. electrical energy applied to destroy glioma cells), and / or targeted therapy. The additional therapy may be administered at the same time as the MRA. For example, the additional therapy may be provided to the subject either in the same composition as the MRA or in a separate composition administered at substantially the same time as the composition comprising the MRA. In some embodiments, the additional therapy may precede or follow the treatment of the therapy with the MRA by time intervals ranging from hours to months.

[0063] In some embodiments, the cancer is a cancer of the central nervous system. A cancer of the central nervous system (e.g. a central nervous system cancer, CNS cancer, CNS tumor, etc.) refers to a tumor within the brain or the spinal cord. In some embodiments, the cancer is a glioma. A glioma refers to a cancer that forms from aberrant growth of glial cells. Gliomas are classified into multiple types. Exemplary glioma types include glioblastomas, astrocytomas,PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 diffuse midline glioma (DMG), ependymomas, mixed gliomas, and oligodendrogliomas. In some embodiments, the cancer is a central nervous system cancer and the subject is an adult subject. In some embodiments, the cancer is a central nervous system cancer and the subject is a pediatric subject. For example, in some embodiments the nervous system cancer is DMG and the subject is a pediatric subject.3. Cholinergic Neuron-Glioma Co-Culture

[0064] In some aspects, provided herein are co-cultures of cholinergic neurons and glioma cells. The term “co-culture” indicates that the cholinergic neurons and glioma cells exist in the same cell culture container (e.g. the same petri dish, culture plate, etc.). In some embodiments, the co-culture comprises cholinergic neurons obtained by a method described below. In some embodiments, the glioma cells are obtained from a patient having a glioma. For example, in some embodiments the glioma cells are obtained from a patient having a diffuse midline glioma. In some embodiments, the cholinergic neurons and the glioma cells are both viable. In some embodiments, the cholinergic neurons and the glioma cells are both viable under the same culture conditions (e.g. using the same culture medium).

[0065] In some aspects, provided herein are methods of producing a cholinergic neuronglioma co-culture. The cholinergic neuron-glioma co-cultures described herein and methods of producing the same are particularly useful for investigating the interaction between neurons (e.g. cholinergic neurons) and glioma cells in vitro. Moreover, the interaction between neurons and glioma cells can be tightly controlled, such as through optogenetic activation of the neurons in the co-culture, to further investigate the interaction between neurons and glioma cells in vitro. In some embodiments, the co-cultures described herein and methods of producing the same are useful for evaluating potential candidate therapeutics for treatment of cancer, such as glioma. In some aspects, provided herein is method of evaluating a potential anti-cancer therapeutic e.g. a potential therapeutic for the treatment of glioma), comprising contacting a cholinergic neuronglioma co-culture with the potential anti-cancer therapeutic and assessing a response to the potential anti-cancer therapeutic in the co-culture. In some embodiments, assessing the response comprises assessing a response in the cholinergic neurons, in the glioma cells, and / or in an interaction between the cholinergic neurons and the glioma cells. For example, potentialPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 therapeutic(s) (e.g. agent(s)) for treatment of glioma can be applied to a cholinergic neuronglioma co-culture described herein and a response in the cholinergic neurons, a response in the glioma cells, and or the interaction between the cholinergic neurons and the glioma cells can be evaluated before and / or after application of the potential therapeutic(s). A modification in the cholinergic neurons, a modification in the glioma cells, and / or a modification in the interaction between the cholinergic neurons and glioma cells after contacting the co-culture with the agent(s) may indicate that agent(s) has potential efficacy for the treatment of cancer (e.g. glioma) in a subject.

[0066] In some embodiments, provided herein is a method comprising culturing cholinergic neurons and glioma cells in a media mixture comprising cholinergic neuron maintenance medium, and a tumor stem cell growth factor-free medium comprising DMEM, neurobasal-A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture. In some embodiments, the method comprises culturing the cholinergic neurons and the glioma cells in the media mixture for at least 24 hours. In some embodiments, the method comprises culturing the cholinergic neurons and glioma cells in the media mixture for at least 48 hours.

[0067] In some embodiments, the cholinergic neuron maintenance medium is Quick- Neuron™ Cholinergic - Maintenance Medium (CH-MM, Elixirgen Scientific, https: / / www.elixirgensci.com / quick-neurontm-cholinergic-maintenance-medium-ch-mm).

[0068] In some embodiments, the tumor stem cell growth factor-free medium comprises DMEM, neurobasal-A, and B27-A and is substantially free of tumor growth factors, such as transforming growth factor (TGF-P), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and the like.

[0069] In some embodiments, the cholinergic neuron maintenance medium and the tumor stem cell growth factor-free medium are present in the media mixture at a ratio of about 0.8: 1.2 to 1.2:0.8. (v / v), respectively. For example, in some embodiments the cholinergic neuron maintenance medium and the tumor stem cell growth factor-free medium are present in the media mixture at a ratio of about 0.8:1.2 (v / v), about 0.85:1.15 (v / v), about 0.9:1.1 (v / v), about 0.95:1.05 (v / v), about 1:1 (v / v / ), about 1.05:0.95 (v / v), about 1.1:0.9 (v / v), about 1.15:0.85 (v / v), or about 1.2:0.8 (v / v). In some embodiments, the cholinergic neuron maintenance medium and the stem cell growth factor-free medium are present in the media mixture at a ratio of about 1:1.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0070] In some embodiments, the cholinergic neurons are obtained by differentiating human induced pluripotent stem cells (hiPSCs) into the cholinergic neurons. hiPSCs may be differentiated into neurons using any suitable differentiation protocol. In some embodiments, differentiating comprises delivering a neurogenic transcription factor to the hiPSCs followed by culturing in a suitable medium for 7 or more days. In some embodiments, the method comprises culturing the hiPSCs in a suitable medium in the presence of one or more neurogenic transcription factors that drive the differentiation of hiPSCs into cholinergic neurons. Exemplary neurogenic transcription factors include, for example, neurogenic differentiation 1 (NeuroDl), homeobox encoding Nkx2.1, Islet-1, Lhx8, Zic4, ER81, and the like. In some embodiments, the neurogenic transcription factor is synthetic or a mixture of synthetic transcription factors. In some embodiments, the neurogenic transcription factor is the synthetic factor CH-SeV (Elixirgen). In some embodiments, the neurogenic transcription factor is delivered virally. For example, in some embodiments the neurogenic transcription factor is a synthetic transcription factor delivered by Sendai virus mediated delivery.

[0071] In some embodiments, the cholinergic neuron-glioma co-culture is used for downstream applications, such as optogenetics. In some embodiments, the cholinergic neurons are transduced with a vector configured for optogenetic manipulation to co-culturing the cholinergic neurons and the glioma cells in the media mixture. In some embodiments, provided herein is a method comprising transducing cholinergic neurons with a vector configured for optogenetic manipulation; culturing the transduced cholinergic neurons and glioma cells in a media mixture comprising cholinergic neuron maintenance medium, and a tumor stem cell growth factor- free medium comprising DMEM, neurobasal-A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture: and performing optogenetic manipulation on the cholinergic neuron-glioma co-culture. Optogenetic manipulation may be performed to activate cholinergic neurons in the co-culture and investigate the interaction between the activated neurons (e.g. activated cholinergic neurons) and the glioma cells in the co-culture.

[0072] The following examples further illustrate aspects of the disclosure, but should not be construed as in any way limiting its scope.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196EXAMPLESExample 1

[0073] METHODS

[0074] Patient-Derived Diffuse Midline Glioma Cells: Diffuse midline glioma cultures were established from patient-derived specimens with informed consent, following a protocol approved by the Stanford University Institutional Review Board (IRB). The utilized patient- derived glioma models included SU-DIPG-1311, SU-DIPG-17, SU-DIPG-19, and SU-DIPG-92. Throughout the culture period, all cultures were subjected to monitoring for authenticity via short tandem repeat (STR) fingerprinting and routine mycoplasma testing was conducted. The glioma cultures were cultivated as neurospheres in serum-free medium composed of DMEM (Invitrogen), Neurobasal(-A) (Invitrogen), B27(-A) (Invitrogen), heparin (2 ng ml-1), human- bFGF (20 ng ml-1) (Shenandoah Biotech), human-bEGF (20 ng ml-1) (Shenandoah Biotech), human-PDGF-AA (10 ng ml-1) (Shenandoah Biotech), and human-PDGF-BB (10 ng ml-1) (Shenandoah Biotech). For in vitro experiments, the neurospheres were dissociated using TrypLE (Gibco) for seeding.

[0075] Animal Models: Homozygous ChAT-IRES-Cre mice (The Jackson Laboratory, strain 006410) were bred with homozygous Ai230 mice. Optogenetic experiments were performed on animals with the genotype ChAT-IRES-Cre+ / wtx Ai230flx / wt, which enabled optogenetic control selectively for cholinergic neurons in immunocompetent mice. All mice used were genotyped at postnatal day 10. For tumour studies, glioma cell implantation was conducted using an electroporated, engineered H3.3 K27- altered MADR model (Kim, G.B., et al. (2019). Cell 119, 251-267). To replicate results obtained from this immunocompetent tumour model in patient-derived glioma cells, viral vectors (specified below) were injected into 4-week-old NSG mice (NOD-SCID-IL2R-gamma chain-deficient, The Jackson Laboratory), allowing optogenetic control of the laterodorsal tegmentum nucleus and pedunculopontine nucleus in immunodeficient mice, followed by xenografting of patient-derived DIPG cells (SU-DIPG-17) after 3 weeks of virus expression. All animal experiments were conducted in accordance with protocols approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) and performed in accordance with institutional guidelines. Animals were housed according to standard guidelines with unlimited access to water and food, under a 12 h light: 12 h dark cycle,PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 a temperature of 21 °C and 60% humidity. For brain tumour allograft or xenograft experiments, the IACUC has a limit on indications of morbidity (as opposed to tumour volume). Under no circumstances did any of the experiments exceed the limits indicated and mice were immediately euthanized if they exhibited signs of neurological morbidity or if they lost 15% or more of their initial body weight.

[0076] Generation of Transgenic Ai230 Mice: Ai230 (full strain name TIGREtm230(TIT2L- XCaMPG-WPRE-ICL-ChRmine-TS-oScarlet-Kv2. l-ER-IRES2-tTA2-WPRE_hyg)Hze) is a new TIGRE2.0 transgenic reporter (Daigle, T.L., et al. (2018). Cell 174, 465-480), which provides Cre-dependent expression of soma-targeted ChRmine-oScarlet and Cre- and tTA- dependent expression of XCaMPG. Ai230 mice contain a modified TIGRE genomic locus that contains: (5' to 3'): A Frt3 site, two tandem copies of the chicken beta-globin HS4 insulator element, a Tet responsive 2 promoter comprised of seven repeats of TRE binding sites and a minimal CMV promoter (based on that in Clontech's pTRE2-hyg vector), a loxP site, a stop cassette (with stops in all three frames linked to a synthetic pA-hGH pA-PGK pA unit), a loxP site, the coding sequence for XCaMP-G, a woodchuck post-transcriptional regulatory element (WPRE), a bGH pA, two tandem copies of the chicken beta-globin HS4 insulator element, a CAG promoter which consists of the CMV enhancer fused to the chicken beta-actin promoter, a lox2272 site, a stop cassette (with stops in all three frames linked to a synthetic pA-hGH pA-TK pA unit), a lox2272 site, the coding sequence of ChRmine, a membrane trafficking signal (TS), oScarlet, Kv2.1, a woodchuck post-transcriptional regulatory element (WPRE), a bGH pA, an IRES2 sequence, tetracycline-transactivator 2 (tTA2), a woodchuck post-transcriptional regulatory element (WPRE), a bGH pA, a PGK promoter, one domain of the hygromycin resistance gene, a mRNA splice donor sequence, a Frt5 site. The locus was generated by recombinase mediated cassette exchange into a previously made docking site integrated into the TIGRE locus.

[0077] Generation of H3.3 K27M MADR cells: The H3.3 K27M MADR tumor cell cultures were generated. Briefly, Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J and Gt(ROSA)26Sortml.l(CAG-EGFP)Fsh / Mmjax mice (J AX Mice) were crossed with wild-type CD1 mice (Charles River) to produce heterozygous mice. Male and female embryos between E12.5 and E15.5 were subjected to in utero electroporations. Pregnant dams were individually housed, and pups remained with their mothers until P21 in the institutional animal facilityPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196(Stanford University). The MADR tumor cell line used here was generated by dissociating and sorting GFP+ tumor cells from female heterozygous mTmG mice. Subsequently, MADR cultures were maintained as neurospheres in serum-free medium composed of DMEM (Invitrogen), Neurobasal(-A) (Invitrogen), B27(-A) (Invitrogen), heparin (2 ng ml-1), human bFGF (20 ng ml-1) (Shenandoah Biotech), human bEGF (20 ng ml-1) (Shenandoah Biotech), human PDGF-AA (10 ng ml-1) (Shenandoah Biotech), insulin (Sigma-Aldrich), and 2- mercaptoethanol (Sigma- Aldrich).

[0078] Stereotaxic Surgery, Ferrule Placement, and Viral Vectors: Mice were anesthetized with 1-4% isoflurane and placed in a stereotaxic apparatus. For all optogenetic experiments, mice were unilaterally implanted with optical fibers above either the laterodorsal tegmentum nucleus (LDT) or the pedunculopontine nucleus (PPN) on the right side. Optical fibers were secured with stainless steel screws (thread size 00-90 x 1 / 16, Antrin Miniature Specialties), C&B Metabond, and light-cured dental adhesive cement (Geristore A&B paste, DenMat). For optogenetic stimulation of NSG mice, AAV-PHPeB-vCHATel-ChRmine-eYFP or AAV- PHPeB-vCHATel-eYFP viral vectors were injected retro-orbital.

[0079] Allografting and Xenografting: Male and female mice were used in cohorts equally. For optogenetic stimulation studies, MADR cultures (H3.3 K27 MADR line l, n = 200.000 cells per mice) or patient-derived DIPG cultures (SU-DIPG-17, n = 400.000 cells per mice) were injected into the thalamus or pontine region. A single-cell suspension of all cultures was prepared in sterile culture medium immediately before surgery. Animals at P28-P35 were anaesthetized with 1-4% isoflurane and placed on stereotactic apparatus. Under sterile conditions, the cranium was exposed via a midline incision and a 31 -gauge burr hole made at exact coordinates. For thalamus injections the coordinates were as follows: AP=- 1.0mm (from bregma), ML=+0.8mm, DV=-3.5mm. For pontine injections coordinates were AP=-0.8 (from lambda), ML=-1.0mm, DV=-5.0mm. Cells were injected using a 31-gauge Hamilton syringe at an infusion rate of 0.5 pl min-1 with a digital pump. At completion of infusion, the syringe needle was allowed to remain in place for a minimum of 5 minutes, then manually withdrawn. The wound was closed using 3M Vetbond (Thermo Fisher Scientific) and treated with Neo- Predef with Tetracaine Powder.

[0080] Viral Vectors: For the optogenetic stimulation of cholinergic midbrain nuclei (LDT and PPN) in immunodeficient NSG mice, expression of channelrhodopsin in each nucleus wasPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 achieved by retroorbital injection of AAV-PHPeB-vCHATel-ChRmine-eYFP (virus t itci- 2.5xl012gc / ml), and eYFP control by injection of AAVPHPeB-vCHATel-ChRmine-eYFP (virus litci- 3.1xl012gc / ml). Both viral vectors were constructed for this study.

[0081] Ontogenetic Stimulation in vivo: Optogenetic stimulations were performed 1 week after optic ferrule implantation. For allograft experiments in ChAT-IRES-Cre+ / wtx Ai230flx / wtmice, freely moving animals were connected to a 595 nm high-power LED system with a monofiber patch cord achieving stimulation of ChRmine. For xenograft experiments in immunodeficient NSG mice, a 473 nm diode-pumped solid-state laser system was used to achieve stimulation of ChR2. Cholinergic neuron stimulation, for both neuronal cell bodies and axon terminals, was performed at 20 Hz, ten 15 ms pulses of 595 nm light delivery every 5 seconds at a light power output of lOmW from the tip of the optic fiber (200 pm core diameter, NA=0.22 - Doric lenses). Optogenetic stimulation session lasted for 30 minutes. Animals were injected intraperitoneally with 40 mg / kg EdU (5-ethynyl-2’-deoxyuridine; Invitrogen, E 10187) before the session, and perfused 3 hours (for OPC response analysis) or 24 hours (for glioma cell proliferation analysis) after the start of the stimulation. The effectiveness of optogenetic stimulation in the ChAT+ / wtx Ai230fbt / wtmodel was validated through cFos staining, as depicted in FIG. 5. In Ai230 models, light delivery results in stimulation. Thus, the group with the "laser on" was designated as the stimulated cohort, while the non- stimulated group did not receive light ("laser off").

[0082] Pharmaceutical Antagonism of Muscarinic Receptors Ml and M3: To assess a potential effect of muscarinic receptors Ml (CHRM1) and M3 (CHRM3) on the proliferative effect of cholinergic neuronal activity in vivo, ChAT-IRES-Cre+ / wtx Ai230flx / wtmice were allografted with the MADR model as above and blind randomized to a treatment group. Four weeks post- allograft, mice were treated with intraperitoneal administration of the Ml blocker VU 0255035 (10 mg kg-1; Tocris) or 4-DAMP (10 mg kg-1; Tocris) and controls treated with equivalent volume of vehicle. Administration of with the drug or vehicle was done 30 minutes before the beginning of the optogenetic stimulation session of each mice. For immunohistological analysis of glioma cell proliferation, mice were perfused 24 hours after optogenetic stimulation.

[0083] Cerebral Slice Conditioned Media of LPT and PPN: ChAT-IRES-Cre+ / wtx Ai230flx / wtmice aged 4 weeks (P28 to P30) were utilized to collect conditioned media fromPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 activated cholinergic neurons in either the LDT or PPN. Brief exposure to isoflurane induced unconsciousness in the mice before immediate decapitation. Extracted brains (cerebrum) were inverted and placed in an oxygenated sucrose cutting solution, then sliced at 300pm to target the region of the LDT or PPN. Slices (n=4 per mouse) were transferred to ACSF and allowed to recover for 30 minutes at 37°C, followed by an additional 30 minutes at room temperature. After recovery, the slices were transferred to fresh ACSF and positioned under a red-light LED using a microscope objective. The optogenetic stimulation paradigm consisted of 20-Hz pulses of red light for 30 seconds on, followed by 90 seconds off, repeated over a period of 30 minutes. Conditioned medium from the surrounding area was collected and stored frozen at -80°C. Stimulated slices were postfixed in 4% paraformaldehyde (PFA) for 30 minutes before cryoprotection in 30% sucrose solution for 48 hours. Successful stimulation of cholinergic neurons in each area was validated through cFos staining.

[0084] EdU Incorporation Assay: EdU staining of glioma monocultures or glioma-neuron co-cultures was performed on glass coverslips in 96-well plates which were pre-coated with poly-l-lysine (Sigma) and laminin (Thermo Fisher Scientific). Neurosphere cultures were dissociated with TrypLE and plated onto coated coverslips with growth factor-depleted medium. Acetylcholine (0.5pM to 5 M, Tocris), VU 0255035 (lOpM, Tocris), 4-DAMP (lOpM, Tocris) and vehicle (DMSO) were added for specified times with 4 pM EdU. After 24 h the cells were fixed with 4% PFA in PBS for 20 min and then stained using the Click-iT EdU kit and protocol (Invitrogen) and mounted using Prolong Gold mounting medium (Life Technologies). Confocal images were acquired on a Zeiss LSM980 using Zen 2011 v8.1. Proliferation index was determined by quantifying the fraction of EdU-labelled cells divided by DAPI-labelled cells (monoculture experiments), or HNA-labeled cells (co-culture experiments) using confocal microscopy at 20x magnification. Quantification of images was performed by a blinded investigator.

[0085] Migration Assay: 3D migration experiments were performed. Briefly, 96-well flat- bottomed plates (Falcon) were coated with 2.5 pg per 50pl laminin per well (Thermo Fisher) in sterile water. After coating, a total of 200pl of culture medium per well was added to each well. A total of lOOpl of medium was taken from 96-well round bottom ULA plates containing ~200pm diameter neurospheres, and the remaining medium including neurospheres was transferred into the pre-coated plates. Images were then acquired using an Evos M5000PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 microscope (Thermo Fisher Scientific) at time zero, 24, 48, and 72 hours after encapsulation. Image analysis was performed using ImageJ by measuring the diameter of the invasive area. The extent of cell migration on the laminin was measured for six replicate wells normalized to the diameter of each spheroid at time zero and the data is presented as a mean ratio for three biological replicates.

[0086] Neuron-Glioma Co-Culture: For EdU incorporation assays, neurons were isolated from CD1 mice (The Jackson Laboratory) at P0 using the Neural Tissue Dissociation Kit Postnatal Neurons (Miltenyi), and followed by the Neuron Isolation Kit, Mouse (Miltenyi) per manufacturer’s instructions. After isolation, 200,000 neurons were plated onto circular glass coverslips (Electron Microscopy Services) pre-coated with poly-l-lysine (Sigma) and mouse laminin (Thermo Fisher Scientific). Neurons were cultured in BrainPhys neuronal medium containing B27 (Invitrogen), BDNF (10 ng ml-1, Shenandoah Biotech), GDNF (5 ng ml-1, Shenandoah Biotech), TRO 19622 (5 pM; Tocris) and p-mercaptoethanol (Gibco). The medium was replenished on days in vitro (DIV) 1 and 3. On DIV 5, fresh medium was added containing 70,000 glioma cells and incubated for 48 h. After 48h incubation, EdU (10 pM) with or without the acetylcholine (0.5-5 pM, Tocris) was added and incubated for a further 24h. Following incubation, the cultures were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and stained for immunofluorescence analysis. For EdU analysis, cells were stained using the Click-iT EdU Cell Proliferation kit (Thermo Fisher Scientific, C10337), before staining with primary antibodies mouse anti-human nuclei clone 235-1 (1:250; Millipore, MAB1281) and rabbit anti-microtubule-associated protein 2 (MAP2; 1:500, EMD Millipore, AB5622), overnight at 4 °C. Following washing, slips were incubated in secondary antibodies, Alexa 488 donkey anti-mouse IgG (1:500, Jackson Immuno Research) and Alexa Fluor 555 donkey anti-rabbit (1:500, Invitrogen) and mounted using ProLong Gold Mounting medium (Life Technologies). Images were collected on a Zeiss LSM980, and proliferation index determined by quantifying percentage EdU-labelled glioma cells over total glioma cells (HNA immunopositivity to identify glioma cells).

[0087] Human iPSC-Derived Cholinergic Neurons: Human induced pluripotent stem cells (iPSCs) of a 12-year-old male healthy donor (CW20032, Elixirgen) maintained under feeder-free conditions in a 96- well plate (20,000 cells per well) were rapidly differentiated to a cholinergic phenotype with Sendai virus mediated delivery of synthetic neurogenic transcription factor (CH-PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196SeV, Elixirgen). A successful morphological differentiation into cholinergic neurons occurred by day 7. Glioma cells (SU-DIPG-17; 20,000 cells per well) were then added and incubated in optimized media for 48 hours. The optimized media consisted of cholinergic neuron maintenance media (CH-MM, Elixirgen) mixed in a 1:1 ratio with tumor stem cell growth factor- free media (DMEM, Neurobasal-A, and B27-A). After 48h incubation, EdU (4 pM) with or without VU 0255035 (lOpM, Tocris) or 4-DAMP (lOpM, Tocris) was added and incubated for a further 24h. Following incubation, the cultures were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and further staining was processed as above described. Primary antibodies mouse anti-nestin (1:500; Abeam, ab6320), rabbit anti-choline acetyltransferase (ChAT; 1:500, Abeam, ab 181023) and goat anti-microtubule-associated protein 2 (MAP2; 1:500, Abeam, ab302488) were used. For synaptic puncta staining, fresh medium was added containing 50,000 glioma cells (“SU-DIPG-13fl”) expressing PSD95-RFP (as generated in Taylor et al.8) and incubated for 72h. After fixation with 4% PFA, cholinergic neuron-to-glioma co-culture coverslips were incubated in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) at room temperature for Ih. Primary antibodies guinea pig anti-synapsinl / 2 (1:500;Synaptic Systems, 106-004), rabbit anti-RFP (1:500; Rockland, 600-401-379), mouse anti-nestin (1:500; Abeam, ab6320), and goat anti-MAP2 (1:500, Abeam, ab302488) diluted in diluent (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated at 4 °C overnight. Following washing, the slides were incubated in secondary antibody (Alexa 555 donkey anti-rabbit IgG, Invitrogen; Alexa 405 donkey anti-guinea pig IgG; Alexa 647 donkey anti-mouse IgG and Alexa 594 donkey anti-goat IgG all used at 1:500, Jackson Immuno Research) overnight at 4 °C. Following washing, coverslips were mounted using ProEong Gold Mounting medium (Fife Technologies). Images were collected on a Zeiss ESM980 confocal microscope using a 63x oilimmersion objective. Co-localization of synaptic puncta images were performed using a custom ImageJ (v.2.1.0 / 153c) processing script. In brief, the quantification determines co-localization of presynaptic synapsin and postsynaptic PDS95-RFP within a defined proximity of 1.5 pm. Background fluorescence is removed using rolling ball background subtraction and peaks detected using imglib2 DogDetection plugin which determines the region of interest for each channel. The percentage of total glioma ROIs that are within 1.5 pm of a neuron ROI is reported. The script was implemented in ImageJ (v.2.1.0 / 153c).PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0088] Optogenetic Stimulation in vitro: At day 7 of human iPSC differentiation, cholinergic neurons were transduced with either AAV-PHPeB-vCHATel-ChRmine-eYFP or AAV-PHPeB-vCHATel-eYFP for 7 days. After successful viral transduction, glioma cells were added and cultured in optimized media as described above. The success of viral transduction was confirmed by eYFP expression visualized using fluorescent microscopy. On day 14 of human iPSC differentiation and 7 days after viral transduction, optogenetic stimulation was performed. Cholinergic neuron-glioma co-cultures were positioned under a red-light LED using a microscope objective. The optogenetic stimulation paradigm consisted of 20-Hz pulses of red light for 30 seconds on, followed by 90 seconds off, repeated over a period of 30 minutes. At the time of stimulation, EdU was added, and cells were fixed 24 hours after optogenetic stimulation.

[0089] Immunohistochemistry: All mice were anesthetized with intraperitoneal injections of 2.5% Avertin (tribromoethanol; Sigma-Aldrich, T48402), and transcardially perfused with 20 ml 0.1M phosphate buffer saline (PBS). Brains were postfixed in 4% paraformaldehyde (PF A) overnight at 4°C before cryoprolcction in 30% sucrose solution for 48 hours. For sectioning, brains were embedded in optimum cutting temperature (OCT; Tissue-Tek) and sectioned coronally at 40 pm using a sliding microtome (Leica, HM450). For immunohistochemistry, brain sections were stained using the Click-iT EdU cell proliferation kit (Invitrogen, C10339 orC 10337) according to manufacturer’s protocol. Tissue sections were then stained with antibodies following an incubation in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in tris buffer saline) at room temperature for 30 minutes. Mouse anti-human nuclei clone 235-1 (1:100; Millipore, MAB1281), rabbit anti-ChAT (1:500; Abeam, ab223346), goat anti-Pdgfra (1:500; R&D Systems, AF1062), rat anti-MBP (1:200; Abeam, ab7349), chicken anti-mCherry (1:1000; Abeam, ab205402), or rabbit anti-efos (1:500; Santa Cruz Biotechnology, sc-52) were diluted in 1% blocking solution (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated overnight at 4°C. All antibodies have been validated in the literature for use in mouse immunohistochemistry. The following day, brain sections were rinsed three times in lx TBS and incubated in secondary antibody solution for 2 hours at room temperature. All secondary antibodies were used at 1:500 concentration including Alexa 488 anti-rabbit, Alexa 488 antimouse, Alexa 488 anti-chicken, Alexa 594 anti-chicken, Alexa 647 anti-goat, Alexa 647 anti-rat, Alexa 647 anti-rabbit, Alexa 405 anti-guinea pig (all Jackson ImmunoResearch), and Alexa 555PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 anti-rabbit (Invitrogen). Sections were then rinsed three times in lx TBS and mounted with ProLong Gold (Life Technologies, P36930).

[0090] Confocal Microscopy and Quantification: All image analysis were performed by experimenters blinded to the experimental conditions or genotype. Cell quantification within allografted or xenografted tumors was conducted by acquiring z-stacks using a Zeiss LSM98O scanning confocal microscope (Carl Zeiss). A l-in-6 series of coronal brain sections were selected, with 4 consecutive slices (40pm thickness) analyzed in the grafted brain area (thalamus, or pons). Brain tissue damaged during perfusion or tissue processing was excluded from histological analysis. Tumor cells were identified as GFP+(MADR allografts) or HNA+(patient- derived xenografts) and quantified in each field to determine the proliferation index, calculated as the percentage of GFP+cells co-labeled with EdU. OPCs were identified by PDGFRa staining and quantified as the percentage of PDGFR+cell co-labeled with EdU.

[0091] Western Blotting: For western blot analysis of NLGN3 in conditioned media, samples were prepared by adding LDS buffer and P-mercaptoethanol, heated, and loaded onto Tris-acetate gels. After electrophoresis and transfer onto PVDF membranes, the membranes were blocked and incubated with primary antibodies against the NLGN3 ectodomain (Abeam #abl92880). Following incubation with secondary antibody and washing, the membranes were developed using chemiluminescent substrate (Thermo Scientific #34580).

[0092] Measurement of BDNF and acetycholine in Conditioned Media: The concentration of BDNF (R&D Systems #DBNT00) and acetylcholine (Sigma- Aldrich # MAK056) in conditioned media was measured using ELISA. We compared the concentrations of conditioned media obtained from non-stimulated, LDT-stimulated, and PPN- stimulated brain slices as described above. Protocols were performed according to the manufacturer’s instructions. The optical density was measured at 450 nm with an absorbance microplate reader (Molecular Devices, SpectraMax M3). The total concentrations were determined as pg / mL from the standard curve. To confirm the specificity of the respective kit, medium and lysis buffer without protein extract were used as negative controls.

[0093] Single-Cell RNA Sequencing Analysis of Published Data

[0094] Cell filtering: Cells with a low number of detected genes were excluded using a cutoff of 2000 genes for smart- seq2 data and a cutoff of 1000 genes for the other types of sequencing data.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0095] Gene filtering: In analyses that necessitated gene filtering, the 7000 genes with the highest mean expression across cells were kept.

[0096] Normalization: UMI counts were converted to counts per million (CPM). Each entryCPM in the expression matrix was then normalized according to E — log2-^ + 1). The same normalization was used for transcripts per million (TPM) values. The values were divided by 10 as the actual complexity is assumed to be in the realm of about 100,000 and not 1 million as implied by the CPM and TPM measures.

[0097] Centering: To illustrate intra-tumor heterogeneity in gene expression (Fig. 8A), centering was performed individually for each sample. This involved subtracting each gene's mean expression value across all cells within the sample. Cells within each sample were then sorted based on their scores for the cholinergic genes using the sigScores function available at https: / / githab.co / jlaffy / sca1op. For the pseudo-bulk data analysis (Figure 4A), expression values were first averaged across malignant cells within each sample. Subsequently, lognormalization was applied, but no centering was performed.

[0098] Correlation with cell states: Utilizing the OPC-like signature outlined by Neftel et al. (Neftel, C., et al. (2019). Cell 178, 835-849), the following analyses were conducted for both H3K27M DGM samples and medulloblastoma samples:

[0099] i. Computed the correlations within each sample between the cells' OPC-score and the expression values of each selected cholinergic gene.

[0100] ii. Calculated the correlations across samples within each study, between the pseudobulk sample OPC scores and the pseudo-bulk gene expression levels.

[0101] These correlation values were then averaged across samples and studieas, respectively (see Figure 4B and Fig. 8B).

[0102] Additionally, correlations across samples in other tumor types using more cell-state signatures were performed (Neftel et al., 2019; Gavish, A., et al. (2023). Nature 618, 598-606) (Figure 4C). Significant correlations (P < 0.05 after FDR correction) were considered.

[0103] Lineage vs sternness analysis: Focusing on the H3K27M DGM data published in the Filbin et al. 201813study, a 'sternness score' for each cell was computed. This score is determined by subtracting either the cell's OC score or its AC score from its OPC score, whichever is higher (the maximum of the two). A 'lineage score' was also calculated for each cell, defined as thePCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 maximum between the OC and AC scores. In cases where the AC score is higher, it is multiplied by -1. If both AC and OC scores are negative, a 0 value is assigned with some jitter.Additionally, cells with a centered CHRM1 value > 1 and cells with a centered CHRM3 value > 1 were identified. All scoring and centering procedures were performed per sample.

[0104] Statistical Analysis: Gaussian distribution was confirmed using the Shapiro- Wilk test. Parametric data were analyzed with unpaired two-tailed Student's t-tests or one-way ANOVA with Tukey’s post hoc tests. Significance was set at p < 0.05. The used statistical test is indicated in the figure legend. GraphPad Prism 10 was used for statistical analyses and data illustrations.Example 2

[0105] Neuronal activity promotes the proliferation of healthy oligodendrocyte precursor cells (OPC) and their malignant counterparts, gliomas. Many gliomas arise from and closely resemble oligodendroglial lineage precursors, including diffuse midline glioma (DMG), a cancer affecting midline structures such as the thalamus, brainstem and spinal cord. In DMG, glutamatergic and GABAergic neuronal activity promotes progression through both paracrine signaling and through bona-fide neuron-to-glioma synapses. However, the putative roles of other neuronal subpopulations - especially neuromodulatory neurons located in the brainstem that project to long-range target sites in midline anatomical locations where DMGs arise - are unknown. Herein it is shown that the activity of cholinergic midbrain neurons modulates both healthy OPC and malignant DMG proliferation in a circuit- specific manner at sites of long-range cholinergic projections. Optogenetic stimulation of the cholinergic pedunculopontine nucleus (PPN) promotes glioma growth in pons, while stimulation of the laterodorsal tegmentum nucleus (LDT) facilitates proliferation in thalamus, consistent with the predominant projection patterns of each cholinergic midbrain nucleus. Reciprocal signaling was evident, as increased activity of cholinergic neurons in the PPN and LDT was observed in pontine DMG-bearing mice. In coculture, hiPSC-derived cholinergic neurons form neuron-to-glioma networks with DMG cells and robustly promote proliferation. Single-cell RNA sequencing analyses revealed prominent expression of the muscarinic receptor genes CHRM1 and CHRM3 in primary patient DMG samples, particularly enriched in the OPC-like tumor subpopulation. Acetylcholine, the neurotransmitter cholinergic neurons release, exerts a direct effect on DMG tumor cells,PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 promoting increased proliferation and invasion through muscarinic receptors. Pharmacological blockade of Ml and M3 acetylcholine receptors abolished the activity-regulated increase in DMG proliferation in cholinergic neuron-glioma co-culture and in vivo. Taken together, these findings demonstrate that midbrain cholinergic neuron long-range projections to midline structures promote activity-dependent DMG growth through Ml and M3 cholinergic receptors, mirroring a parallel proliferative effect on healthy OPCs.

[0106] RESULTS

[0107] Cholinergic neuronal activity increases OPC proliferation: It was first investigated whether cholinergic neuronal activity modulates healthy OPC proliferation. In vivo optogenetic stimulation was used to specifically target cholinergic midbrain neurons in either the pedunculopontine nucleus (PPN) or the laterodorsal tegmentum nucleus (LDT) (Fig. 1A), which respectively project to the pons and thalamus. To stimulate cholinergic neurons, ChAT-IRES- Cre mice were cross-bred with Ai230 mice, a Cre-dependent reporter expressing the opsin ChRmine (ChAT-IRES-Cre+ / wtx A i230ll / wl), thereby enabling stimulation of cholinergic neuronal activity using a potent opsin specifically expressed in cholinergic neurons (FIG. 5A).

[0108] An optical ferrule was placed within either the LDT or PPN to selectively stimulate cholinergic neurons confined to a single cholinergic nucleus. Light stimulation of the PPN or LDT at 20 Hz increased cFos expression in cholinergic neurons after 90 minutes, demonstrating successful optogenetic stimulation (Fig. 5B-5D). As cholinergic projections originating from the PPN and LDT target distinct brain regions, optogenetic stimulation of each nucleus was performed and OPC proliferation in response to cholinergic neuronal activity was evaluated by administering the thymidine analogue EdU at the time of each stimulation session. Predominant projections from LDT to thalamus (LDT->thalamus) and from PPN to pons (PPN->pons) were visualized utilizing Cre-dependent AAV tracing of cholinergic neurons. In line with the anatomy of these projections, stimulation of cholinergic neurons in the LDT increased OPC proliferation in the thalamus (Fig. IB), whereas PPN stimulation increased OPC proliferation in the pons (Fig. 1C), as indicated by quantification of EdU+ / Pdgfra+cells (Fig. ID). Taken together, these results illustrate circuit- specific OPC proliferation consistent with target sites of midbrain cholinergic axonal projections.

[0109] Cholinergic axons from LDT and PPN also project to a variety of neuroanatomical sites. LDT stimulation resulted in notably increased OPC proliferation in the cortex (Fig. IE),PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 with no changes in OPC proliferation observed in the ventral tegmental area (Fig. IF), nucleus accumbens (Fig. 1G), or hippocampus (Fig. 1H). Despite the known functional and topographical organization of cholinergic projections to these regions, these data demonstrate a brain region- specific response of OPCs to cholinergic neuronal activity.

[0110] Cholinergic neuronal activity modulates glioma growth: Given the similarities between OPCs and DMG, and the observed cholinergic circuit-specific thalamic and pontine OPC responses to cholinergic neuronal activity, it was next evaluated whether DMG cells are equally responsive to long-range cholinergic neuronal activity in these midline structures. This was addressed by combining the genetically engineered optogenetic mouse model (ChAT-IRES- Cre+ / wtx Ai230flx / wt) with an in utero electroporation-induced genetic mouse model of H3K27M- DMG that leverages dual recombinase-mediated cassette exchange to express mutations in H3f3a, Tp53 and Pdgfra in neural precursor cells (MADR). This model faithfully recapitulates an H3.3-K27M DMG and allows allografting into immunocompetent mice. The OPC-like characteristics of DMGs described in the MADR model were re-confirmed by Pdg ra-positivity in GFP+-tumour cells (Fig. 51).

[0111] Allografting into either the thalamus or pons was performed in 4-week-old mice (postnatal day (P) 28-30), with optical ferrule placement in the PPN or LDT three weeks later (P51). Optogenetic stimulation of cholinergic neurons was conducted after 4 weeks of tumour growth (P58), with administration of EdU at the time of the stimulation session. Mice were perfused 24-hours after optogenetic stimulation (Fig. 2A). As observed for OPCs, stimulation of cholinergic neurons in the LDT increased tumour cell proliferation in thalamic allografts more than 2-fold (Fig. 2B,D) ; no discernible effect was observed in the pons after LDT stimulation (Fig. 2C), consistent with the anatomy of cholinergic axon projections. Concordantly, stimulation of PPN cholinergic neurons increased DMG tumour cell proliferation in pontine allografts (Fig. 2C), but not in thalamic allografts (Fig. 2B-C).

[0112] Patient-derived orthotopic xenograft models of DMG are complimentary to genetically engineered mouse models. The effects of cholinergic neuronal activity on patient- derived pontine H3.3-K27M DMG orthotopically xenografted to the pons was next investigated. Viral vectors (AAV-Efla-DIO-ChR2::YFP or AAV-Efla-DIO-YFP) were injected into the LDT or PPN of 4-week-old NOD-SCID-IL2-gamma chain-deficient (NSG) mice, followed by xenografting of patient-derived DMG cells (SU-DIPG-17) into the pons. Optogenetic stimulationPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 was performed after 6 weeks of tumour engraftment, with perfusion conducted 24 hours later (Fig. 6A). Consistent with the results above, stimulation of the PPN increased proliferation of patient-derived diffuse intrinsic pontine glioma / pontine diffuse midline glioma cells xenografted to the pons, whereas this effect was not observed after LDT stimulation (Fig. 2E-F). These findings mirror the observed OPC response in vivo and highlight cholinergic neuronal activity as a driver of DMG proliferation in a brain circuit- specific and midbrain nucleus-dependent manner.

[0113] The role of secreted factors in cholinergic activity-regulated DMG proliferation was next assessed. Conditioned medium (CM) was collected from midbrain explants containing either PPN or LDT nuclei (Fig. 6B). Ex vivo optogenetic stimulation of cholinergic neuronal cell bodies in either PPN or LDT explants generated CM that increased patient-derived H3K27M DMG (SU-DIPG-17) proliferation in vitro (Fig. 6C-D). The conditioned medium was fractionated by molecular size, which revealed that the proliferative effect of the midbrain explant CM is attributable to macromolecules with a molecular weight between 10 to 100 kDa (Fig. 6E). ANA- 12 (a specific inhibitor of the BDNF-receptor TrkB) abolished the proliferative effect of cholinergic neuronal activity-regulated factors in PPN- or LDT-CM while the addition of Neurexin (to sequester NLGN3) only minimally decreased proliferation (Fig. 6F). Elevated BDNF levels were found in LDT-CM (Fig. 6G) and PPN-CM (Fig. 6H), while NLGN3 was only mildly increased in midbrain cholinergic nuclei CM by western blot analysis (Fig. 61), evidencing that BDNF is the chief paracrine growth factor released as a result of midbrain cholinergic neuronal activity in this experimental paradigm. Acetylcholine levels were mostly unchanged in midbrain explant CM following cholinergic neuronal activity (Fig. 6G-H). These midbrain cholinergic nuclei explants contain cholinergic cell bodies but not axon terminals, which likely explains the lack of acetylcholine release into CM in this experimental paradigm. Taken together, these findings indicate that activity-regulated neurotrophin signaling from midbrain cholinergic neurons to TrkB on DMG cells contributes to the growth-promoting effects of cholinergic neuronal activity, at least locally in the midbrain.

[0114] DMG cells increase cholinergic midbrain neuronal activity: It was next investigated whether midbrain cholinergic neurons may be reciprocally influenced by DMGs. Either H3K27M DMG cells (MADR model) or vehicle control (buffered saline, HBSS) into the pons of 4-week-old immunocompetent mice (ChAT-IRES-Cre+ / wtx Ai230flx / wt) and cholinergic neuronalPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 activity was assessed within both midbrain cholinergic nuclei after 4 weeks of tumor growth (Fig. 2G). Neuronal activity was measured by expression of the immediate early gene cFos in tumor- or saline vehicle control-injected mice and compared to the cFos expression of 4-week- old healthy littermate controls. Increased midbrain cholinergic neuronal activity was observed within both nuclei in DMG tumor-bearing mice, which was not observed in saline vehicle control-injected mice (Fig. 2H). These findings indicate bidirectional interactions between pontine DMG and cholinergic midbrain neurons whereby cholinergic neurons increase DMG growth and DMG cells increase cholinergic neuronal activity.

[0115] Acetylcholine directly affects DMG growth and migration: To assess the effects of human cholinergic neurons on DMG, patient-derived DMG cells were co-cultured with cholinergic neurons derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived cholinergic neurons from a healthy 12-year-old male (Fig. 3A) were matched with a pontine DMG cell culture from an 8-year-old male (SU-DIPG-17) (Fig. 3B). Co-culture resulted in a two-fold increase in the glioma proliferation rate (Fig. 3C). hiPSC-derived cholinergic neurons and glioma cells formed a dense and extensive neuron-to-glioma network (Fig. 3B, 3D). Using a patient-derived DMG cell culture (SU-DIPG-13FL) expressing postsynaptic PSD95-RFP, cholinergic neuron-to-DMG synaptic structures were evident by pre- and post-synaptic puncta co-localization between presynaptic neurons and post-synaptic glioma cells within these networks (Fig. 3D).

[0116] The tumor growth-promoting effects of cholinergic neurons in vivo and in vitro suggest that acetylcholine release at axon terminals may directly act on DMG cells. To assess the direct effects of acetylcholine on DMG cells, the proliferation rate of patient-derived H3K27M DMG cell cultures (n = 4 distinct patient-derived DMG cultures, Table 1) exposed to varying concentrations of acetylcholine in vitro was evaluated. These experiments revealed a dosedependent increase in DMG cell proliferation following exposure to acetylcholine, which plateaued at higher concentrations (Fig. 3E-F and Fig. 7A-C). Muscarinic receptor blockers abrogated the effects of acetylcholine, while nicotinic receptor blockers had no effect on the proliferation caused by acetylcholine (Fig. 3G). Neither muscarinic nor nicotinic receptor blockers affected glioma cell proliferation in the absence of acetylcholine (Fig. 3G). Furthermore, muscarine (a direct muscarinic receptor agonist) induced glioma cell proliferation to the same extent as acetylcholine, while nicotine (a direct nicotinic receptor agonist) exerted noPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 effect (Fig. 3G). Taken together, these data indicate that the proliferative effect of acetylcholine is mediated by muscarinic receptors rather than nicotinic receptors.

[0117] Table 1: Characteristics of Patient-Derived Diffuse Midline Glioma Cell Cultures Used

[0118] It was next investigated whether acetylcholine alters the proliferative effect of cortical neuron co-culture on glioma cells. Early postnatal mouse pup (P0-P1) cortical preparation generated mixed glutamatergic (Vglut+) and GABAergic (GAD65+) cortical neuron cultures (Fig. 7D). Cortical neuron-DMG co-culture markedly increased the proliferation rate of DMG cells, tested in two independent patient-derived cultures (SU-DIPG-17 and SU-DIPG-92; Fig. 7E-G). Addition of acetylcholine substantially augmented this effect, further increasing the glioma cell proliferation rate in a dose-dependent manner (Fig. 7F-G). Acetylcholine exposure increased the migration of DMG cells in vitro in a dose-dependent manner (Fig. 3H-I). Taken together, these findings highlight a direct effect of acetylcholine on proliferation and infiltration in DMG, representing a second mechanism - in addition to the cholinergic neuronal activity- regulated neurotrophin signaling described above - by which cholinergic neuronal activity modulates DMG pathophysiology.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0119] CHRM1 and CHRM3 are therapeutic targets in DMG: The proliferation-promoting effects of acetylcholine in vitro and cholinergic long-range projections in vivo led to further investigation of the cholinergic receptor profile of DMG cells. Receptor gene expression was evaluated in multiple single-cell and single-nucleus RNA sequencing (sc / snRNAseq) datasets from primary patient tumor samples across various central nervous system tumors, including 54 DMG samples. Cholinergic receptor genes exhibited varying levels of expression across malignant cells within DMG tumors (Fig. 8A), reflecting intratumoral heterogeneity. To highlight intertumoral heterogeneity pseudo-bulk expression in malignant cells was compared across samples (Fig. 4A). Notably, the muscarinic receptor CHRM3 was found to be highly expressed across all glioma subtypes, ependymoma, and medulloblastoma, but not in neuroendocrine pituitary tumors (Fig. 4A). It was next investigated how intratumor heterogeneity and cell states are correlated to cholinergic receptor gene expression. The OPC- like cell state was initially evaluated, which reflects the cancer stem cell / tumor-initiating cell subpopulation and is particularly prevalent in DMG tumors compared to other glioma subtypes. Examining differentially expressed genes associated with either muscarinic or nicotinic receptor genes across all tumor samples revealed a specific association between muscarinic receptor genes and PDGFRA - a hallmark gene for OPCs - exclusively in DMG. Correlations between cholinergic receptor gene expression and OPC scores were further evaluated, both within tumor cells and across samples at the pseudo-bulk level. The Ml muscarinic receptor gene CHRM1 exhibited the highest values on both measures (Fig. 4B), stronger than but similar to the values for the M3 muscarinic receptor gene CHRM3 (Fig. 4B). Both CHRM1 and CHRM3 expression are enriched in the OPC-like cell state of DMG (Fig. 4C-D). Notably, in medulloblastoma samples, which is an embryonal, non-glioma tumor entity, no correlation between any cholinergic receptor gene and an OPC score was observed (Fig. 8B).

[0120] Further comparisons of intertumoral correlations of cholinergic receptor gene expression across various tumor types and with different cell states underscored the association between CHRM1 and OPC-like states in DMG using the Neftel et al and a Gavish et al. pancancer study-derived cell states as references (Fig. 4C). While not the most highly expressed cholinergic receptor gene in glioma, CHRM1 seems to play a unique role in OPC-like DMG cells, distinguishing it from other CNS tumor entities such as glioblastoma, ependymoma, and medulloblastoma. By contrast, CHRM3 appears to be important in multiple other tumors,PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 including glioblastoma (Sun, Y. et al. (2024). Brain-wide neuronal circuit connectome of human glioblastoma. Preprint, 10.1101 / 2024.03.01.583047 10.1101 / 2024.03.01.583047; Tetzlaff, S.K., et al. (2024). Characterizing and targeting glioblastoma neuron-tumor networks with retrograde tracing. Preprint, 10.1101 / 2024.03.18.585565 10.1101 / 2024.03.18.585565).

[0121] Since CHRM1 and CHRM3 appear to play significant roles in DMG, their involvement in DMG proliferation was investigated both in vitro and in vivo. In a co-culture model, delivery of both Ml receptor (VU0255035) and M3 receptor (4- DAMP) antagonists was shown to abolish the proliferation-inducing effects of cholinergic neurons on DMG (Figures 9A and 9B). The reduction in the DMG proliferation rate by Ml / M3 antagonists was further observed following in vitro optogenetic stimulation of cholinergic neurons (Figure 9C). Blocking M1 / M3 also led to a decrease in the number of synaptic structures between cholinergic neurons and DMG cells (Figure 9D). In vivo experiments confirmed that blocking both receptors reduced DMG cell proliferation in the thalamus following LDT stimulation (Figures 10A and 10B). Fiber photometry recordings of GCaMP-transduced DMG cells, combined with local drug delivery to the glioma cells, revealed a robust decrease in calcium signaling during ongoing optogenetic LDT stimulation with M1 / M3 blockade (Figure 10C). Since muscarinic receptors can be present at both pre- and post-synaptic sites, which may be blocked by pharmacological inhibition, a CRISPR-based knockout of both muscarinic receptors was performed in a patient-derived DMG xenograft model to specifically block the muscarinic receptors Ml and M3 in the glioma cells. This experiment validated the findings above, as the effect of cholinergic neuronal activity on glioma cell proliferation in the pons was completely abrogated in the M1 / M3 knockout DMG cells following PPN cholinergic neuronal stimulation in vivo (Figures 10D and 10E). Taken together, these findings highlight the critical and redundant role of muscarinic receptors Ml and M3 in activity-dependent cholinergic neuron-DMG interactions.DISCUSSION

[0122] H3K27M-altered diffuse midline gliomas are aggressive brain cancers that occur in midline structures, chiefly the pons (also called DIPG), thalamus and spinal cord. Herein, the effects on normal OPCs and malignant OPC-like tumor cells of cholinergic neurons located in the midbrain, a structure located between the pons and thalamus that is frequently invaded by both pontine and thalamic DMGs, was investigated. The axon projections of cholinergic neuronsPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 in the two distinct cholinergic nuclei of the midbrain - LDT and PPN - project robustly to the thalamus and pons, respectively. It was found that both normal OPCs and DMG cells in pons and thalamus proliferate in response to cholinergic neuronal activity in a circuit- specific manner, with acetylcholine acting on muscarinic Ml and M3 acetylcholine receptors enriched in the OPC-like DMG cellular subpopulation.

[0123] Cholinergic long-range projections were found to similarly influence both normal and malignant cell proliferation. It was also demonstrated that cholinergic neuronal activity promotes OPC proliferation in midline brain regions and in cortex, but not in other regions like the hippocampus and VTA. The tumor growth-promoting effects of cholinergic neuronal activity on DMG cells are mediated by both acetylcholine and BDNF release, although these two mechanisms are spatially enriched at different locations. The proliferative effects of long-range cholinergic neuron projections to thalamus and pons are chiefly mediated through acetylcholine signaling via Ml and M3 receptors, as the pro-tumor effects of cholinergic neuronal activity are blocked by Ml or M3 antagonists in vitro and in vivo. Furthermore, cholinergic neurons influence DMG cells through BDNF-TrkB signaling, which is known to promote proliferation and malignant synaptic plasticity in gliomas. BDNF release appears to occur closer to the neuronal soma, as explants of cholinergic midbrain nuclei containing neuronal soma and local neuronal projections such as short-range axons and dendrites - but not longer axons - exhibited activity-regulated BDNF release that promoted DMG proliferation.

[0124] A role for acetylcholine in driving glioma was also identified herein. Acetylcholine can function as a paracrine growth factor in DMG, but also may be signalling at cholinergic neuron-to-glioma synapses. Evidence of cholinergic neuron-to-DMG cell synaptic structures was found herein.

[0125] Interactions between neurons and glioma are bidirectional, and glioma cells can affect neuronal function by increasing neuronal excitability and functionally remodeling neural circuits. An effect of DMG on the baseline neuronal activity of cholinergic midbrain neurons was observed in the experiments conducted herein. This raises the possibility that tumor-induced dysregulation of cholinergic neurons - and possibly other neuromodulatory neuron types - in the brainstem may contribute to the cognitive, behavioral and emotional symptoms that DMG patients frequently experience.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196

[0126] Integration of primary tumor single-cell / single-nucleus datasets revealed the muscarinic receptor CHRM3 as the most highly expressed cholinergic receptor gene across various CNS tumours, including DMG. Further detailed analysis identified CHRM1 as a unique target, upregulated solely in the OPC-like cell state within DMG. The in vitro and in vivo findings herein highlight the potential of these two receptors as therapeutic targets for DMG. Blocking Ml and M3 muscarinic receptors using tool compounds demonstrate the therapeutic promise of targeted muscarinic receptor inhibition for DMG.

[0127] Taken together, the results presented here implicate cholinergic brainstem neurons as an important driver of diffuse midline glioma pathophysiology, and that blocking Ml or M3 muscarinic receptors is an effective strategy for treatment of central nervous system cancer, including DMG.

[0128] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0129] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0130] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196CLAIMS:

1. A method of treating a central nervous system cancer in a subject, comprising providing to the subject at least one muscarinic acetylcholine receptor antagonist (MRA), wherein the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist and / or a muscarinic acetylcholine receptor M3 antagonist.

2. The method of claim 1, wherein the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist.

3. The method of claim 1, wherein the at least one MRA comprises a muscarinic acetylcholine receptor M3 antagonist.

4. The method of claim 1, wherein the at least one MRA comprises a muscarinic acetylcholine receptor Ml antagonist and a muscarinic acetylcholine receptor M3 antagonist.

5. The method of claim 1, wherein the at least one MRA is an antagonist of muscarinic acetylcholine receptor Ml and muscarinic acetylcholine receptor M3.

6. The method of any one of claims 1-5, wherein the nervous system cancer is a glioma.

7. The method of claim 6, wherein the glioma is a diffuse midline glioma.

8. The method of claim 7, wherein the subject is a pediatric subject.

9. The method of claim 6, wherein the glioma is a glioblastoma.

10. The method of claim 9, wherein the subject is an adult subject.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-19611. The method of any one of claims 1-10, wherein the at least one MRA penetrates the blood-brain barrier.

12. The method of any one of claims 1-11, wherein the at least one MRA is selected from diclyomine, scopolamine, biperiden, trihexyphenidyl, clemastine, PIPE-359, <V [3-oxo-3- [4-(4-pyridinyl)-l-piperazinyl]propyl]-2,l,3-benzothiadiazole-4-sulfonamide (VU0255035), dicycloverine, benztropine, muscarinic toxin 7 (MT7), 4-DAMP, darifenacin, procyclidine, zamifenacin, solifenacin, fesoterodine, tramadol, hyoscyamine, diphenhydramine, cyproheptadine, mequitazine, oxybutynin, tolterodine, dicylomine, atropine, mecamylamine, olanzapine, amitriptyline, desipramine, desmethyldespiramine, dosulepin, doxepin, femoxetine, imipramine, lofepramine, nortriptyline, paroxetine, sertraline, chlorprothixene, chlorpromazine, clozapine, cymamazine, N- dexmethylclozapine, flurperlapine, loxapine, mesoridazine, olanzapine, thoridazine, and zotepine.

13. A muscarinic acetylcholine receptor antagonist (MRA) for use in a method of treating a central nervous system cancer in a subject.

14. A muscarinic acetylcholine receptor antagonist (MRA) for use in the manufacture of a medicament for the treatment of a central nervous system cancer in a subject.

15. The MRA of claim 13 or claim 14, wherein the MRA comprises a muscarinic acetylcholine receptor Ml antagonist.

16. The MRA of claim 13 or claim 14, wherein the MRA comprises a muscarinic acetylcholine receptor M3 antagonist.

17. The MRA of claim 13 or claim 14, wherein the MRA comprises a muscarinic acetylcholine receptor Ml antagonist and a muscarinic receptor M3 antagonist.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-19618. The MRA of claim 13 or claim 14, wherein the MRA is an antagonist of muscarinic acetylcholine receptor Ml and muscarinic acetylcholine receptor M3.

19. The MRA of any one of claims 13-18, wherein the nervous system cancer is a glioma.

20. The MRA of claim 19, wherein the glioma is a diffuse midline glioma.

21. The MRA of claim 20, wherein the subject is a pediatric subject.

22. The MRA of claim 19, wherein the glioma is a glioblastoma.

23. The MRA of claim 22, wherein the subject is an adult subject.

24. The MRA of any one of claims 13-23, wherein the MRA penetrates the blood-brain barrier.

25. The MRA of claim 24, wherein the MRA is selected from diclyomine, scopolamine, biperiden, trihexyphenidyl, clemastine, PIPE-359, A'-[3-oxo-3-[4-(4-pyridmyl)-l- piperazinyl]propyI]-2,l,3-benzothiadiazole-4-sulfonamide (VU0255035), dicycloverine, benztropine, muscarinic toxin 7 (MT7), 4-DAMP, darifenacin, procyclidine, zamifenacin, solifenacin, fesoterodine, tramadol, hyoscyamine, diphenhydramine, cyproheptadine, mequitazine, oxybutynin, tolterodine, dicylomine, atropine, mecamylamine, olanzapine, amitriptyline, desipramine, desmethyldespiramine, dosulepin, doxepin, femoxetine, imipramine, lofepramine, nortriptyline, paroxetine, sertraline, chlorprothixene, chlorpromazine, clozapine, cymamazine, N-dexmethylclozapine, flurperlapine, loxapine, mesoridazine, olanzapine, thoridazine, and zotepine.

26. A co-culture comprising cholinergic neurons and glioma cells.

27. A method of evaluating a potential anti-cancer therapeutic, comprising contacting the coculture of claim 26 with the potential anti-cancer therapeutic and assessing a response toPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 the potential anti-cancer therapeutic in the co-culture.

28. The method of claim 27, wherein assessing the response comprises assessing a response in the cholinergic neurons, in the glioma cells, and / or in an interaction between the cholinergic neurons and the glioma cells.

29. A method comprising: a) culturing cholinergic neurons and glioma cells in a media mixture comprising: i. cholinergic neuron maintenance medium, and ii. and a tumor stem cell growth factor-free medium comprising DMEM, neurobasal- A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture.

30. The method of claim 29, comprising culturing the cholinergic neurons and glioma cells in the media mixture for at least 24 hours.

31. The method of claim 29, comprising culturing the cholinergic neurons and glioma cells in the media mixture for at least 48 hours.

32. The method of any one of claims 29-31, wherein the cholinergic neuron maintenance medium and the tumor stem cell growth factor- free medium are present in the media mixture at a ratio of about 0.8: 1.2 to 1.2:0.

8. (v / v), respectively.

33. The method of claim 32, wherein the cholinergic neuron maintenance medium and the stem cell growth factor-free medium are present in the media mixture at a ratio of about 1:1.

34. The method of any one of claims 29-33, wherein the cholinergic neurons are obtained by differentiating human induced pluripotent stem cells (hiPSCs) into the cholinergic neurons, wherein the differentiating comprises delivering a neurogenic transcriptionPCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-196 factor to the hiPSCs followed by culturing for 7 or more days.

35. The method of claim 34, wherein the neurogenic transcription factor is delivered virally.

36. The method of claim 35, wherein the cholinergic neurons are transduced with a vector configured for optogenetic manipulation to co-culturing the cholinergic neurons and the glioma cells in the media mixture.

37. A method comprising: a) transducing cholinergic neurons with a vector configured for optogenetic manipulation; b) culturing the transduced cholinergic neurons and glioma cells in a media mixture, the media mixture comprising: i. cholinergic neuron maintenance medium, and ii. and a tumor stem cell growth factor-free medium comprising DMEM, neurobasal- A, and B27-A, thereby obtaining a cholinergic neuron-glioma co-culture; and c) performing optogenetic manipulation on the cholinergic neuron-glioma co-culture.

38. The method of claim 37, comprising culturing the transduced cholinergic neurons and the glioma cells in the media mixture for at least 24 hours.

39. The method of claim 38, comprising culturing the transduced cholinergic neurons and the glioma cells in the media mixture for at least 48 hours.

40. The method of any one of claims 37-39, wherein the cholinergic neuron maintenance medium and the tumor stem cell growth factor- free medium are present in the media mixture at a ratio of about 0.8: 1.2 to 1.2:0.

8. (v / v), respectively.PCT / US25 / 44446 02 September 2025 (02.09.2025)Atty. Docket: STDU2-43525.601 Client Ref. S24-19641. The method of claim 40, wherein the cholinergic neuron maintenance medium and the stem cell growth factor-free medium are present in the media mixture at a ratio of about 1:1.

42. The method of any one of claims 37-41, wherein the cholinergic neurons are obtained by differentiating human induced pluripotent stem cells (hiPSCs) into the cholinergic neurons, wherein the differentiating comprises delivering a neurogenic transcription factor to the hiPSCs followed by culturing for 7 or more days.

43. The method of claim 42, wherein the neurogenic transcription factor is delivered virally.

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