Treatment of cancer of the central nervous system

Abstract

The present invention relates to treatment of a cancer of the central nervous system, in particular the use of Wallerian degeneration inhibitors in the treatment of CNS cancer. Also provided are in vivo and in vitro methods of screening Wallerian degeneration inhibitors as anti-CNS cancer therapeutic compounds.

Description

[0001] Treatment of Cancer of the Central Nervous System

[0002] This application claims priority from GB2408068.1 filed 6 June 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present invention relates to the fields of Wallerian degeneration inhibitors and their use in the treatment of cancer, in particular cancer of the central nervous system.

[0005] Background

[0006] Gliomas are a heterogenous group of cancers originating in the glial cells of the brain and spinal cord. They represent the most common type of malignant primary central nervous system tumours in adults and are classified according to the type of cell they arise from (e.g. astrocytomas, oligodendrogliomas, ependymomas). Glioblastoma, previously known as glioblastoma multiforme (GBM), is the most prevalent type of brain cancer in adults. The median survival time is just 12 to 18 months, with fewer than 7% of patients surviving for 5-years, representing a significant unmet clinical need. Generally, glioblastoma is diagnosed at an already advanced stage, which is largely due to nonspecific early signs and symptoms (including headaches and nausea). Advanced stage glioblastoma is extremely difficult to treat, and presents with a range of severe symptoms, including neurological impairments that are often debilitating for patients.

[0007] Due to the lack of early diagnosis, research around glioblastoma has generally relied on advanced-stage tumour samples. This means there is a significant gap in our knowledge around how glioblastoma initiates and progresses. A limited number of case studies have investigated patients with early-stage glioblastoma and found that lesions may have a prolonged latency before progressing to advanced stage of disease. This may indicate that glioblastoma progression includes a latent pre-clinical phase, which progresses to advanced disease following extrinsic signalling (Kawauchi etal. Neurosurgery (2022) 91 (5):741 -748; Aggarwal et al. Br J Neurosurg. (2015) 29(4):520-523; Toh et al. AJNR Am J Neuroradiol. (2017) 38(2):288-293; Nishi etal. Neurol Med Chir. (2009) 49(1):8-12).

[0008] Wallerian degeneration and inhibitors of Wallerian degeneration have been discussed in Cancers, vol. 16, no. 11 , 2024, Perryman Richard et al., Distinct Capabilities in NAD Metabolism Mediate Resistance to NAMPT Inhibition in Glioblastoma; Journal of Medicinal Chemistry, vol. 67, no. 11 , 2024, Wen Fei et al., Discovery of Novel Dual Inhibitors Targeting Mutant IDH1 and NAMPT for the Treatment of Glioma with IDHIMutation; Child's Nervous System, vol. 6, no. 1 , 1990, Tamaki N et al., Germ cell tumors of the thalamus and the basal ganglia, pages 3-7; Pediatric Radiology, vol. 36, no. 4, 2006, Ozelame Rodrigo V et al., Basal ganglia germinoma in children with associated ipsilateral cerebral and brain stem hemiatrophy; Brain, vol. 144, no. 10, 2021 , Bosanac Todd et al., Pharmacological SARM1 inhibition protects axon structure and function in paclitaxel-induced peripheral neuropathy, pages 3226-323;

[0009] Neuron, vol. 110, no. 22, 2022, Bratkowski Matthew et al..Uncompetitive, adduct-forming SARM1 inhibitors are neuroprotective in preclinical models of nerve injury and disease., pages 3711-3726; US 2024 / 0102018 A1 (COHEN et al.) and Neurobiology of Disease, vol. 171 , 2022, Alexandris Athanasios S. et al., Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons, article no. 105808.

[0010] Furthermore, it has been shown that phenotypically ‘normal’ brain tissue contains driver mutations, indicating that driver mutations alone are insufficient to initiate tumour growth. Instead, these driver mutations may act to ‘prime’ cells, which are then triggered to transform following a cell-extrinsic stimulus, known as a promoter. A variety of stimuli may constitute a promoter, including a number of pro- inflammatory signals, such as infection, extrinsic injury, chronic inflammation, and ageing. Together these known promoters highlight a critical role for tissue damage and inflammation in tumour initiation.

[0011] The initiation and progression of gliomas, in particular glioblastoma, remains poorly understood, representing a vital avenue for research for the development of new therapeutic targets.

[0012] The present invention has been devised in light of the above considerations.

[0013] Summary of the Invention

[0014] Broadly, the present inventors have discovered an improved approach to treating and / or preventing cancer of the central nervous system. The inventors have discovered that axonal injury drives tumorigenesis by activating Wallerian degeneration, a major active programme of neuronal death, which leads to increased inflammation and tumour proliferation. Inactivation of Wallerian degeneration preserves axonal integrity and shows anti-cancer, as well as neuroprotective, effects.

[0015] In a first aspect, the present invention provides a Wallerian degeneration inhibitor for use in a method of treating or preventing a cancer of the central nervous system.

[0016] In a second aspect, the present invention provides the use of a Wallerian degeneration inhibitor in the manufacture of a medicament for use in a method of treating or preventing a cancer of the nervous system.

[0017] In a third aspect, the present invention provides a method of treating or preventing a cancer of the nervous system comprising administering a therapeutically or prophylactically effective amount of a Wallerian degeneration inhibitor to a subject in need thereof.

[0018] In some embodiments of the first to the third aspect, the Wallerian degeneration inhibitor is a SARM1 inhibitor.

[0019] In some embodiments of the first to the third aspect, the Wallerian degeneration inhibitor is a nucleic acid.

[0020] In some embodiments, the Wallerian degeneration inhibitor is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide is complementary to a region of a SARM1 mRNA. In some embodiments, the antisense oligonucleotide is complementary to a region of the SARM1 mRNA encoded by the nucleotide sequence of NCBI reference sequence: NM_015077, v4.

[0021] In some embodiments, the Wallerian degeneration inhibitor is a small interfering RNA.

[0022] In some embodiments of the first to the third aspect, the Wallerian degeneration inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is selected from the list comprising: CHS828, isoquinolines, isothiazole, zinc pyrithione; tryptoline acrylamides and FK866.

[0023] In some embodiments of the first to the third aspect, the cancer of the central nervous system is selected from the list comprising: glioblastomas, gliomas, craniopharyngioma, brain stem glioma, medulloblastoma, meningioma, astrocytomas, pituitary adenomas, oligodendroglioma, ependymal tumors, pineal parenchymal tumors and brain metastases. In some embodiments, the cancer of the central nervous system is glioblastoma. In some embodiments, the cancer is an early-phase cancer.

[0024] In some embodiments of the first to the third aspect, the cancer is an early-phase CNS cancer, and the method prevents or slows progression of the CNS cancer to a later stage.

[0025] In some embodiments of the first to the third aspect, the method comprises reducing glioblastoma- associated neurological symptoms of the subject. In some embodiments, the glioblastoma-associated neurological symptoms are selected from the list including: seizure (including focal seizure and / or generalised seizure), loss of consciousness, syncope, aphasia, headache, visual deficit ( / .e. dysfunction or loss), gait disturbance, memory impairment, paralysis, paresis, plegia, weakness, sensory dysfunction, language dysfunction, and cognitive impairment.

[0026] In some embodiments of the first to the third aspect, the method comprises administering the inhibitor of Wallerian degeneration to a subject in conjunction with, or immediately following, tumour resection or chemotherapy.

[0027] In some embodiments of the first to the third aspect, the subject is considered at risk of developing glioblastoma.

[0028] In a fourth aspect, the present invention provides a method of screening therapeutic candidates as anti- CNS cancer therapeutic compounds. In some embodiments, the method of screening comprises a step of identifying if the therapeutic candidate is an inhibitor of Wallerian degeneration.

[0029] In some embodiments, the method is an in vitro method. In some embodiments, the present invention provides an in vitro method of screening therapeutic candidates as anti-CNS cancer therapeutic compounds, the method comprising:

[0030] (i) selecting Wallerian degeneration inhibitors as therapeutic candidates

[0031] (ii) providing organotypic brain slice cultures derived from both healthy and tumour-bearing brains; (iii) adding therapeutic candidates to the organotypic brain slice culture media;

[0032] (iv) analysing the organotypic brain slice culture to determine the impact on tumour progression.

[0033] In some embodiments, the method of screening is an in vivo method. In some embodiments, the present invention provides an in vivo method of screening therapeutic candidates as anti-CNS cancer therapeutic compounds, the method comprising:

[0034] (i) selecting Wallerian degeneration inhibitors as therapeutic candidates

[0035] (ii) providing genetically engineered mouse models of CNS cancer;

[0036] (iii) delivering therapeutic candidates to the tumour, cerebrospinal fluid reservoir, or tumour resection cavity via implantable osmotic pumps;

[0037] (iv) analysing the mouse model to determine the anti-cancer effect of the therapeutic candidates.

[0038] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0039] Brief Description of the Figures

[0040] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0041] Figure 1. (a) Representative images of white matter bundles in the tumour-involved (Tumour) and contralateral (tumour-free; Contra) striatum of ThylYFP mice bearing intermediate npp tumours. Arrowheads exemplify a heavily- (yellow), moderately- (white) and a lowly- (blue) tumour infiltrated bundle. Scale bar=100 pm. (b) Quantification of YFP mean fluorescence intensity (MFI) as a function of tumour cell density in tumour-involved striatal white matter of npp tumour-bearing mice from a. Turquoise line and blue band indicate mean contralateral fluorescence intensity and SD, respectively. Pearson R. n=6. (c) Representative electron micrographs of tumour-involved and contralateral (tumour-free; Contra) white matter bundles in the striatum of WT and Sarml- / - mice bearing intermediate npp tumours. Yellow arrows point to axons with signs of degeneration. Scale bar = 2.5 pm. (d) Quantification of the percentage of degenerating neurons over total number of neurons in tumour-involved striatal white matter of WT and Sarml- / - npp tumour-bearing mice from c. Each dot represents a white matter bundle. Two-way ANOVA with Tukey’s multiple comparisons. WT n= 3, Sarml- / - n= 3. (e) G-ratios of tumour-involved striatal white matter of WT and Sarml- / - mice bearing intermediate npp tumours. Each dot represents a white matter bundle. Two-way ANOVA with Tukey’s multiple comparisons. WT n=3, Sarml- / - n=3. (f) Representative images of neurofilament staining (NF, yellow) of tumour-involved (tumour) or contralateral (tumour-free, Contra) striatal white matter bundles in WT and Sarml- / - mice bearing intermediate npp tumours (10.5 weeks post-electroporation). Scale bar=50 pm. (g) Quantification of neurofilament (NF) mean fluorescence intensity (MFI) in tumours depicted in a. Individual white matter bundles are shown. Two- way ANOVA with Tukey’s multiple comparisons. WT n= 3, Sarml- / - n= 3. (h) Representative images correlating confocal microscopy images and electron micrographs of intermediate tumours (10.5 weeks post-electroporation) induced in WT (i-iv) and Sarml- / - mice (v-viii); i, overview image of npp tumours in WT and iv, Sarml- / - mice (bottom). Scale bar=200 pm. ii and vi, Corresponding representative electron micrographs of fluorescence images from i and v. Scale bar = 200 pm. Dashed boxes indicate the striatal white matter bundle depicted at higher magnification on the right (iii, iv, vii and viii). Dashed yellow boxes indicates region shown in Figure 1 c. Scale bar=50 pm. (i) Representative images of GFAP+ astrocytes surrounding tumour-involved striatal white matter bundles in mice bearing intermediate npp tumours. Contralateral tumour-free striatum (Contra) is shown on the right. Scale bar=100 pm. (j) Representative images of CD68+ / lba1 + microglia in tumour-involved striatal white matter bundles in mice bearing intermediate npp tumours. Contralateral tumour-free striatum (Contra) is shown on the right. Scale bar = 100 pm. (k) Quantification of GFAP+ astrocyte density as a function of tumour cell density in tumour- involved striatal bundles in mice bearing intermediate npp tumours. Each point represents a bundle, >5 bundles in tumour-involved striatum per mouse were quantified. Turquoise line and blue band indicate mean contralateral GFAP+ cell density and SD, respectively. Pearson R. n=6. (I) Quantification of CD68 integrated intensity (IntDen) as a function of tumour cell density in in tumour-involved striatal bundles in mice bearing intermediate npp tumours. Each point represents a bundle, >5 bundles in tumour-involved striatum per mouse were quantified. Turquoise line and blue band indicate mean contralateral CD68 integrated density and SD, respectively. ±1 SD range. Pearson R. n=4. (m) Representative superresolution confocal images of tumour-involved (left and middle top panels) and contralateral striatal bundles (right top panel) of Thy1 YFP mice bearing intermediate npp tumours. tdTomato+ tumour cells are in red, axons in green, Lower panels: Examples of varicosities, a hallmark of physical injury to axons, seen in tumour-infiltrated bundles. White box indicates area depicted in adjacent higher magnification images. Yellow arrowheads indicate varicosities. Scale bar=10 pm.

[0042] Figure 2. (a) Schematic of experimental outline. 8.5 weeks after tumour induction in wildtype (WT) or Sarml- / - mice, corpus callosum axons ipsilateral to the tumour were injured (Injury) by surgical transection or left intact (Sham). Analysis was performed 2 weeks later following a 2h Edll pulse, (b) Representative image of tdTomato+ (red) sham and injured WT tumours stained for Edll (grey) and DAPI (blue). Dashed boxes indicate representative regions proximal (P) and distal (D) to the injury site in the tumour-ipsilateral hemisphere that are magnified in e and f. Scale bar=500 pm. (c) Quantification of the percentage of proliferating Edll+-tdTomato+ tumour cells over total number of tdTomato+ tumour cells in WT and Sarml- / - npp tumours from (b), measured in all tumour regions either proximal or distal to the injury. Two-way ANOVA with Tukey’s multiple comparisons. WT Sham n=7, WT Injury n=8, Sarm1-Z- Sham n=7, Sarml- / - Injury n=6. (d) Quantification of the percentage of tdTomato+ cells in tumour- involved white (WM; turquoise spots) and grey matter (GM; purple spots) in the striatum of WT mice bearing npp tumours and subjected to Sham or Injury. Tumour- and injury-ipsilateral striatum was quantified. Two-way ANOVA with Tukey’s multiple comparisons. n=7 per condition, (e, f) Representative images of tdTomato (red), GFAP (turquoise), CD68 (yellow) and Iba1 (magenta) staining in npp tumours in Sham and Injury WT mice; P (e) and D (f) regions from (b) are shown. Scale bar=100 pm. (g, h) Quantification of GFAP area as a readout of astrocyte reactivity (g) and CD68 intensity (IntDen) as a readout of microglia activation (h) within npp tumours generated in WT and Sarml - / - mice; tumour regions proximal and distal to the injury site were quantified separately and normalised to Sham in each genotype. Two-way ANOVA with Tukey’s multiple comparisons. WT Sham n=7, WT Injury n=6, Sarml- / - Sham n=7, Sarml- / - Injury n=8. (i) Representative images of neurofilament staining (NF, yellow) of intermediate npp tumours generated in WT and Sarml- / - mice, 2 weeks following Sham injury or transection of tumour-ipsilateral corpus callosum axons (Injury, see timeline in (a)). Scale bar=200 pm. White box indicates the injury site region depicted at high magnification on the right. Note significant axonal protection in Sarml - / - animals even at the site of injury demarcated by the star symbol. Scale bar=200 pm. (j, k) Representative images of intermediate-stage npp tumours generated in Sarml - / - animals 2 weeks following Sham injury or transection of tumour-ipsilateral corpus callosum axons (Injury, see timeline in (a)). tdTomato+ cells are in red; sections were stained for EdU (grey) and DAPI (blue) in (j) and GFAP (turquoise), I ba1 (magenta) and CD68 (yellow) in (k). Tumour regions distal to the injury site in the tumour-ipsilateral hemisphere are shown. Note that unlike in distal WT tumour regions there is no increase in tumour cell proliferation or inflammation following axonal transection. Corpus callosum is indicated by dashed line. Scale bar=100 pm. (I), Representative images of tdTomato (red) and GFP (green) fluorescence in npp tumours injected intraventricularly with GFP or SarmDN-GFP AAVs at the time of tumour induction (P2), subjected to Sham (left panel) or Injury (right panel) at intermediate stage and stained for EdU (grey) and DAPI (blue). Scale bar=200mm. (m), Quantification of the percentage of proliferating EdU+ / tdTomato+tumour cells over total number of tdTomato* tumour cells in tumours from Figure 2(l). Mean±SEM. Multiple unpaired t tests. GFP Sham n=5, GFP Injury n=5, SarmDN Sham n=4, SarmDN Injury n=5. (n) Representative images of the injury site in npp tumour-bearing brains transduced with GFP or SarmDN AAVs via intraventricular injection at the time of tumour induction (P2, see Figure 4g) and subjected to axonal transection at intermediate disease stage. Tissue was stained for neurofilament (grey). GFP (green) denotes successful neuronal transduction. Note axonal protection in the SarmDN-transduced brains. Scale bar=200mm. (o-p) Quantification of GFAP area (h) and CD68 intensity (IntDen, i) within npp tumours injected intraventricularly with GFP or SarmDN-GFP AAVs at the time of tumour induction (P2) and subjected to Sham or Injury at intermediate stage; injury site and the rest of the tumour area were quantified separately and normalised to Sham in each genotype.

[0043] Mean±SEM. Multiple unpaired t tests. GFP Sham n=7, GFP Injury n=5, SarmDN Sham n=4, SarmDN Injury n=5. (q) Representative image of tdTomato (red) and GFP (green) fluorescence in WT npp tumours injected intratumourally with GFP or SarmDN-GFP AAVs at intermediate disease stage and stained for EdU (grey), (r), Quantification of percentage EdU+ / tdTomato+tumour cells over total number of tdTomato* tumour cells in tumours from q. Mean±SEM. Unpaired t test. GFP n=4, SarmDN n=4. (s), as in q for tumours injected with AAVs at late stage. Scale bar=200mm. (t), Quantification of percentage EdU tdTomato* tumour cells over total number of tdTomato* tumour cells in tumours from k. Mean±SEM. Unpaired t test. GFP n=5, SarmDN n=4. (u), Kaplan Meier survival curves of npp tumour-bearing WT mice subjected to Sham (WT Sham) or Injury (WT Injury) at intermediate disease stage. n= 15 for both groups. Figure 3. (a) Representative images of terminal npp tumours in WT and Sarml- / - mice. Scale bar=1000 pm. (b) Quantification of total brain area covered by tdTomato+ cells in WT and Sarm 1- / - tumour mice. Paired t test. WT n=10, Sarml- / - n=6. (c) UMAP representation of scRNA-seq data from npp tumours in WT and Sarml- / - mice tumour cells. Cell type labels were assigned to clusters using published signatures and marker genes: neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like), mesenchymal like (MES-like) and active neural stem cell-like (aNSC-like). (d) As in c for microenvironmental cells: choroid plexus cells (CP), astrocytes (Astro), inflamed glia, oligodendrocyte progenitor cells (OPC), transient amplifying progenitors / neuroblasts (TAP / NB), active neural stem cells (aNSC), ependymal cells (EpC), endothelial cells (EC), pericytes, tumour associated macrophages (TAMs), monocytes (Mn), and T cells, (e, f) Proportion of different tumour and microenvironmental cell types. Data were down-sampled to equal number of cells of both genotypes in either tumour (n=12591) or microenvironment cells (n=4176). The dashed line at 0.5 denotes no changes between genotypes. Cell types with PPearson's chi-squared test < 0.01 and relative difference > 3% were considered significantly different (§). (g, h, i, j, k) Flow cytometry analysis of indicated immune populations in terminal npp tumours generated in WT and Sarml - / -mice. Unpaired t test. WT n= 5, Sarml- / - n= 5. (I) Kaplan Meier survival curves of npp tumour-bearing WT (grey line) and Sarml- / - (turquoise line) mice. Median survival WT=18 weeks, Sarm1- / -=21 weeks. Log-rank test. WT n=22, Sarml- / - n=18. (m) Assessment of motor function. Neuroscores of npp tumour-bearing WT (grey spots) and Sarml- / - (turquoise spots) mice assessed at early (8 weeks) and advanced (<2 weeks prior to death) disease stage. Two-way ANOVA with uncorrected Fisher’s LSD test. WT n=5, Sarml- / - n=5. (n) UMAP representation of scRNA-seq data from microenvironmental cells identified as TAMs in Fig. 3d. Two clusters are labelled in red (1) and blue (2). (o) Heatmap of Iog2 expression ratios (logFC) between cluster 1 and 2 for microglia markers, (p) as in o for macrophage markers, (q) Proportion of Microglia or Macrophages in tumours from WT or Sarml- / - mice. The dashed line represents equal proportion in both genotypes. Cell types with PPearson's chi-squared test < 0.01 and relative difference > 3% were considered significantly different (§). (r) LIANA Ligand-receptor analysis based on ligands from microenvironmental cells (Sender) and receptors from tumour cells (Receiver) in tumours from WT animals. Numbers on the plot denote the numbers of significant interactions, (s) As in r for Sarml - / - animals, (t) LIANA Ligand-receptor analysis based on ligands from microenvironmental cells (Sender, cell types at the top of the graph) and receptors from tumour cells (Receiver, cell types at the bottom of the graph) in tumours from WT animals. The diameter of the dots represents the specificity of the interaction and their colour the expression magnitude as defined in the LIANA package. Ligand-receptor pairs are listed on the left of the graph, (u) As in t for Sarml- / - animals, (v), Kaplan Meier survival curves of npp tumour-bearing Sarm1Jil(WT, grey line) and Sarrn1em1 1 Tftc(turquoise line) mice. Median survival WT=17 weeks, Sarm1em1 1Tftc-=22 weeks. Log-rank test. WT n=11 , Sarm1em1 1Tftcn=12. (w), Neuroscores of npp tumour-bearing WT (grey spots) and Sarrn1em1 1Tftc(turquoise spots) mice assessed at early (8 weeks) and advanced (<2 weeks prior to death) disease stage. Two-way ANOVA with Tukey’s multiple comparisons. Sarml WT early n=8, advanced n = 6, Sarrn1em1 1 Tftcearly n=13, advanced n=11 . (x) Representative fluorescence images of npp tumours generated in Sarm 1em1 1 Tftcand genetic background matched Sarm1wtmice (WT). Images show tdTomato+tumour cells (red) and nuclei are counterstained with DAPI (blue), (y), Quantification of number of tumour cells per mm2in tumours shown in d. Mean±SD. Unpaired t test. n=4 for both genotypes.

[0044] Figure 4. Example plasmids used in glioblastoma somatic tumour generation.

[0045] Detailed Description

[0046] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0047] Wallerian degeneration

[0048] Wallerian degeneration (or ‘WD’) as used herein refers to the process following traumatic injury, or other non-traumatic injury (including impairment of axonal transport), referred to as ‘Wallerian-like’ injury, wherein part of the axon distal to the site of injury degenerates. The process of Wallerian degeneration is associated with neurodegenerative disorders, such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis and glaucoma.

[0049] Wallerian degeneration occurs in both the peripheral nervous system and the central nervous system, although some of the exact molecular processes differ between the two. The process of WD can be divided into four phases: the acute response, latency, degeneration, and clearance. Numerous factors involved in the Wallerian degeneration pathway have been identified, including SARM1 , NMNAT, MAPKs, NAD+ and NMN. Factors or components that are involved in the Wallerian degeneration pathway are referred to herein as ‘Wallerian degeneration factors’. These factors may be Wallerian degeneration promoting factors ( / .e. the activity of these components promotes the Wallerian degeneration process, or the inhibition of these components inhibits the Wallerian degeneration process), or Wallerian degeneration inhibiting factors ( / .e. the inhibition of these components promotes the Wallerian degeneration process, or the activity of these components inhibits the Wallerian degeneration process). The cellular and molecular mechanisms of Wallerian degeneration are reviewed, for example, in Coleman etal. Nat Rev Neurosci. (2020) 21 :183-96.

[0050] SARM1 (also known as Sarml , NAD(+) hydrolase SARM1 , and sterile alpha and TIR motif containing 1) is a member of the Toll / I nterleuki n receptor-1 (TIR) family. The TIR domain of SARM1 is highly conserved and has intrinsic NADase activity, which hydrolyses NAD+ to adenosine diphosphate ribose (ADPR), cyclic ADPR and nicotinamide. In mammals, SARM1 is highly expressed in neurons, in both cell bodies and axons. SARM1 NADase activity is activated following axon injury, causing hydrolysis of NAD+and so reduction in NAD+levels, which is considered a major event in the Wallerian degeneration pathway. SARM1 is considered a Wallerian degeneration promoting factor. The role of SARM1 is reviewed, for example, in Waller et al. Front Cell Neurosci. (2022) 16:958900. SARM1 as described herein includes any forms, homologues or variants of SARM1 . For example, the SARM1 may be a SARM1 of any mammalian species. In some embodiments, SARM1 is human SARM1 , the amino acid sequences of which is disclosed at UniProt ID: Q6SZW1 (entry version: 158; release number: 2024_03 / 2024_03).

[0051] SARM1 according to the present invention is understood to encompass monomeric, dimeric or multimeric forms of SARM1.

[0052] NMNATs (also known as Nicotinamide / nicotinic acid mononucleotide adenylyltransferase, nicotinamidenucleotide adenylyltransferase and nicotinate-nucleotide adenylyltransferase) are a family of conserved enzymes that catalyse NAD+synthesis from nicotinamide mononucleotide (NMN) and ATP. There are three human isofroms, NMNAT1, NMNAT2 and NMNAT3, with differing subcellular localization. NMNAT1 is a nuclear protein, whereas NMNAT2 is localized to the Golgi complex and the axon, and NMNAT3 has a mitochondrial location. Following axonal injury, protein levels of NMNAT, and particularly NMNAT2, decrease, leading to reduced biosynthesis of NAD+, and buildup of the NAD+precursor NMN. Both factors are considered important in the process of Wallerian degeneration. NMNAT is considered a Wallerian degeneration inhibiting factor. The role of NMNATs is reviewed, for example in Brazil et al. Curr Opin Genet Dev. (2018) 44:156-162. The NMNAT as described herein includes any forms, homologues or variants of NMNAT. For example, the NMNAT may be a NMNAT of any mammalian species. In some embodiments, the NMNAT according to the present invention is NMNAT2. In some embodiments, the NMNAT is human NMNAT2, the amino acid sequence of which is disclosed at UniProt ID: Q9BZQ4 (entry version: 170; release numbers: 2024_03 / 2024_03). NMNAT according to the present invention is understood to encompass monomeric, dimeric or multimeric forms of NMNAT.

[0053] NAD (also known as nicotinamide adenine dinucleotide) is a coenzyme that is central to metabolism and found in all living cells. NAD exists in an oxidised form (NAD+) and a reduced form (NADH). NAD+ can be produced through salvage reactions from nicotinamide mononucleotide (NMN). NAD+ depletion and NMN accumulation are both implicated as key processes in Wallerian degeneration, as described in, for example, Gerdts et al. Neuron. (2016) 89(3):449-460.

[0054] Inhibitors of Wallerian degeneration

[0055] Aspects of the present invention involve inhibition ( / .e. antagonism) of the process of Wallerian degeneration.

[0056] Herein, ‘inhibition’ refers to a reduction, decrease or lessening relative to a control condition. For example, inhibition of Wallerian degeneration by a Wallerian degeneration inhibitor refers to a reduction, decrease or lessening of the extent / degree of Wallerian degeneration in the presence of the Wallerian degeneration inhibitor.

[0057] Inhibition may herein also be referred to as neutralisation or antagonism. A Wallerian degeneration inhibitor (e.g. an antagonist of a factor that promotes Wallerian degeneration or an agonist of a factor that inhibits Wallerian degeneration) may be said to be a ‘neutralising’, ‘antagonistic’ or ‘agonistic’ agent with respect to the relevant function or process. For example, a Wallerian degeneration inhibitor ( / .e. an agent which is capable of inhibiting Wallerian degeneration) may be referred to as an agent which is capable of neutralising Wallerian degeneration or may be referred to as an antagonist of a factor that promotes Wallerian degeneration or may be referred to as an agonist of a factor that inhibits Wallerian degeneration.

[0058] The skilled person may identify Wallerian degeneration inhibitors by appropriate assay to determine the level of Wallerian degeneration in samples treated with candidate inhibitors compared against samples treated with appropriate control reagents. The skilled person is able to identify appropriate control conditions for a given assay.

[0059] In embodiments of the present invention, inhibition of Wallerian degeneration may encompass the following features:

[0060] • Inhibition of axonal degeneration (for example as measured through neurofilament staining, as described in Example 1 herein)

[0061] • Inhibition of demyelination (for example as measured through immunohistochemistry, as described in Example 1 herein)

[0062] • Inhibition of white matter atrophy (for example, as measured through immunohistochemistry as described in, e.g. Marion et al. Experimental Neurology. (2019) 321 :113040).

[0063] • Inhibition of neuroinflammation (for example as measured through detecting expression of inflammatory cytokines, including IFN-a, IFN-p, IFN-y, I L-1 p, MIP-1a, and TNF-a)

[0064] Wallerian degeneration signalling pathways offer multiple routes for inhibition of signalling. A Wallerian degeneration inhibitor may be an agonist ( / .e. an activator) of a factor that inhibits Wallerian degeneration (for example, NMNAT). A Wallerian degeneration inhibitor may be an antagonist ( / .e. an inhibitor) of a factor that promotes Wallerian degeneration (for example, SARM1).

[0065] For example, inhibition of Wallerian degeneration may be achieved by inhibition of the activity of SARMI . In some embodiments, the Wallerian degeneration inhibitor according to the present invention is a SARM1 inhibitor. In some embodiments, the Wallerian degeneration inhibitor inhibits the NADase activity of SARMI .

[0066] Inhibition of Wallerian degeneration may be achieved by manipulation of gene or protein expression of a Wallerian degeneration factor. An agent capable of manipulating the expression of one or more Wallerian degeneration factors may do so e.g. through inhibiting or enhancing transcription of the gene encoding the Wallerian degeneration factor, inhibiting or enhancing post-transcriptional processing of RNA encoding the Wallerian degeneration factor, manipulating the stability of RNA encoding the Wallerian degeneration factor, promoting or inhibiting degradation of RNA encoding the Wallerian degeneration factor, manipulating post-translational processing of the Wallerian degeneration factor polypeptide, manipulating the stability of the Wallerian degeneration factor polypeptide or promoting or inhibiting degradation of the Wallerian degeneration polypeptide. In some embodiments, the Wallerian degeneration inhibitor reduces the gene or protein expression of SARMI .

[0067] Wallerian degeneration inhibitors may be of any kind, but in some embodiments the inhibitor may be a nucleic acid, an oligonucleotide, an aptamer, a small molecule, an antigen-binding molecule e.g. an antibody or an antigen-binding fragment thereof, a polypeptide or a peptide. The agents may be provided in isolated or purified form or may be formulated as a pharmaceutical composition or medicament.

[0068] Antigen-binding molecules

[0069] In some embodiments, the Wallerian degeneration inhibitor according to the present invention may be an antibody, or an antigen-binding fragment thereof, e.g. an anti-SARM1 antibody or antigen-binding fragment thereof. An “antibody” is used herein in the broadest sense, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they display binding to the relevant target molecule.

[0070] In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982).

[0071] Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments may also be used / provided as can genetically engineered antibodies and antibody fragments. Antibodies and antigen-binding fragments according to the present disclosure comprise the complementarity-determining regions (CDRs) of an antibody which is capable of binding to the relevant target molecule (e.g. SARM1 / a SARM1 containing complex).

[0072] The skilled person is familiar with techniques for producing antibodies suitable for therapeutic use in a given species / subject. For example, procedures for producing antibodies suitable for therapeutic use in humans are described in Park and Smolen Advances in Protein Chemistry (2001) 56: 369-421 (hereby incorporated by reference in its entirety). Furthermore, the skilled person is aware of methods and techniques for targeting the central nervous system ( / .e. crossing the blood-brain barrier). Such methods and techniques are reviewed in, for example, Zhao et al. Antib. Ther. (2022) 5(4):311-331 and Banks, Nat Rev Drug Discov. (2016) 15(4):275-292.

[0073] Nucleic acids

[0074] In some embodiments, the Wallerian degeneration inhibitor is a nucleic acid. Nucleic acids according to the present invention include antisense oligonucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNAs (miRNA) and double-stranded RNA (dsRNA).

[0075] In some embodiments, the Wallerian degeneration inhibitor may be an antisense oligonucleotide, or small interfering RNA. In some embodiments, the Wallerian degeneration inhibitor may be shRNA, dsRNA, miRNA or siRNA. In some embodiments, the Wallerian degeneration inhibitor is an antisense oligonucleotide. For example, in some embodiments, the Wallerian degeneration inhibitor is an antisense oligonucleotide targeting SARM1 RNA.

[0076] An antisense oligonucleotide is an oligonucleotide, preferably single-stranded, that targets and binds, by complementary sequence binding, to a target oligonucleotide, e.g. mRNA. Where the target oligonucleotide is an mRNA, binding of the antisense to the mRNA may block translation of the mRNA and expression of the gene product. Antisense oligonucleotides may be designed to bind sense genomic nucleic acid and inhibit transcription of a target nucleotide sequence. Alternatively, antisense oligonucleotides binding to a target mRNA may enhance expression of the gene product, for example, by the antisense oligonucleotide targeting the 3’ or 5’UTRs of the target mRNA. Antisense oligonucleotide technology for enhancing protein expression is reviewed in, for example, Khorkova et al. Nat Rev Drug Discov. (2023) 22(7):539-561 .

[0077] An antisense oligonucleotide may target the coding sequence (CDS) of a target mRNA. An antisense oligonucleotide may target the 5’UTR or the 3’UTR of a target mRNA.

[0078] In view of the known nucleic acid sequences for Wallerian degeneration factors (e.g. the known mRNA sequences available, for example: NCBI reference sequences: NM_015077, version 4 (v4) (human SARM1); NM_015039, v4 and NM_170706, v4 (human NMNNAT2); NM_001297778, v1 , NM_001297779, v2, NM_022787, v4 (human NMNAT1); NM_001200047, v3 (human NMNAT3); oligonucleotides may be designed to manipulate the expression of a Wallerian degeneration factor. Such antisense oligonucleotides may repress or silence the expression of a target Wallerian degeneration factor (e.g. a Wallerian degeneration promoting factor). Alternatively, antisense oligonucleotides may enhance expression of a target Wallerian degeneration factor ( / .e. a Wallerian degeneration inhibiting factor), for example, by targeting translation inhibitor elements in 5’UTRs.

[0079] Such oligonucleotides may have any length, but may preferably be short, e.g. less than 100 nucleotides, e.g. 10-40 nucleotides, or 20-50 nucleotides, and may comprise a nucleotide sequence having complete- or near- complementarity (e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity) to a sequence of nucleotides of corresponding length in the target oligonucleotide, e.g. the mRNA of a Wallerian degeneration factor, e.g. SARM1. The complementary region of the nucleotide sequence may have any length, but is preferably at least 5, and optionally no more than 50, nucleotides long, e.g. one of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0080] Repression of expression of a Wallerian degeneration promoting factor will preferably result in a decrease in the quantity of a Wallerian degeneration promoting factor expressed by a cell / tissue / organ / organ system / subject. For example, in a given cell the repression of a Wallerian degeneration promoting factor by administration of a suitable nucleic acid will result in a decrease in the quantity of a Wallerian degeneration promoting factor expressed by that cell relative to an untreated cell. Repression may be partial. Preferred degrees of repression are at least 50%, more preferably one of at least 60%, 70%, 80%, 85% or 90%. A level of repression between 90% and 100% is considered a ‘silencing’ of expression or function.

[0081] Enhancement of expression of a Wallerian degeneration inhibiting factor will preferably result in an increase in the quantity of a Wallerian degeneration inhibiting factor expressed by a cell / tissue / organ / organ system / subject. For example, in a given cell the enhancement of a Wallerian degeneration inhibiting factor by administration of a suitable nucleic acid will result in an increase in the quantity of a Wallerian degeneration promoting factor expressed by that cell relative to an untreated cell. Enhancement of expression may be partial. Preferred degrees of enhancement are at least 10%, more preferably one of at least 20%, 30%, 40%, 45% or 50%.

[0082] In some embodiments, the Wallerian degeneration inhibitor according to the present invention is an antisense oligonucleotide targeting SARM1 . That is, an antisense oligonucleotide being complementary to a region of SARM1 mRNA. In some embodiments, the antisense oligonucleotide is complementary to a region of the SARM1 mRNA encoded by the nucleotide sequence of NCBI ref: NM_015077, v4. In some embodiments, the antisense oligonucleotide is complementary to a region of a 5’ untranslated region (UTR) of a SARM1 mRNA. In some embodiments, the antisense oligonucleotide is complementary to a coding region of a SARM1 mRNA. In some embodiments, the antisense oligonucleotide is complementary to a region of a 3’ UTR of a SARM1 mRNA. In some embodiments, the antisense oligonucleotide is complementary to a region of the 5’ UTR of SARM1 mRNA, encoded by positions 1 to 339 of the nucleotide sequence of NCBI ref: NM_015077, v4. In some embodiments, the antisense oligonucleotide is complementary to a region of the coding sequence of SARM1 mRNA, encoded by positions 340 to 2514 of NCBI ref: NM_015077, v4. In some embodiments, the antisense oligonucleotide is complementary to a region of the 3’ UTR of SARM1 mRNA, encoded by positions 2515 to 10277 of NCBI ref: NM_015077, v4. In some embodiments, the Wallerian degeneration inhibitor is an antisense oligonucleotide selected from the antisense oligonucleotides described in WO2021108602A1 .

[0083] The nucleic acids of the invention may be introduced into mammalian cells in vitro or in vivo using known techniques to suppress expression of a Wallerian degeneration promoting factor (e.g. SARM1).

[0084] In some embodiments, the Wallerian degeneration inhibitor is a vector. In some embodiments, the Wallerian degeneration inhibitor is a viral vector, such as a lentiviral vector, an adenovirus vector, a retroviral vector, or an adeno-associated vector.

[0085] AA V vectors

[0086] Adenoviral vectors have been described for use in human gene therapy (Rosenfeld et al, Cell 68:143 (1992)). Advantages of adenovirus vectors include their potential to carry larger insert polynucleotide sequences than retroviral vectors, very high viral titres, ability to infect non-replicating cells, and suitability for infecting tissues in situ, especially in the lung. In some embodiments, the Wallerian degeneration inhibitor comprises an adeno associated virus (AAV) vector comprising a nucleic acid encoding a dominant negative SARM1 . Typically, the AAV vector is a recombinant AAV vector. The AAV vector may comprise an AAV1 , AAV2, AAV5, AAV6, AAV8 or AAV9 vector. It will be appreciated that an AAV vector will comprise inverted terminal repeats (ITRs), typically 5’ and 3’. The AAV vector may further comprise a mammalian promoter and a terminator. The AAV vector may comprise, from 5’ to 3’, the 5’ ITR, the mammalian promoter, the nucleic acid encoding the dominant negative SARM1 , the terminator and a 3’ ITR. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the neuron-specific synapsin promoter.

[0087] As used herein, a dominant negative SARM1 is a mutant SARM1 protein that interferes with the function of the wild type SARM1 protein to inhibit SARM1 activity. In some embodiments, the dominant negative SARM1 has 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence encoded by the nucleotide sequence of NCBI ref: NM_015077, v4. In some embodiments, the dominant negative SARM1 comprises amino acid substitutions at amino acid positions 193 and 685, numbered relative to the amino acid sequence encoded by the nucleotide sequence of NCBI ref: NM_015077, v4. In some embodiments, the amino acid substitutions are K193R and H685A.

[0088] In some embodiments, the Wallerian degeneration inhibitor is an adeno associated virus (AAV) vector comprising a nucleic acid encoding a dominant negative SARM1 as described in Geisler et al. (2019) J Exp Med 216, 294-303 (the entire content of which is incorporated herein by reference).

[0089] Small molecules

[0090] In some embodiments, the Wallerian degeneration inhibitor is a small molecule. The Wallerian degeneration inhibitor may be a small molecule targeting any Wallerian degeneration factor.

[0091] In some embodiments, the Wallerian degeneration inhibitor according to the present invention is small molecule inhibitor of SARM1 . In some embodiments, the Wallerian degeneration inhibitor is a small molecule inhibitor of SARM1 NADase activity.

[0092] Small molecule inhibitors of SARM1 include isoquinolines (as described in Hughes et al. Cell Rep. (2021) 34(1):108588 in particular compounds 1 to 6 of Figure 1 B, in particular DSRM-3716), isothiazole (for example as described in Bosanac et al. Brain. (2021) 144(10):3226-3238), NB series compounds NB-1 , NB-2, NB-3, NB-4, NB-5, NB-6, and NB7 (described in Bratkowski et al. Neuron. (2022) 110(22):3711 - 3726. e16), zinc pyrithione (for example as described in Loring et al. (2020)); tryptoline acrylamides (including EV-99, described in Feldman et al. Proc Natl Acad Sci USA. (2022) 119(35):e2208457119); TK compounds TK106, TK142, TK174, TK198, TK210, TK222 and TK138 (described in Khazma et al. Cell Mol Life Sci. (2022) 80(1):16); FK866 (Sigma-Aldrich, catalog #F8557); CHS828 (Hjarnaa et al. Cancer Res. (1999) 59(22):5751-7); LY3873862 (Eli Lily, clinical trials.gov: NCT05492201); NB-4746 (Nura Bio Inc.). In some embodiments, the Wallerian degeneration inhibitor is selected from: Apomorphine, bismuth subsalicylate, Bronopol, Cisplatin, D-phenylalanine, Epiestriol, Erythromycin, Merbromin, Metyropone, phenylmercuric acetate, pimethixene maleate, pyrithione zinc, thiram, NSC2805, NSC1152, NSC22806, NSC34879, NSC92937, NSC645330, NSC661221 , NSC641396, NSC70931 , NSC727038, NSC228155, NSC228150, NSC48443, NSC90749, NSC98363, NSC16339, NSC62208, NSC622689, as described in WO2018057989.

[0093] In some embodiments, the Wallerian degeneration inhibitor is an isoquinoline derivative as shown below and described in Hughes et al. Cell Rep. (2021) 34(1):108588.

[0094] In particular, in some embodiments the Wallerian degeneration inhibitor is DSRM-3716, as shown below and described in Hughes et al. Cell Rep. (2021) 34(1):108588.

[0095] The molecular characteristics of DSRM-3716 — moderate molecular weight, predicted blood-brain barrier permeability, and neutral / weakly basic charge — suggest strong potential for CNS penetration and favourable pharmacokinetics. Methods of screening

[0096] The present invention also provides methods of screening therapeutic candidates as anti-CNS cancer therapeutic compounds. In some embodiments, the method of screening comprises a step of identifying if the therapeutic candidate is an inhibitor of Wallerian degeneration. In some embodiments, the method of screening comprises a step of selecting an agent for testing as a therapeutic candidate for treating and / or preventing CNS cancer if the agent is, or is suspected to be, a Wallerian degeneration inhibitor.

[0097] In some embodiments, the method comprises an in vitro method. In some embodiments, the method comprises the steps of:

[0098] (i) selecting Wallerian degeneration inhibitors as therapeutic candidates

[0099] (ii) providing organotypic brain slice cultures derived from both healthy and tumour-bearing brains;

[0100] (iii) adding therapeutic candidates to the organotypic brain slice culture media;

[0101] (iv) analysing the organotypic brain slice culture to determine the anti-cancer effect of the therapeutic candidates.

[0102] In some embodiments, the Wallerian degeneration inhibitors are known Wallerian degeneration inhibitors. In some embodiments, the Wallerian degeneration inhibitors are suspected Wallerian degeneration inhibitors. That is, in some embodiments, the Wallerian degeneration inhibitors selected as therapeutic candidates may be agents suspected of being Wallerian degeneration inhibitors. In some embodiments, the Wallerian degeneration inhibitors selected as therapeutic candidates are Sarml inhibitors.

[0103] In some embodiments, the organotypic brain slice cultures are derived from human brain tissue. In some embodiments, the organotypic brain slice cultures are derived from mouse brain tissue. In some embodiments, analyse of the organotypic brain slice culture is carried out via immunohistochemistry and / or flow cytometry, as described herein.

[0104] In some embodiments, determining an anti-cancer effect of the therapeutic candidates is achieved by investigating the impact on the tumour. For example, by assessing tumour progression, tumour survival, tumour proliferation and / or tumour invasion. In some embodiments, determining an anti-cancer effect of the therapeutic candidates is achieved by investigating the impact on the tumour microenvironment. For example, by assessing immune response and / or neuronal / axonal survival, and / or neuronal / axonal connectivity.

[0105] In some embodiments, the method comprises an in vivo method. In some embodiments, the method comprises an in vivo method using mouse models of CNS cancer. In some embodiments the method comprises the steps of:

[0106] (i) selecting Wallerian degeneration inhibitors as therapeutic candidates

[0107] (ii) providing genetically engineered mouse models of CNS cancer;

[0108] (iii) delivering therapeutic candidates to the tumour, cerebrospinal fluid reservoir, or tumour resection cavity via implantable osmotic pumps;

[0109] (iv) analysing the mouse model to determine the anti-cancer effect of the therapeutic candidates. In some embodiments, analysis of mouse model is carried out via analysis of mouse behaviour, for example, by determining the mouse motor score as described in Example 1 herein. In some embodiments, analysis of mouse model is carried out via analysis of tumour biopsy, for example, as described in Example 1 herein.

[0110] In some embodiments, the mouse model of CNS cancer is a genetically engineered model. In some embodiments, the mouse model is a patient derived xenograft model. In some embodiments, the mouse model of CNS cancer is a mouse model of glioblastoma. In some embodiments, the mouse model of CNS cancer is an npp model, ( / .e. mouse transformed in situ via inactivation of the tumour suppressors Nf1 , Pten and Trp53, as described herein).

[0111] Cancer of the central nervous system (CNS cancer)

[0112] The present disclosed is concerned with the treatment and / or prevention of a cancer of the central nervous system.

[0113] While cancers encompass a hugely heterogenous group of diseases, the pathology of carcinogenesis can generally be divided into four steps: tumour initiation, tumour promotion, malignant conversion and tumour progression.

[0114] Tumour initiation refers to the accumulation of mutations in DNA that increase cancer risk. These mutations ‘prime’ a cell to become cancerous. The mutations may accumulate in genes controlling the cell cycle and cell proliferation. Following ‘initiation’ cells are susceptible to cancer ‘promoters’. Promoters are generally non-mutagenic and considered not carcinogenic in isolation. Cancer promoters may be chemical agents, or events causing injury or trauma, such as chronic wounding. Such promoters provide initiated cells with a selective advantage, triggering initiated cells to expand. This can trigger the development of a cancer from an earlier stage to a more advanced / later stage. The progression of cancer is reviewed in, Compton, C. (2020). Cancer Initiation, Promotion, and Progression and the Acquisition of Key Behavioral Traits. In: Cancer: The Enemy from Within. Springer, Cham. In some embodiments, the cancer is an early-stage cancer; that is a cancer that is in the ‘initiation’ stage. In some embodiments, the cancer has not undergone cancer promotion.

[0115] As used herein, “cancer of the central nervous system” (CNS cancer) refers to any cancer associated with any cell, tissue, organ, organ system of the central nervous system.

[0116] CNS cancer encompasses brain and spinal cord tumours. CNS cancer includes gliomas, glioneuronal tumours and neuronal tumours, choroid plexus tumours, embryonal tumours, pineal tumours, cranial and paraspinal nerve tumours, meningiomas, mesenchymal, non-meningothelial tumours, melanocytic tumours, hematolymphoid tumours, germ cell tumours, tumours of the sellar region, and metastases to the CNS (e.g. brain metastases). In some embodiments, the cancer is selected from the group comprising: glioblastomas, gliomas, craniopharyngioma, brain stem glioma, medulloblastoma, meningioma, astrocytomas, pituitary adenomas, oligodendroglioma, ependymal tumors, pineal parenchymal tumors and brain metastases. CNS cancers are reviewed in, for example, Louis et al. Neuro Oncol. (2021) 23(8):1231- 1251 .

[0117] In some embodiments, the cancer of the central nervous system as described herein is a glioma. In some embodiments, the cancer of the central nervous system is selected from the list comprising: astrocytoma, oligodendroglioma and glioblastoma. In some embodiments, the cancer of the central nervous system is a glioblastoma (also known as GBM, glioblastoma multiforme, or glioblastoma IDH wildtype). The cancer may be a primary ( / .e. may occur de novo) or secondary ( / .e. may arise from a pre-existing astrocytoma) glioblastoma.

[0118] A subject having a cancer of the central nervous system may have been diagnosed as having a cancer of the central nervous system. A subject may satisfy the diagnostic criteria for the diagnosis of a cancer of the central nervous system. The diagnosis of cancers of the central nervous system is described in WHO classification of CNS tumours, 5thed. (2021) International Agency for Research on Cancer.

[0119] A cancer of the central nervous system may be a primary cancer. Alternatively, a cancer of the central nervous system may be a cancer that has spread from the original (primary) tumour. That is, a cancer of the central nervous system may be a CNS metastasis.

[0120] Cancers according to the present invention may be classified into ‘early’ or ‘advanced’ phase cancer. Histological and molecular profiles of CNS cancers are described in, for example, Bhattacharya et al. J Lab Physicians. (2023) 15(1):38-44; Forjaz et al. Neurooncol Adv. (2021) 3(1): vdaa175; and Kristensen et al. Ann Oncol. (2019) 30(8):1265-1278. In some embodiments, the cancer according to the present invention is an early-phase cancer.

[0121] In some embodiments, the cancer according to the present invention is glioblastoma. Glioblastoma may be identified / diagnosed according to diagnostic criteria known to the skilled person, as described in, for example, WHO classification of CNS tumours, 5thed. (2021) International Agency for Research on Cancer. In some embodiments, the cancer according to the present invention is early phase glioblastoma. Early phase glioblastoma can be difficult to diagnose due to non-specific or absent symptoms. Nonetheless, early-phase glioblastoma may be identified by methods known to the skilled person, including, for example magnetic resonance imaging. Early-stage glioblastoma lesions may present as hyperintensities in T2-weight or FLAIR MRI. The lesions may be ill-defined. The lesions may appear without, or with little, mass effect. The lesions may appear without central necrosis. The lesions may appear with little or no contrast enhancement. The diagnosis of early-stage glioblastoma is discussed, for example, in Ideguchi et al. J Neurooncol. (2015) 123(2):289-297 and Toh et al. AJNR Am J Neuroradiol. (2017) 38(2):288-293. Subject

[0122] The mammalian subject in accordance with aspects of the present disclosure may be any mammal or human. A subject may therefore be a rat, mouse, feline, canine, equine, porcine, ovine, bovine, primate or human. The subject is preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease / condition described herein requiring treatment, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0123] In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure herein is a subject having, or at risk of developing, a cancer of the nervous system, e.g. a cancer of the nervous system as described herein. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterization for certain markers of such disease / condition.

[0124] In some embodiments, the subject is a human patient having cancer of the nervous system. In particular, the subject is a human patient having cancer of the central nervous system. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is selected from the group comprising: glioblastomas, gliomas, craniopharyngioma, brain stem glioma, medulloblastoma, meningioma, astrocytomas, pituitary adenomas, oligodendroglioma, ependymal tumors, pineal parenchymal tumors and brain metastases. In some embodiments, the subject is a human patient having glioblastoma.

[0125] In some embodiments, the subject is a human patient having a cancer of the central nervous system, where the cancer is glioma. In some embodiments, the cancer of the central nervous system is selected from the list comprising: astrocytoma, oligodendroglioma and glioblastoma. In some embodiments, the cancer of the central nervous system is a glioblastoma (also known as GBM, glioblastoma multiforme, or glioblastoma IDH wildtype). The cancer may be a primary ( / .e. may occur de novo) or secondary ( / .e. may arise from a pre-existing astrocytoma) glioblastoma.

[0126] In some embodiments, the subject is a human patient having a cancer of the central nervous system, wherein the cancer of the central nervous system is not a primary cancer. That is, in some embodiments, the cancer of the central nervous system according to the present invention is a cancer that has spread from the original (primary) tumour to the central nervous system. In some embodiments, the subject according to the present invention is a human patient having a cancer of the central nervous system, wherein the cancer is a CNS metastasis.

[0127] In some embodiments, the subject is a human patient having glioblastoma. In some embodiments, the subject is a human patient having early phase glioblastoma. Early-phase glioblastoma may be identified by methods known to the skilled person, including, for example magnetic resonance imaging. Early-stage glioblastoma lesions may present as hyperintensities in T2-weight or FLAIR MRL The lesions may be ill- defined. The lesions may appear without, or with little, mass effect. The lesions may appear without central necrosis. The lesions may appear with little or no contrast enhancement. The diagnosis of early- stage glioblastoma is discussed, for example, in Ideguchi et al. J Neurooncol. (2015) 123(2):289-297 and Toh et al. AJNR Am J Neuroradiol. (2017) 38(2):288-293.

[0128] In some embodiments, the subject is considered ‘at risk’ of developing a cancer as described herein. A subject may be considered ‘at risk’ of developing a cancer as described herein if the subject:

[0129] • Has one or more genetic markers predictive of cancer (genetic markers in CNS cancers are reviewed in, for example, Ostrom et al. Epilepsia. (2014) 54(9): 10.1111 ; and Jelski et al. Int J Mol Sci. (2021) 22(13):7039).

[0130] • Undergoes an event associated with tumour promotion. Events associated with tumour promotion include axonal injury or trauma, tumour resection, chemotherapy (in particular, chemotherapy associated with axonal injury, degeneration or trauma), medical radiation.

[0131] • Has one or more risk factors associated with developing cancer. Particular risk factors include age ( / .e. a human subject being over 70 years of age), obesity and traumatic brain injury.

[0132] • Has a family history of cancer, in particular a family history of CNS cancer.

[0133] • Has genetic conditions or syndromes associated with developing a brain tumour. Particular syndromes include: neurofibromatosis (NF) type 1 and type 2, tuberous sclerosis (TSC), Li- Fraumeni syndrome, Von Hippel-Lindau syndrome (VHL), Turner syndrome, Turcot syndrome, Gorlin syndrome.

[0134] In some embodiments, the subject has experienced axonal injury or trauma. Axonal injury or trauma may be intrinsic or extrinsic. In some embodiments, the subject has experienced extrinsic axonal injury or trauma. In some embodiments, the subject has undergone tumour resection. In some embodiments the subject has received chemotherapy. In some embodiments, the subject has received chemotherapy resulting in axonal injury or trauma. In some embodiments the subject has received radiotherapy. In some embodiments, the subject has received radiotherapy resulting in axonal injury or trauma.

[0135] In some embodiments, the subject is experiencing neurological symptoms. Neurological symptoms may include: seizure (including focal seizure and / or generalised seizure), loss of consciousness, syncope, aphasia, headache, visual deficit ( / .e. dysfunction or loss), gait disturbance, memory impairment, paralysis, paresis, plegia, weakness, sensory dysfunction, language dysfunction, and cognitive impairment.

[0136] In some embodiments of the present invention, the subject is not undergoing chemotherapy. In some embodiments of the present invention, the subject is not experiencing, has not been diagnosed with and / or is not considered at risk of chemotherapy induced peripheral neuropathy (CIPN).

[0137] Prophylaxis / treatment

[0138] The present invention provides methods and articles (agents and compositions) for the treatment / prevention of cancer e.g. as described herein.

[0139] Accordingly, the present disclosure provides a Wallerian degeneration inhibitor for use in a method of treating or preventing a cancer of the central nervous system. Also provided is the use of a Wallerian degeneration inhibitor in the manufacture of a medicament for use in a method of treating or preventing a cancer of the central nervous system. Also provided is a method of treating or preventing a cancer of the central nervous system, the method comprising administering a therapeutically- or prophylactically- effective amount of a Wallerian degeneration inhibitor.

[0140] Treatment and / or prevention is achieved by inhibition of Wallerian degeneration. That is, the present invention provides for the treatment / prevention of cancer through inhibition of Wallerian degeneration, in e.g. a cell, tissue / organ / organ system / subject.

[0141] It will be clear to the person skilled in the art that the therapeutic and prophylactic utility of the present invention extends to essentially any disease / condition which would benefit from a reduction in Wallerian degeneration. The therapeutic and prophylactic utility of the present invention extends to any subject suffering from cancer of the nervous system.

[0142] In some embodiments, the present invention provides for the treatment / prevention of diseases / conditions that are caused / exacerbated by Wallerian degeneration, for example a cancer of the central nervous system that may be caused / exacerbated by Wallerian degeneration. In some embodiments, there is provided the treatment / prevention of diseases / conditions in a subject in which Wallerian degeneration provides a poor prognosis, for example a cancer of the central nervous system in which Wallerian degeneration provides a poor prognosis.

[0143] Treatment may be effective to prevent progression of cancer, e.g. to reduce / delay / prevent worsening of, or to reduce / delay / prevent development of, cancer. In some embodiments treatment may lead to an improvement, e.g. a reduction in the severity of, and / or a reversal of, the symptoms or clinical features of cancer. In some embodiments treatment may increase survival. In some embodiments treatment is effective to reverse the effects and / or symptoms and / or clinical features of cancer.

[0144] In some embodiments, the cancer to be treated and / or prevented according to the present invention is a cancer of the nervous system. In particular, the cancer to be treated and / or prevented is a cancer of the central nervous system. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is selected from the group comprising: glioblastomas, gliomas, craniopharyngioma, brain stem glioma, medulloblastoma, meningioma, astrocytomas, pituitary adenomas, oligodendroglioma, ependymal tumors, pineal parenchymal tumors and brain metastases.

[0145] In some embodiments, the cancer to be treated and / or prevented according to the present invention is glioma. In some embodiments, the cancer of the central nervous system to be treated and / or prevented is selected from the list comprising: astrocytoma, oligodendroglioma and glioblastoma. In some embodiments, the cancer of the central nervous system to be treated and / or prevented according to the present invention is a glioblastoma (also known as GBM, glioblastoma multiforme, or glioblastoma IDH wildtype). The cancer may be a primary ( / .e. may occur de novo) or secondary ( / .e. may arise from a pre-existing astrocytoma) glioblastoma. In some embodiments, the cancer of the central nervous system is not a primary cancer. That is, in some embodiments, the cancer of the central nervous system according to the present invention is a cancer that has spread from the original (primary) tumour to the central nervous system. In some embodiments, the cancer to be treated and / or prevented according to the present invention is a CNS metastasis.

[0146] Cancers according to the present invention may be classified into ‘early’ or ‘advanced’ phase cancer, as described herein. Prevention may refer to prevention of development of cancer, and / or prevention of worsening of cancer, e.g. prevention of progression of cancer from an early stage to a later stage. In some embodiments, prevention may be in the context of treatment of a subject considered “at risk” of cancer developing from an early to a later stage. For example, a patient diagnosed with cancer of the central nervous system, the subject having undergone chemotherapy, or tumour resection, or the subject undergoing chemotherapy, or tumour resection.

[0147] In some embodiments, treatment or prevention according to the present invention comprises treating or preventing neurological symptoms of the subject. Neurological symptoms associated with cancer of the nervous system include, but are not limited to: seizure (including focal seizure and / or generalised seizure), loss of consciousness, syncope, aphasia, headache, visual deficit ( / .e. dysfunction or loss), gait disturbance, memory impairment, paralysis, paresis, plegia, weakness, sensory dysfunction, language dysfunction, and cognitive impairment. Neurological symptoms may be assessed via neurological examination, for example, motor exam, sensory exam, gait assessment and reflex examination. Neurological symptoms may be measured according to appropriate scales, for example the Glasgow Coma Scale, or MRC motor scale.

[0148] In some embodiments, the Wallerian degeneration inhibitor according to the present invention is not for use in the treatment of chemotherapy induced peripheral neuropathy (CIPN).

[0149] Dosages and method of administration

[0150] Administration of the Wallerian degeneration inhibitor, or a pharmaceutical composition comprising the Wallerian degeneration inhibitor according to the present invention is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show benefit to the subject.

[0151] In some embodiments, the inhibitor of Wallerian degeneration of the present invention, or pharmaceutical compositions thereof are administered prior to the cancer developing to a later stage. The inhibitor of Wallerian degeneration or pharmaceutical compositions thereof may be administered in anticipation of the cancer developing to a later stage. The inhibitor of Wallerian degeneration or pharmaceutical compositions thereof may be administered to prevent / slow development of cancer to a later stage. In some embodiments, the agent / agents are administered to a subject considered at risk of cancer developing to a later stage, as described herein.

[0152] In some embodiments, the Wallerian degeneration inhibitor, or a pharmaceutical composition thereof may be administered before, in conjunction with, or after the subject undergoes axonal injury or trauma. Axonal injury may result from intrinsic or extrinsic events. Extrinsic events that may result in axonal injury or trauma include tumour resection or chemotherapy. In some embodiments, the Wallerian degeneration inhibitor is administered before, in conjunction with, or after the subject undergoes tumour resection. In some embodiments, the Wallerian degeneration inhibitor is administered before, in conjunction with, or after the subject undergoes chemotherapy. For example, chemotherapy known to cause axonal injury or trauma.

[0153] In some embodiments, the Wallerian degeneration inhibitor, or a pharmaceutical composition thereof may be administered before, in conjunction with, or after the subject undergoes axonal injury or trauma. In some embodiments the Wallerian degeneration inhibitor according to the present invention is administered within a specified time after the subject undergoes axonal injury or trauma. In some embodiments the Wallerian degeneration inhibitor is administered within 1 month after a subject undergoes axonal injury or trauma, e.g. within 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour or 30 minutes after an event which may cause axonal injury or trauma.

[0154] In some embodiments the Wallerian degeneration inhibitor according to the present invention is administered within a specified time before the subject undergoes axonal injury or trauma. In some embodiments the Wallerian degeneration inhibitor is administered within 1 month before a subject undergoes axonal injury or trauma, e.g. within 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour or 30 minutes before an event which may cause axonal injury or trauma.

[0155] In some embodiments the Wallerian degeneration inhibitor according to the present invention is administered to the subject in conjunction with the event causing axonal injury or trauma. In some embodiments, administering the Wallerian degeneration inhibitor ‘in conjunction with’ the event causing axonal injury or trauma includes administering the Wallerian degeneration inhibitor within 1 week, e.g. within 6 days, 5 days, 4 days, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes of or simultaneously with an event which may cause axonal injury or trauma.

[0156] Preferably, the inhibitor of Wallerian degeneration of the present invention, or a pharmaceutical composition comprising the inhibitor of Wallerian degeneration according to the present invention is administered via injection. Such administration may be both via infusion (continuous) or bolus (discreate) administration. The method of administration via injection may be, for example, directly into the tumour cavity, subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intra-orbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intra- sternal injection. Preferably, the administration is by intravenous infusion or intravenous injection (bolus administration). More preferably, the administration is by intravenous infusion.

[0157] Administration of the articles according to the present disclosure is preferably in a ‘therapeutically- effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic / prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0158] Multiple doses of the Wallerian degeneration inhibitor according to the present invention, or pharmaceutical composition thereof, may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0159] Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

[0160] Pharmaceutical compositions

[0161] The inhibitor of Wallerian degeneration according to the present disclosure may be provided in the form of a composition comprising the relevant agent. Such compositions may be pharmaceutical compositions or medicaments suitable for clinical use, and may additionally comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.

[0162] A pharmaceutical composition according to the present invention may comprise, in addition to inhibitor of Wallerian degeneration as described herein, one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to: pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, pH modifiers, preservatives, anti-oxidants, bacteriostats, stabilisers, suspending agents, solubilisers, surfactants (e.g., wetting agents), colouring agents, and isotonicising solutes ( / .e., which render the formulation isotonic with the blood, or other relevant bodily fluid, of the intended recipient).

[0163] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0164] The pharmaceutical compositions / medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, a pharmaceutical composition / medicament may be formulated for parenteral, systemic, intravascular, intravenous, intra-arterial, intramuscular, topical, intracavitary, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0165] In some embodiments, Wallerian degeneration inhibitors according to the present disclosure may be formulated and / or modified to facilitate delivery to, and / or uptake by, cells / tissues / organs of the central nervous system ( / .e. crossing the blood brain barrier).

[0166] ***

[0167] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0168] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0169] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0170] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0171] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0172] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0173] Examples

[0174] Example 1 - Experimental methods

[0175] Animals

[0176] All animal procedures were carried out in accordance with the Animal Scientific Procedures Act, 1986 and approved by the UCL Animal Welfare and Ethical Review Body (AWERB) in accordance with local ethical and care guidelines and the International guidelines of the Home Office (UK). Mice used in this study were wildtype C57BL / 6NCrl (Charles River), sterile alpha and TIR Motifl - / - (Sarml- / -) featuring mutation Chr11 :78472330-78497754 (-) and Sarm+ / +1and B6.Cg-Tg(Thy1-YFP)16Jrs / J (Jax laboratories (003709)2. Genetically and aged matched animals were used as controls for Sarml- / - experiments. NOD.CB17-Prkdcscid / NCrCrl (NSG Charles River) were used for generation of the PDX models through orthotopic injections of patient derived GBM cell lines.

[0177] Generation of somatic and orthotopic PDX models

[0178] Somatic tumours were generated as previously reported45. Briefly, plasmids were injected into the right ventricle of isoflurane-immobilized pups at postnatal day 2 using an Eppendorf Femtojet microinjector (Eppendorf, 5247000030) followed by electroporation (5 square pulses, 50 msec / pulse at 100V, with 850 msec intervals). EF1 a-tdTomato only plasmid (tdTom) was generated by SnaBI and Pmel digestion of npp plasmid to remove Nf1 , Pten and Trp53 guide RNAs before re-ligation. piggyBase (hGFAPMIN- SpCas9-T2A-PBase, 1 mg / ml) and piggyBac vector U6-Nf1 ,Pten,Trp53-EF1a-tdTomato (npp, 0.564 mg / ml) or EF1a-tdTomato (0.423 mg / ml) were diluted in saline (0.9% NaCI) and mixed at a molar ratio of 1 :1 . 0.1% fast green (Sigma, F7258) was added to the mix to visualise the injection.

[0179] To pharmacologically inhibit Sarml protein selectively in neurons from npp tumour initiation, an AAV encoding a dominant negative SARM1 (as described in Geisler et al. 201944)was added to the piggyBase / npp piggyBac plasmid mix prior to intraventricular injection and electroporation, as above. The AAV construct comprises a nucleic acid encoding SARM1 -compound dominant negative, with mutations at K193 and H685, under the control of the neuron-specific synapsin promoter. AAV8-Syn-GFP (Addgene #50465- AAV8 0.745x1013vg / ml) was used as a control. To investigate Sarml function following tumour initiation, WT mice bearing tumours were injected with either 2.5pl of the AAV construct encoding a dominant negative SARM144(1 x1013vg / ml) or AAV8-Syn-GFP (1 x1013vg / ml) at intermediate or late disease stage. Briefly, mice were anaesthetized and mounted on a stereotaxic frame. A small craniotomy was performed on the tumour ipsilateral right side of the skull 1 .7mm lateral to bregma and -0.5mm anterior to bregma. Virus was injected through a 5ul Hamilton syringe attached to a pump (Pump 11 Elite Nanomite 70-4507, Harvard Apparatus) at a speed 0.3pl / minute to a depth of 2.4mm. The virus was injected continuously as the needle was introduced and removed. The wound was sutured, prior to recovery of the mice. Four weeks after injection, mice were given an intraperitoneal injection of EdU (5mg / kg) 2 hours before brains were collected following transcardial perfusion with 4% paraformaldehyde (PFA) under terminal anaesthesia. Orthotopic PDX models were generated as previously described45.

[0180] All tumour bearing mice were monitored daily and sacrificed at required time points or when they began to show signs of disease and reached humane endpoints.

[0181] Injury induction in tumour-bearing mice

[0182] Brain injury experiments were carried out on tumour-bearing mice at 8.5 weeks post-electroporation with PiggyBase / npp PiccyBac plasmids. Mice were anaesthetized and mounted on a stereotaxic frame. A small craniotomy was performed on the tumour-ipsilateral right side of the skull 1 .7mm medial to bregma and extending from 1 .Omm anterior of bregma. A 25G needle with the bore facing to the right was introduced into the brain through the craniotomy to a depth of 2.5mm. The needle was moved anterior and posterior three times across a 1 .0mm distance to sever the axons of the corpus callosum. Sham mice underwent the same analgesia and anaesthesia protocol but were not mounted on the stereotaxic frame. Two weeks post injury mice were given an intraperitoneal injection of EdU (5mg / kg) 2 hours before brains were collected following transcardial perfusion with 4% paraformaldehyde (PFA) under terminal anaesthesia. Brains were fixed overnight in 4% PFA at 4oC, transferred to PBS prior to vibratome sectioning (50mm sections) and storage in cryobuffer (ethylene glcol:glycerol:PBS 1 :1 :2).

[0183] Behavioural assessment

[0184] Behavioural neuroscores were determined as follows. A ledge test was carried out observing mice walking along the edge of a cage and lowering themselves into the cage and scored as follows: 0 = confident walk and good landing, 1 = trips and wobbles while walking, 2 = trips and wobbles, slips from ledge but recovers; 3 = unable to walk along ledge. A hindlimb clasping test was carried out and scored as follows: 0 = hindlimbs consistently pointing outward away from abdomen, 1 = hindlimbs pulled in slightly towards body for more than 50% of the time, 2 = hindlimbs pointed downwards towards abdomen for more than 50% of the time, 3 = hindlimbs entirely retracted and touching the abdomen for more than 50% of the time. A gait test was carried out and scored as follows: 0 = mouse moves normally, 1= slight tremor observed, slightly raised pelvis or slight waddle, 2 = severe tremor, raised pelvis or pronounced waddle, 3 = movements disjointed, stuttering with raised pelvis and severe waddle. A Kyphosis test was carried out and scored as follows: 0 = easily able to straighten its spine as it walks, 1 = mild kyphosis (curvature of the spine) but mostly able to straighten itself as it walks, 2 = unable to straighten spine completely and maintains mild but persistent kyphosis, 3 = maintains pronounced kyphosis as it walks or while it sits. Scores from each test were combined to give an overall neuroscore between 0 and 12. Mice were tested at 8 weeks for early disease stages and at 14 weeks and 16 weeks for advanced disease stages for WT and Sarml- / - mice, respectively (Figure 3m).

[0185] Tissue preparation and immunohistochemistry

[0186] Animals were perfused (4% paraformaldehyde in PBS; Merck P6148) under terminal anaesthesia, brains collected, post-fixed overnight at 4oC in PFA (4%) before transferring to PBS. Vibratome sections (50pm) were prepared and stored in cryopreservative (glycerol:ethylene glycol; PBS 1 :1 :2) prior to immunohistochemistry. For staining, floating sections were permeabilized overnight (1% Triton X-100, 10% serum in PBS) at 4oC, incubated in primary antibody overnight (1% Triton X-100, 10% serum in PBS) at 4oC and for 3 h in secondary antibody (0.5% Triton X-100, 10% serum in PBS) containing DAPI counterstain (Insight Biotechnology, sc3598). Sections were mounted with antifade mounting solution (Prolong gold antifade mountant, Thermo Fisher, P36934) prior to imaging on a 3i confocal spinning disk. For imaging of axonal damage, tumour-involved and tumour-free contralateral bundles from ThylYFP mice were imaged using the Airyscan function of the LSM 880 confocal microscope.

[0187] The following antibodies were used: rabbit anti-Ki67 (1 :250; Abeam, ab16667), goat anti-GFAP (1 :1 ,000; Abeam, ab53554), rat anti-CD68 (1 :500; Abeam, ab53444), rabbit anti-lba1 (1 :1 ,000; Wako, 019-19741), L0159), mouse anti-neurofilament H (1 :1000 Enzo ENZ-ABS219-0100), mouse anti-myelin basic protein (1 :1000 Covance SMI-99). For detection of EdU, sections were stained with Click-it EdU Alexa Fluor 647 Imaging Kit (Invitrogen, C10340) following manufacturer’s guidelines.

[0188] Image analysis

[0189] Analysis of tumour cell localisation and proliferation (Figure 2c, d) was performed in Imaris 10.1.0 on single z plane images from 3i Spinning disk microscope. Spots segmentation was first performed on tdTomato / GFP channel, before being filtered for intensity median or centre on DAPI to segment tumour cells. Tumour cells were then classified as EdU / Ki67+ / -. Surfaces were manually drawn for the subventricular zone (SVZ), necrotic regions, striatum, and injury sites, and tumour cells within SVZ and necrotic regions were filtered out. White matter bundle surfaces were generated using the machine learning function. Percentage of white matter area was calculated by dividing the area of white matter bundles in tumour infiltrated striatum by the total area of tumour infiltrated striatum.

[0190] Analysis of ThylYFP (Figure 1 b) and neurofilament (Figure 1g) mean fluorescence intensity, as well as GFAP+ cell density (Figure 1i and k) and CD68 integrated density (Figure 1j and I) was performed in Imaged on maximum intensity projection (MIP) images from 3i Spinning disk confocal using a custom script. Individual bundle ROIs were manually drawn and tdTomato+ and GFAP+ cells manually counted. ROI area and mean fluorescence intensity was measured using the Measure function. Mean fluorescence intensity was normalised to the average of mean fluorescence intensities in contralateral bundles (>5 bundles per animal). CD68 integrated density was measured by first thresholding CD68 channel with Li autothreshold, and integrated density (IntDen) was quantified with AnalyzeParticles function.

[0191] Analysis of GFAP area (Figure 2g) and CD68 intensity (Figure 2h) was performed in Imaged on maximum intensity projection (MIP) images from 3i Spinning disk confocal using a custom script. Triangle threshold was used on tdTomato image to generate a tdTomato ROI, which was used for “Sham”. ROIs were manually drawn for injury site in Imaged. “Injury Proximal” ROI was generated from the overlap of tdTomato and injury site ROIs. “Injury Distal” ROI was generated from the tdTomato ROI excluding the injury site ROI. GFAP area was calculated by thresholding GFAP channel using Imaged Triangle threshold, and measuring the area covered within each ROI using the Imaged AnalyzeParticles function. CD68 analysis was calculated by thresholding CD68 channel with Triangle or Li autothreshold, and integrated density (IntDen) was quantified with AnalyzeParticles function. For both, quantification was normalised to its own genotypes Sham control. Analysis of white matter phenotypes was carried out in the tumour-ipsilateral striatum of tumour-bearing mice. Analysis of injury-responses in Figure 2 was carried out across all areas occupied by tdTomato+ tumour cells.

[0192] Single cell RNA preparation

[0193] Mouse brains were collected into ice-cold HBSS media and dissected into 1 mm coronal sections using a brain matrix (WPI, RBMS200C). Tumour regions were dissected out and mechanically dissociated into small pieces. Cells were isolated by papain dissociation (as above) and RNA libraries prepared using Chromium Next GEM Chip G Single Cell Kit (10x genomics; 1000127) and sequenced on Nova Seq X Plus PE 150. scRNA-sequence data analysis

[0194] Read selection and mapping

[0195] Reads were pre-processed and mapped to the mm10-2020-A mouse genome using 10x Genomics Cell Ranger 7.0.1 .The tdTomato sequence, expressed by transformed cells, was added to the reference genome.

[0196] Cells and genes filtering

[0197] Cells with zero UMI count of the 4 red blood cell markers Hbb-bs, Hba-a1 , Hba-a2 and Hbb-bt and with either tdTomato expression 2 (microenvironment cells) or tdTomato 5 (tumour cells) were kept for further analysis. For all analysis, cells from wild type mice were down sampled so each genotype had -20000 cells. Cells with i) proportion of mitochondria genes below 0.25, ii) Iog2 total counts between 9 and 15 were kept for further analysis. Genes with non-zero UMI counts in at least 0.5% of cells were kept for further analysis. As a result, the dataset presented in this study consists of 19886 and 21378 cells for the wild type and Sarml- / - samples respectively, and of a total of 14842 genes.

[0198] Identification of high-confidence tumor cells

[0199] Two rounds of data filtering were used to identify high-confidence tumor cells. First, the Harmony package12was used to integrate the WT and Sarml- / - datasets from this study with scRNA-seq datasets from normal mouse brain1522. Tumor-associated macrophages (TAMs)23and from a mouse GBM model which contains annotated tumour cell24. Specifically, data from each study were normalised using the sctransform package8and merged after selection of common features using “SelectlntegrationFeatures” function from Seurat13. Then, the batch correction function “RunHarmony” from the Harmony package12was applied to the data in PCA space (generated with the “RunPCA” function in Seurat)13.

[0200] High-confidence tumour cells were defined as either: i) expressing at least 5 tdTomato UMI count; ii) being predicted to be aneuploid using the copyKat package25 iii) being predicted to be tumour cells using the integrated dataset annotation from12 and a random forest approach in Harmony space. Specifically, labels from references1522 24were used for training a random forest model26, which was then applied to predict cell labels in the scRNA-seq data from this study13. Finally, tumour cells with UMI count for the Ptprc (Cd45) and Cd68 genes > 0 were discarded as these are considered to be immune cell specific markers. In a second round of filtering, tumor cells were again integrated and clustered using the Harmony package12 and the Louvain approach27 both in Harmony space (same procedure as above, but this time the integration was done using scRNA-seq from this study only). Cells identified to be TAM or EC based on markers from1522were removed from the high-confidence list.

[0201] Identification of high-confidence non-tumor cells

[0202] Cells with tdTomato UMI counts 2 and predicted to be diploid using the copyKat package25 were called high-confidence.

[0203] Cell type annotation of high-confidence tumour and non-tumour cells

[0204] High-confidence tumour and non-tumour cells were integrated (between genotypes) and clustered separately using Harmony and Seurat12 13. Clustering was performed using the Louvain approach in Harmony space with resolution = 0.227. Clusters were annotated using lineage markers and the gene enrichment analysis package fgsea28. Cluster annotation was finally checked manually for accuracy.

[0205] Cell type annotation of unassigned cells

[0206] Unassigned cells are cells that are not part of the two high-confidence lists. First, all the cells from this study were integrated using Harmony as above12. Second, a random forest model was trained using high- confidence tumour and non-tumour cells (training dataset) and used to identify and annotate tumour cells in the list of unassigned cells. Third, a new round of clustering was applied on tumour or non-tumour cells separately. Cell type labels were then assigned using random forest and the cell type annotation from high-confidence tumour or non-tumour cells.

[0207] Re-clustering of TAMs

[0208] TAM cells were re-clustered separately and Macrophage / Microglia markers from two studies2930were used for annotation (Figure 3n).

[0209] Proportion test

[0210] To perform proportion tests on equal numbers of cells in both genotypes tumour and non-tumour cells, were down-sampled to: 12591 tumour cells and 4176 cells respectively. The “prop.test” function from R was used to test the significance of difference in proportion of cell types between two the genotypes. P- value below 0.01 and absolute proportion difference above 0.03 was called significant (Figure 3e, f, Figure 3q)

[0211] Differential gene expression and gene enrichment analysis

[0212] Differentially expressed genes between Sarml- / - and WT cells in each cell type (pWilcoxon <0.01 and auc value above 0.99 quantile of fitted Gaussian distribution on the auc values reported from “wilcoxauc" of R package “presto”) 19 were analysed for GO enrichment using the “enricher” function from the R package clusterProfiler31 32. Ligand receptor analysis

[0213] The cellphoneDB method33from the LIANA package34 was used to identify significant pairs of ligandreceptors in each genotype (Figure 3r to u).

[0214] Flow Cytometry Analysis

[0215] Brains were collected into ice-cold HBSS media and dissected into 1 mm coronal sections using a brain matrix (World Precision Instruments, RBMS200C). Tumour regions were dissected out and mechanically dissociated into small pieces, followed by enzymatic dissociation using Liberase TL (Roche, 05401119001) supplemented with DNAse I (Merck, 11284932001) for 30 min at 37oC. Following addition of EDTA to stop the enzymatic reaction, cells were washed with PBS and filtered through a 70mm cell strainer (Falcon, 352350) to remove large debris. Samples were blocked on ice for 20 min (BioXCell blocking buffer; BE0307) prior to incubation in antibodies and fixable viability dye eFluor780 (eBioscience, 65-0865-18, 1 :1000) at 4°C for 20 min. To detect immune cells within the tumour population the following antibodies were used rat anti-LY6G-BUV563 (1 : 100, Clone IA8, BD, 612921), rat anti-CD11 b-BUV661 (1 :400, Clone M1 / 70, BD, 612977), rat anti-MHC Class II-BB700 (1 :800, Clone M5 / 114.15.2, BD, 746197), mouse anti-CD45-BUV805 (1 :400, Clone 30-F11 , BD, 748370), mouse anti-CD64-BV421 (1 :100, Clone X54-5 / 7.1 , Biolegend, 139309), mouse anti-CX3CR1-BV510 (1 :400 Clone SA011f11 , Biolegend 139309), rat anti-LY6C-BV605 (1 :200, Clone AL-21 , BD 563011), rat anti-CD19-BV650 (1 :50, Clone ID3, BD 563235), hamster anti-CD11C-BV785 (1 :100, Clone N418, Biolegend 117336), rat anti- CD49d-APC (1 :200, Clone R1-2, Biolegend 103622), rat anti-F4 / 80-AF700 (1 :100, Clone BM8, Biolegend 123130), mouse anti-Ki67-BUV395 (1 :100, Clone B56, BD 564071), rat anti-CD3-BUV737 (1 :300, Clone 17A2, BD564380), rat anti-CD206-AF488 (1 :100, Clone C068C2, Biolegend 141710). Data was analyzed using Flowjo (v10.7.1 ; RRID:SCR_008520). Data was compensated, fluorescence minus one controls were generated, and only viable singlets were used for downstream analysis.

[0216] Targeted electron microscopy

[0217] Brains were perfused with EM grade 4% formaldehyde immersion fixed overnight, embedded in 4% agarose, and sectioned on a vibrating microtome (100 pm).

[0218] Sections were stained with DAPI and imaged using confocal microscopy (x20 objective) to map the tdTomato+ tumour cells and identify regions of interest. These regions were prepared for electron microscopy by processing, ultrathin sectioning and imaging on a scanning electron microscope (SEM) 35,36. All EM analysis was conducted on >50 axons per bundle in >3 bundles (n = 3). Degenerating axons were identified as those exhibiting any of the following features of axonal pathology: condensed / dark axoplasm, organelle accumulation, axonal swelling, vacuolisation (Figure 1c and d). For quantitative analysis of demyelination, only axons with no obvious myelin decompaction or vacuolisation were analysed. Inner diameter, outer diameter, myelin thickness, and corresponding g-ratios of myelinated axons were semiautomatically calculated using the software program MyelTracer37. Feret diameters were used to account for the imperfect circularity of axons6(Figure 1 e). Statistical Analysis

[0219] Statistical analysis was performed in Prism 10 or R. All data are expressed as mean±SEM, unless otherwise stated. Significance is stated as follows: p>0.05 (ns), p<0.05 (*), p<0.01 (**), p<0.001 (***). Significance was calculated as indicated in the figure legends. No statistical method was used to predetermine sample size. Sample size was determined based on existing literature and previous experience.

[0220] Example 2 - Axonal injury is an early event in qliomaqenesis

[0221] Axonal integrity was examined in the context of tumour development from endogenous NSCs by inducing npp tumours in Thy1-YFP16 reporter mice, in which a subset of neurons are labelled with YFP allowing detailed histological analysis of axonal processes. Intermediate disease stages (8-12 weeks post induction) were focused on, a time-window immediately prior to the switch to rapid proliferation during which tumour promotion mechanisms would be predicted to be most active. A significant decrease in YFP fluorescence in tumour-bearing white matter was found, which is indicative of axonal loss, which already began in areas of low tumour infiltration and became progressively more pronounced as the numbers of tumour cells increased (Figure 1a and b). A similar response was detected in wildtype npp tumours by immunostaining for the axonal marker neurofilament, ruling out potential non-specific loss of the endogenous YFP reporter in tumour regions (Figure 1f and g). Furthermore, correlative light and electron microscopy (CLEM) of sparsely tumour-infiltrated white matter of npp tumours 10.5 weeks post-induction showed extensive axonal damage, including hallmarks of degeneration (e.g. axonal swelling, vacuolisation, organelle accumulation, and presence of condensed / dark axoplasm), but no frank demyelination, as judged by comparable g-ratios between tumour-involved and contralateral white matter (Figure 1c-e and h). This indicates that degeneration is caused by direct injury to the axons, rather than being a secondary response to demyelination. In keeping with this idea, super-resolution confocal imaging of npp tumours in Thy1 YFP mice revealed that compared to contralateral regions white matter bundles were expanded in tumour-involved areas, and frequently contained axons with hallmarks of physical injury in areas adjacent to tumour cells, including varicosities and blebbing (Figure 1 m). This suggests that compression and mechanical stress caused by infiltrating tumour cells contribute to the axonal loss we detected in early tumours.

[0222] Axonal injury was also accompanied by pronounced white matter inflammation, which again increased progressively with tumour density (Figure 1i to I). Indeed, immunohistochemical analysis revealed extensive astrocyte reactivity, with GFAP+cells forming glial scar-like structures around and within tumour white matter (Figure 1i and k). Microglia activation was also increased, though to a lesser extent, and confined to myelinated fibres (Figure 1j and I). Thus, axonal injury is an early event in gliomagenesis, which is triggered by neural progenitor cells that upon acquisition of mutations are rerouted to the white matter, causing injury and neuroinflammation. Example 3 - Axonal injury drives GBM progression through Wallerian degeneration

[0223] In the central nervous system injury to axons typically results in loss of the distal portion of the axon, which impairs neuronal connectivity and function and can also lead to death of the whole neuron. Several pathways and pathophysiological processes underlie axonal degeneration and associated neuronal death, the most prevalent of which is Wallerian degeneration (WD). WD is an active programme of anterograde axonal degeneration mediated by the downstream executioner sterile alpha and TIR motifcontaining 1 (SARM1) protein. Genetic inactivation of Sarml suppresses Wallerian degeneration and preserves neuronal integrity and function for extended time periods after injury. This can be weeks to months, depending on injury type and severity, with axons being preserved even after complete axonal disconnection. Consistently, pharmacological inhibitors of SARM1 are being actively developed for the treatment of neurodegenerative diseases.

[0224] Therefore, whether WD might also be responsible for the axonal loss observed in early tumours was investigated. Npp tumours were induced in Sarml - mice and axonal integrity examined at 10.5 weeks post-electroporation using neurofilament staining and CLEM as described above. Both approaches showed robust neuroprotection with much reduced axonal loss in tumour-infiltrated white matter, confirming that WD is a key mediator of axonal degeneration in GBM (Figure 1 c to h).

[0225] Next, whether white matter axonal injury plays a causative role in tumour promotion was investigated. Furthermore, whether inactivating SARM1 would be an effective strategy for reversing these effects, was also explored. This was achieved using genetic perturbation of the pathway. Npp tumours were generated in wild type (WT) or Sarml ' mice and gain-of-function experiments were performed by subjecting the animals to an experimental injury paradigm and assessing its impact on tumourigenesis. Eight and a half weeks post-induction, a stage that precedes progression in the npp model, corpus callosum axons in the tumour ipsilateral hemisphere were surgically transected and analysis performed two weeks later by immunohistochemistry (Figure 2a). Aged-matched sham-operated npp tumours of both genotypes were used as controls (Figure 2a).

[0226] It was found that axonal transection significantly increased tumour cell proliferation in npp tumours in WT, but not Sarml7mice, in which axonal degeneration was suppressed and proliferation remained at baseline sham levels (Figure 2b, c, i, and j). Notably, proliferation was not increased at the wound site itself in WT samples, but rather in all distal tumour areas, including grey matter regions into which tumour cells began to expand (Figure 2b, d and f), reminiscent of late-stage npp tumours. These effects are consistent with axonal degeneration, which occurs distal to the injury site, playing a key role in promoting tumorigenesis. Consistently, astrocyte reactivity and microglial activation also increased throughout the tumour mass in WT npp tumours, although astrocyte reactivity was more pronounced than microglia activation in distal areas (Figure 2e to h) in npp tumours generated in Sarml - mice, injury-induced inflammation was significantly reduced compared to WT in areas proximal to the injury and fully abolished distally (Figure 2e to h and k).

[0227] Importantly, these effects were not due to Sarml -independent strain-specific phenotypes (mixed 129 / C57BI6 background)4041or non-neuronal roles of Sarml4243because a similar rescue of injury- induced progression was observed using an AAV-mediated gene therapy approach to inactivate Sarml specifically in neurons and in a pure C57BI6 background (Figure 2l-m)44. Indeed, intraventricular administration of AAVs constructs encoding dominant negative Sarml driven by the human Synapsin promoter at the time of tumour induction resulted in axonal protection and reversed effects of transection injury at intermediate disease stage, relative to AAV8-Syn-EGFP-injected controls (Figure 2n-p).

[0228] In contrast, no significant changes were observed in proliferation or neuroinflammation relative to sham in either genotype when transection injury was performed in late tumours. Thus, experimental injury accelerates progression of latent (early and intermediate stage) WT lesions via WD, but has little impact on advanced tumours (late stage), which have already undergone progression pre-transection within the main tumour mass and display pronounced proliferation, neuroinflammation and axonal degeneration at baseline. Consistent with WD being a key driver of progression, intratumoural inactivation of Sarml by administration of the AAV-mediated gene therapy44(but not AAV8-Syn-EGFP) suppressed tumour cell proliferation at intermediate but not late tumour stage (Figure 2q-t).

[0229] To assess the impact of axonal transection injury at latent stage on long term tumourigenesis, survival studies were carried out. The inventors found that corpus callosum transection in mice bearing intermediate npp tumours significantly accelerated tumourigenesis and decreased survival relative to sham controls (Figure 2u).

[0230] These findings indicate that axonal injury and ensuing WD increases inflammation and drives tumour cells to rapid proliferation, thus acting as a GBM promoter, a process that is rescued by inactivation of Sarml.

[0231] Example 4 - Blocking Wallerian degeneration delays GBM progression and reduces disease severity

[0232] These results so far suggest that inhibition of SARM1 may counteract tumour promotion during GBM development and as such represent a potential therapeutic target for suppressing disease progression. To test this more directly, genetic perturbation of the Sarml pathway was again used as a tool to assess the impact of Sarml inactivation on long-term tumourigenesis.

[0233] Npp tumours were generated in WT and Sarml - mice (as above) and immunohistochemistry used to examine their phenotypes at terminal disease stage. Strikingly, in the absence of Sarml, the tumours appeared less dense than wildtype controls, occupying on average a greater area of the brain (Figure 3a and b). To understand the mechanisms involved, scRNA-seq analysis was performed. Transcriptomes from a total of 19,886 and 21 ,378 cells were analysed from tumours in WT and Sarm T / _mice, respectively. After pre-processing and filtering, cells were clustered and cluster labels defined using published scRNA-seq datasets (Figure 3c, d, and methods). Tumour cells were then identified and separated from normal cells of the microenvironment based on their gene expression profile, aneuploid state and expression of tdTomato (Figure 3c, d, and methods). As previously reported, WT npp tumours reflected canonical transcriptomic Neftel states38, encompassing both neurodevelopmental-like (NPC-like, OPC-like and AC-like cells) and mesenchymal / injured-like populations (MES-like cells), as well as an actively proliferating state resembling active NSCs (aNSC- like). The same states were also found in Sarm7tumours, but in different proportions, with a particularly marked increase in NPC-like cells and a reduction in MES-like cells (Figure 3c and e). Pronounced differences in the tumour microenvironment were also detected; WT tumours contained greater proportions of tumour associated microglia and macrophages (TAMs), endothelial cells, pericytes and glial cells of mixed astrocytic and oligodendrocytic fate with markers of high interferon signalling (hereon termed inflamed glia, Figure 3d and f).

[0234] Furthermore, differential expression analysis between genotypes across normal cell populations, suggested that WT endothelial cells and pericytes upregulated signature of angiogenesis (e.g. cell migration, adhesion, positive regulation of smooth muscle cell proliferation, angiogenesis), whereas astrocytes upregulated signatures linked to reactivity (e.g. cell adhesion, cell population proliferation). Reclustering of the TAMs population alone indicated that cells more closely resembling anti-inflammatory macrophages of the tumour core were enriched in WT relative to tumours in Sarm 17mice (Figure 3n to q). In contrast, Sarml-- TAMs expressed higher levels of pro-inflammatory microglia markers characteristics of infiltrative tumour regions (Figure 3n to q).

[0235] Immune profiling of late-stage tumours by flow cytometry confirmed this result, revealing that npp tumours in WT mice were overall more immune infiltrated, containing higher proportions of macrophages and T- cells, whereas microglia dominated in tumours generated in Sarm7mice (Figure 3g to k). Furthermore, Liana analysis revealed that increased heterotypic signalling occurred between tumour cells and their microenvironment in WT relative to Sarm7tumours (Figures 3r to u). Together, these findings demonstrate that inhibition of the SARM1 pathway significantly slowed tumour progression to dense, angiogenic, and immune-suppressive lesions as well as the accompanying transition of tumour cells to MES-like / injured states; instead, it led to the development of less inflamed tumours that more closely mirrored normal neurodevelopmental lineages.

[0236] These results prompted investigation into whether Sarm l loss might affect the course of the disease more broadly in two sets of complementary experiments. First, tumour latencies in survival studies were compared, and it was found that Sarm l deletion resulted in a significant extension of survival (Figure 31, median survival 18 weeks in WT and 21 weeks in Sarm l ). Second, given the prolonged preservation of axonal integrity observed in Sarm l7tumours (Figure 1 c and d), neurological function was assessed in mice with advanced tumours (corresponding to < 2 weeks before death) using motor score testing (Figure 3m). Strikingly, whereas severe deterioration of motor function was detected in tumour bearing WT animals, motor function was maintained at near normal level in Sarm l7mice, indicative of neuroprotection. These effects were again Sarml -specific because induction of npp tumours in a second independent mouse model based on CRISPR / Cas9-mediated Sarml gene knock out50also resulted in more diffuse terminal tumours with extended survival and preservation of motor function relative to background-matched controls (Figure 3v-y). To conclude, these results demonstrate that Wallerian degeneration represents a key GBM promoter, which could be targeted via inhibition of Sarml to suppress tumour progression and ameliorate disease course and its symptoms.

[0237] It is hypothesized that targeting WD in the context of brain tumours may lead to delays in tumour progression, reduced neurological deficits (thus preserving function and quality of life), and increased progression-free and overall survival.

[0238] As described in, for example, Hughes et al. Cell Rep. (2021) 34(1 ): 108588, small molecule inhibitors of SARM1 have been shown to effectively recapitulate the effects seen in SARM1 (SARM1 knockout) models, with respect to neuroprotection. Since the impact on cancer progression seen in the present examples in SARM1 models depends on the neuroprotective effect of knocking out SARM1, it is expected that SARM1 inhibitors will similarly recapitulate the anti-cancer results demonstrated here.

[0239] Example 5 - Effects of Wallerian degeneration inhibitors in human and mice glioblastoma models

[0240] Next, the effect of inhibiting Wallerian degeneration / Wallerian-like degeneration in patients with brain tumours, in particular intrinsic brain tumours such as high-grade gliomas (glioblastoma) is investigated.

[0241] Inhibitory Strategies and Agents:

[0242] The inhibition of Wallerian degeneration using pharmacological agents aimed at the Wallerian degeneration pathway is investigated, focusing on modulating the function of key proteins such as Sarml , NMNAT2, PHR1 , WLDs, Ube4b, and SCG10.

[0243] Pharmacological agents tested for inhibition of Wallerian degeneration include those described herein. This includes: antisense oligonucleotides targeting Sarml transcripts, such as those described in WO2021108602A1 , direct Sarml inhibitors, such as those described in WO2021142006A1 , and compounds such as CHS828 (Hjarnaa et al. Cancer Res. (1999) 59(22):5751-7). Additionally, a broad spectrum of agents will be evaluated, including: Apomorphine, bismuth subsalicylate, Bronopol, Cisplatin, D-phenylalanine, Epiestriol, Erythromycin, Merbromin, Metyropone, phenylmercuric acetate, pimethixene maleate, pyrithione zinc, thiram, NSC2805, NSC1152, NSC22806, NSC34879, NSC92937, NSC645330, NSC661221 , NSC641396, NSC70931 , NSC727038, NSC228155, NSC228150, NSC48443, NSC90749, NSC98363, NSC16339, NSC62208, NSC622689, as described in WO2018057989.

[0244] Experimental Models and Evaluative Methods

[0245] Genetically engineered mouse models of glioblastoma, and patient derived xenograft models are used in combination with drug / molecule delivery methods including implantable infusion pumps to deliver the therapeutic agents into the tumour / tumour resection cavity / cerebrospinal fluid reservoir. Organotypic brain slice cultures and organoids / tumour spheroids derived from both healthy and tumourbearing brains are used as a human or mouse tissue derived model system to investigate how effects seen in murine models translate to human models.

[0246] The effects of the described agents on tumour viability, proliferation, invasion, gene expression, neuroinflammation, and microenvironmental responses, including axonal and neuronal degeneration, responses from microglia and astrocytes, and changes related to myelin and oligodendrocytic function, as well as tumour hypoxia, ischemic responses, and the development of necrosis is investigated.

[0247] For in vivo models, survival outcomes and metrics of neurological function including motor scores and cognition is investigated.

[0248] Materials and methodology Animals

[0249] Wildtype C57BL / 6NCrl (Charles River), sterile alpha and TIR MotifTA(SarmT featuring mutation Chr11 :78472330-78497754 (-)). Sarm1+ / + 1and B6.Cg-Tg(Thy1-YFP)HJrs / J (ThylYFP; Jax laboratories (003782) maintained as a homozygous / C57BL / 6NRL cross. NOD.CB17-Pr / <c / csc'd / NCrCrl (NSG Charles River) are used for orthotopic injections of patient derived glioma stem cells.

[0250] Tumour generation in mice

[0251] Somatic tumours are generated as previously reported45, Figure 4. Plasmids are injected into the ventricle of isoflurane-immobilized pups at postnatal day 2. EF1 a-tdTomato plasmid is generated by SnaBI and Pmel digestion of npp plasmid to remove Nf1 , pten and Trp53 guide RNAs before re-ligation. PDX models are generated as previously described by stereotaxic injection of glioma stem cells39. Tumour bearing mice are monitored daily and sacrificed at required time points or when they began to show signs of disease and reached humane endpoints.

[0252] Tumour resection in tumour mice

[0253] Tumour resection in tumour mice is carried out on tumour-bearing mice at 6-12 weeks postelectroporation with npp plasmids. Mice are anaesthetized and mounted on a stereotaxic frame. A small craniotomy is performed on the right side of the skull 1 .7 mm medial to bregma and extending from 1 .0 mm anterior of bregma. Resection is undertaken with suction and microinstrumentation. This may be fluorescent assisted with either transgenic fluorescent reporter of 5ALA. Sham mice may undergo the same analgesia and anaesthesia protocol without mounting on the stereotaxic frame. Post injury mice may be given an intraperitoneal injection of EdU (5 mg / kg) 2 hours before brains were collected following transcardial perfusion with 4% paraformaldehyde (PFA) under terminal anaesthesia. Brains are fixed overnight in 4% PFA at 4°C, transferred to PBS prior to vibratome sectioning (50 mm sections) and storage in cryobuffer (ethylene glcol:glycerol:PBS 1 :1 :2).

[0254] Infusion pump / Continuous administration of substances directly into the brain through a slow release device To investigate the delivery of novel pharmacological agents, including drugs and antisense oligonucleotides (ASOs), implantable osmotic pumps are used in mice. All procedures were performed in accordance with the UK Home Office regulations and institutional approval. For continuous delivery of activating or inhibiting or control substances alone or in combination, a slow-release osmotic pump (Azlet® osmotic pumps) or pellet will be implanted in the mouse.

[0255] Under aseptic conditions the mouse is mounted on a stereotaxic frame. Following Visoctear application and disinfection, local anaesthetic will be delivered subcutaneously over the skull and analgesia by subcutaneous injection into the hind limb. An incision is made in the skin of the head large enough to accommodate the cannula of the pump and to allow positioning of the pump subcutaneously on the back of the mouse. Using bregma as a guide a small hole is drilled in the skull at the appropriate coordinates. The skull is cleaned and dried using sterile reagents and the cannula is positioned above the hole. The pump is positioned under the skin of the back and the cannula implanted into the skull using sterile superglue to hold it in place. The skin is sutured over the cannula and catheter, the wound disinfected and the mouse allowed to recover following a bolus of saline subcutaneously in a heated box. Analgesia is delivered for the following 72 hours through analgesia dosed jelly cubes. Mice are acclimatised to eating jelly cubes for 48 hours prior to surgery.

[0256] In some cases, the pumps will need to be replaced or removed. This is done under general anaesthesia and aseptic conditions by performing a small incision on the skin of the back of the mouse and replacing or removing the pump through the incision. The incision is closed by suturing and appropriate analgesia is delivered during and following the procedure. Pumps will only be replaced on one occasion. Mice are allowed to recover following a bolus of saline subcutaneously in a heated box. Post-operative analgesia is delivered the following day and for a further 48 hours through the drinking water.

[0257] Behavioural assessment

[0258] Behavioural neuroscores are determined as described in Example 1.

[0259] Tissue preparation and immunohistochemistry

[0260] Tissue preparation and immunohistochemistry is carried out as described in Example 1.

[0261] Image analysis

[0262] Analysis of tumour cell localisation and proliferation is performed in Imaris 10.1.0 on single z plane images from 3i Spinning disk microscope.

[0263] Flow Cytometry Analysis

[0264] Flow cytometry is carried out as described in Example 1 .

[0265] Derivation and culture of cell lines

[0266] Cell lines were derived from the CRUK glioma cellular genetics resources. GBM2 was derived independently as previously described3. All patient lines were cultured adherently in serum-free GSC media (N2 (1 / 200), B27 (1 / 100) (Life Technologies), 1 mg / mL laminin (Merck L2020), 10 ng / mL EGF (Biotechne NBP2 35176), 10ng / ml FGF-2 (Biotechne NBP2 35152), 1x MEM NEAA (Fisher Scientific 12084947), 0.1 mM 2-mercaptoethanol (Fisher Scientific 31350010), 0.012% BSA (ThermoFisher 15260- 037), 0.2 g / L glucose (Merck G8769), 1000 U / ml penicillin-streptomycin (Merck P0781).

[0267] Organotypic human slices and organoids

[0268] Annotated fresh human brain tissue / tumour tissue slices are obtained from appropriately consented patients. The brain slice is transferred to a vibratome (Leica VT1200 S) platform and immediately fixed to this device by embedding in low melting point agarose and fixing with a drop of superglue. 200-350 pm thick slices are cut with a maximal speed of 0.2 mm / s. Multiple slices are gathered and transferred onto permeable interfaces for organotypic culture. The brain slice medium is composed of a combination such as:

[0269] Base Media

[0270] • 50% (vol / vol) Advanced DMEM / F-12, Thermo Fisher Scientific (Life Technologies), cat no. 11320033

[0271] • 50% (vol / vol) Neurobasal medium, Thermo Fisher Scientific (Life Technologies), cat no. 21103049

[0272] • 1x GlutaMAX supplement (100x), Thermo Fisher Scientific (Life Technologies), cat no. 35050061

[0273] • 1x MEM-NEAAs solution (100x), Thermo Fisher Scientific (Life Technologies), cat no. 11140050

[0274] • 1x HyClone™ Pen / Strep Solution (100x), Fisher Scientific, cat no. SV30010

[0275] • 1x N2 (100x), Thermo Fisher Scientific (Life Technologies), cat no. 17502048

[0276] • 1x B-27 minus vitamin A (50x), Thermo Fisher Scientific (Life Technologies), cat no. 12587010 Complete Culture media, add fresh:

[0277] • 2.5 pg Human Insulin Solution (4000x stock), Merck cat no. 19278

[0278] • 50 nM 2-mercaptoethanol (50 mM stock), Thermo Fisher Scientific

[0279] The tissue is incubated at 37 °C and 5% CO2 Medium is removed from the filter and 1 mL of fresh brain slice medium was added below. The medium was refreshed after 18-24 h and then every other day. To prevent dehydration the tissue is moistened with a drop of medium every 1-2 days, and remaining excess medium removed. Viability is regularly checked. Pharmacological and biological agents of interest are tested in this system by direct addition to the organotypic media. Analysis includes immunohistochemistry and flow cytometry as described.

[0280] Statistical Analysis

[0281] Statistical analysis was performed in Prism 10 and R. All data are expressed as mean±SEM, unless otherwise stated. Significance is stated as follows: p>0.05 (ns), p<0.05 (*), p<0.01 (**), p<0.001 (***) and calculated using appropriate tests. No statistical method was used to predetermine sample size. Sample size was determined based on existing literature.

[0282] Context of Therapy-Associated Injury:

[0283] The role of the test pharmacological compounds in mitigating tumour progression within the context of therapy-associated injuries, including surgical interventions (biopsy, debulking, resection), radiotherapy, and chemotherapy with agents such as Temozolomide, PCV, Carboplatin, Lomustine, Cisplatin, Carmustine, and Bevacizumab is investigated.

[0284] The impact of these agents in the context of immunotherapy, vaccine-based treatments, and targeted therapies like the isocitrate dehydrogenase inhibitor Vorasidenib is also investigated.

[0285] References

[0286] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0287] 1. Gilley, J., Ribchester, R. R. & Coleman, M. P. Sarml Deletion, but Not Wld(S), Confers Lifelong Rescue in a Mouse Model of Severe Axonopathy. Cell Rep 21 , 10-16 (2017). https: / / doi.org:10.1016 / j.celrep.2017.09.027

[0288] 2. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41-51 (2000). https: / / doi.org:10.1016 / s0896-6273(00)00084-2

[0289] 3. Pollard, S. M. et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4, 568-580 (2009). https: / / doi.org:10.1016 / j.stem.2009.03.014

[0290] 4. Garcia-Diaz, C. et al. Glioblastoma cell fate is differentially regulated by the microenvironments of the tumor bulk and infiltrative margin. Cell Rep 42, 112472 (2023). https: / / doi.org:10.1016 / j.celrep.2023.112472

[0291] 5. Clements, M., Simpson Ragdale, H., Garcia-Diaz, C. & Parrinello, S. Generation of immunocompetent somatic glioblastoma mouse models through in situ transformation of subventricular zone neural stem cells. STAR Protoc 5, 102928 (2024). https: / / doi.org:10.1016 / j.xpro.2024.102928

[0292] 6. Brooks, L. J. et al. The white matter is a pro-differentiative niche for glioblastoma. Nat Commun 12, 2184 (2021). https: / / doi.org:10.1038 / s41467-021-22225-w

[0293] 7. Tang, W. et al. bayNorm: Bayesian gene expression recovery, imputation and normalization for singlecell RNA-sequencing data. Bioinformatics 36, 1174-1181 (2020). https: / / doi.org:10.1093 / bioinformatics / btz726

[0294] 8. Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20, 296 (2019). https: / / doi.org:10.1186 / s13059- 019-1874-1

[0295] 9. Palla, G. et al. Squidpy: a scalable framework for spatial omics analysis. Nat Methods 19, 171-178 (2022). https: / / doi.org:10.1038 / S41592-021-01358-2

[0296] 10. Zhao, E. et al. Spatial transcriptomics at subspot resolution with BayesSpace. Nat Biotechnol 39, 1375-1384 (2021). https: / / doi.org:10.1038 / s41587-021 -00935-2

[0297] 11. Wang, Q. et al. The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas. Cell 181 , 936-953. e920 (2020). https: / / doi.org:10.1016 / j.cell.2020.04.007

[0298] 12. Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16, 1289-1296 (2019). https: / / doi.org:10.1038 / s41592-019-0619-0 13. Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587. e3529 (2021). https: / / doi.org:10.1016 / j.cell.2021.04.048

[0299] 14. Kleshchevnikov, V. et al. Cell2location maps fine-grained cell types in spatial transcriptomics. Nat Biotechnol 40 , 661-671 (2022). https: / / doi.org:10.1038 / s41587-021-01139-4

[0300] 15. Ximerakis, M. et al. Single-cell transcriptomic profiling of the aging mouse brain. Nat Neurosci 22, 1696-1708 (2019). https: / / doi.org:10.1038 / s41593-019-0491-3

[0301] 16. Aibar, S. et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods 14, 1083-1086 (2017). https: / / doi.org:10.1038 / nmeth.4463

[0302] 17. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545-15550 (2005). https: / / doi.org:10.1073 / pnas.0506580102

[0303] 18. Ravi, V. M. et al. Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma. Cancer Cell 40, 639-655. e613 (2022). https: / / doi.org:10.1016 / j.ccell.2022.05.009

[0304] 19. Korsunsky, L, Nathan, A., Millard, N. & Raychaudhuri, S. Presto scales Wilcoxon and auROC analyses to millions of observations. bioRxiv, 653253 (2019). https: / / doi.org:10.1101 / 653253

[0305] 20. Hipel, K. W. & McLeod, A. I. Time series modelling of water resources and environmental systems. (Elsevier, 1994).

[0306] 21. Libiseller, C. & Grimvall, A. Performance of partial Mann-Kendall tests for trend detection in the presence of covariates. Environmetrics 13, 71-84 (2002). https: / / doi.org:https: / / doi.org / 10.1002 / env.507

[0307] 22. Kalamakis, G. et al. Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain. Cell 176, 1407-1419.e1414 (2019). https: / / doi.org:10.1016 / j.cell.2O19.01 .040

[0308] 23. Pombo Antunes, A. R. et al. Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization. Nat Neurosci 24, 595-610 (2021). https: / / doi.org:10.1038 / s41593-020-00789-y

[0309] 24. Yeo, A. T. et al. Single-cell RNA sequencing reveals evolution of immune landscape during glioblastoma progression. Nat Immunol 23, 971-984 (2022). https: / / doi.org:10.1038 / s41590-022-01215-0

[0310] 25. Gao, R. et al. Delineating copy number and clonal substructure in human tumors from single-cell transcriptomes. Nat Biotechnol 39 , 599-608 (2021). https: / / doi.org:10.1038 / s41587-020-00795-2

[0311] 26. Breiman, L. Random Forests. Machine Learning 45, 5-32 (2001). https : / / doi .org: 10.1023 / A: 1010933404324

[0312] 27. Blondel, V. D., Guillaume, J.-L., Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment 2008, P10008 (2008). https : / / doi .org: 10.1088 / 1742-5468 / 2008 / 10 / P10008

[0313] 28. Korotkevich, G. et al. Fast gene set enrichment analysis. bioRxiv, 060012 (2021). https: / / doi.org:10.1101 / 060012

[0314] 29. Darmanis, S. et al. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma. Cell Rep 21 , 1399-1410 (2017). https: / / doi.org:10.1016 / j.celrep.2017.10.030

[0315] 30. Muller, S. et al. Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment. Genome BioI S, 234 (2017). https: / / doi.org:10.1186 / s13059-017-1362-4

[0316] 31. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics 16, 284-287 (2012). https: / / doi.org:10.1089 / omi.2011 .0118 32. Wu, T. et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb) 2, 100141 (2021). https: / / doi.org:10.1016 / j.xinn.2021.100141

[0317] 33. Efremova, M., Vento-Tormo, M., Teichmann, S. A. & Vento-Tormo, R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat Protoc 15, 1484-1506 (2020). https: / / doi.org:10.1038 / s41596-020-0292-x

[0318] 34. Dimitrov, D. et al. Comparison of methods and resources for cell-cell communication inference from single-cell RNA-Seq data. Nat Commun 3, 3224 (2022). https: / / doi.org:10.1038 / s41467-022-30755-0

[0319] 35. Pocratsky, A. M. et al. Pathophysiology of Dyt1-Tor1a dystonia in mice is mediated by spinal neural circuit dysfunction. Sci Transl Med 15, eadg3904 (2023). https: / / doi.org:10.1126 / scitranslmed.adg3904

[0320] 36. Deerinck, T. et al. Enhancing Serial Block-Face Scanning Electron Microscopy to Enable High

[0321] Resolution 3-D Nanohistology of Cells and Tissues. Microscopy and Microanalysis 16, 1138-1139 (2010). https: / / doi.org:10.1017 / s1431927610055170

[0322] 37. Kaiser, T. et al. MyelTracer: A Semi-Automated Software for Myelin g-Ratio Quantification. eNeuro 8 (2021). https: / / doi.org:10.1523 / eneuro.0558-20.2021

[0323] 38. Neftel, C. et al. An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma. Cell

[0324] 178, 835-849. e821 (2019). https: / / doi.org:10.1016Zj.cell.2019.06.024

[0325] 39. Krusche B, Ottone C, Clements MP et al. EphrinB2 drives perivascular invasion and proliferation of glioblastoma stem-like cells. Elife. 2016;5:1-32.

[0326] 40. Doran, C. G. et al. CRISPR / Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages. J Biol Chem 297, 101417 (2021). https: / / doi.org:10.1016 / j.jbc.2021.101417

[0327] 41. Uccellini, M. B. et al. Passenger Mutations Confound Phenotypes of SARM1 -Deficient Mice. Cell Rep

[0328] 31 , 107498 (2020). https: / / doi.org:10.1016 / j.celrep.2020.03.062

[0329] 42. Jin, L. et al. Astrocytic SARM1 promotes neuroinflammation and axonal demyelination in experimental autoimmune encephalomyelitis through inhibiting GDNF signaling. Cell Death Dis 13, 759 (2022). https: / / doi.org:10.1038 / S41419-022-05202-z

[0330] 43. Mukherjee, P., Woods, T. A., Moore, R. A. & Peterson, K. E. Activation of the innate signaling molecule MAVS by bunyavirus infection upregulates the adaptor protein SARM1 , leading to neuronal death. Immunity 38, 705-716 (2013). https: / / doi.org:10.1016 / j.immuni.2013.02.013

[0331] 44. Geisler, S. et al. Gene therapy targeting SARM1 blocks pathological axon degeneration in mice. J Exp Med 216, 294-303 (2019). https: / / doi.org:10.1084 / jem.20181040

[0332] 45. Karschnia, P. et al. A framework for standardised tissue sampling and processing during resection of diffuse intracranial glioma: joint recommendations from four RANO groups. Lancet Oncol 24, e438-e450 (2023). https: / / doi.org:10.1016 / S1470-2045(23)00453-9

[0333] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

Claims:

1. A Wallerian degeneration inhibitor for use in a method of treating or preventing a cancer of the central nervous system (CNS) in a mammalian subject.

2. The Wallerian degeneration inhibitor for use according to claim 1 , wherein the Wallerian degeneration inhibitor is a SARM1 inhibitor.

3. The Wallerian degeneration inhibitor for use according to claim 1 or 2, wherein the Wallerian degeneration inhibitor is a nucleic acid.

4. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 3, wherein theWallerian degeneration inhibitor is an adeno-associated virus vector comprising a nucleic acid encoding a dominant negative SARM1 .

5. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 3, wherein theWallerian degeneration inhibitor is an antisense oligonucleotide.

6. The Wallerian degeneration inhibitor for use according to claim 5, wherein the antisense oligonucleotide is complementary to a region of a SARM1 mRNA.

7. The Wallerian degeneration inhibitor for use according to claim 5 or 6, wherein the antisense oligonucleotide is complementary to a region of the SARM1 mRNA encoded by the nucleotide sequence of NCBI reference sequence: NM_015077, v4.

8. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 3, wherein theWallerian degeneration inhibitor is a small interfering RNA.

9. The Wallerian degeneration inhibitor according to claim 1 or claim 2, wherein the Wallerian degeneration inhibitor is a small molecule inhibitor.

10. The Wallerian degeneration inhibitor according to claim 9, wherein the small molecule inhibitor is selected from the list comprising: isoquinolines e.g DSRM-3716, isothiazole compounds, tryptoline acrylamides, NB3, NB7, TK106, TK138, LY3873862, and NB-4746. .11 . The Wallerian degeneration inhibitor for use according to any one of claims 1 to 10 wherein the cancer of the central nervous system is selected from the list comprising: glioblastomas, gliomas, craniopharyngioma, brain stem glioma, medulloblastoma, meningioma, astrocytomas, pituitary adenomas, oligodendroglioma, ependymal tumors, pineal parenchymal tumors and brain metastases.

12. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 11 , wherein the cancer is glioblastoma.

13. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 12, wherein the cancer is an early-phase cancer.

14. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 13, wherein the cancer is an early-phase CNS cancer, and the method prevents or slows progression of the CNS cancer to a later stage.

15. The Wallerian degeneration inhibitor for use according to any one of claims 1 to 14, wherein the method comprises reducing glioblastoma-associated neurological symptoms of the subject.

16. The Wallerian degeneration inhibitor for use according to claim 15, wherein the neurological symptoms are selected from the list comprising: seizure (including focal seizure and / or generalised seizure), loss of consciousness, syncope, aphasia, headache, visual deficit ( / .e. dysfunction or loss), gait disturbance, memory impairment, paralysis, paresis, plegia, weakness, sensory dysfunction, language dysfunction, and cognitive impairment.

17. The inhibitor of Wallerian degeneration for use according to any one of claims 1 to 16, wherein the method comprises administering the Wallerian degeneration inhibitor to a subject in conjunction with, or immediately following, tumour resection or chemotherapy.

18. The inhibitor of Wallerian degeneration for use according to any one of claims 1 to 17 wherein the subject is considered at risk of developing glioblastoma.

19. An in vitro method of screening therapeutic candidates as anti-CNS cancer therapeutic compounds, the method comprising:(i) selecting Wallerian degeneration inhibitors as therapeutic candidates;(ii) providing organotypic brain slice cultures derived from both healthy and tumour-bearing brains;(iii) adding therapeutic candidates to the organotypic brain slice culture media;(iv) analysing the organotypic brain slice culture to determine the anti-cancer effect of the therapeutic candidates.

20. An in vivo method of screening therapeutic candidates as anti-CNS cancer therapeutic compounds, the method comprising:(i) selecting Wallerian degeneration inhibitors as therapeutic candidates(ii) providing genetically engineered mouse models of CNS cancer;(iii) delivering therapeutic candidates to the tumour, cerebrospinal fluid reservoir, or tumour resection cavity via implantable osmotic pumps;(iv) analysing the mouse model to determine the anti-cancer effect of the therapeutic candidates.

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