Regulation of delta-2 tubulin for treating chemotherapy-induced peripheral neuropathy

Abstract

The subject matter provides methods and compositions for preventing or treating chemotherapy-induced peripheral neuropathy (CIPN) by modulating delta-2 tubulin accumulation in sensory neurons. The therapeutic approaches targeting enzymes that regulate delta-2 tubulin formation, such as cytosolic carboxypeptidases (CCP1 and CCP6), or enhancing tubulin re-tyrosination via tubulin tyrosine ligase (TTL).

Description

[0001] Docket No:070050.6974_CU18162

[0002] PCT Application

[0003] REGULATION OF DELTA-2 TUBULIN FOR TREATING CHEMOTHERAPY- INDUCED PERIPHERAL NEUROPATHY

[0004] CROSS-CONFERENCE INFORMATION

[0005] This International PCT Application claims the priority of U.S. provisional application Ser. No. 63 / 721,882, filed on November 18, 2024, the entire contents of which are incorporated by reference herein.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under NS 120076 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] The disclosed subject matter relates to t methods and compositions for preventing or treating chemotherapy -induced peripheral neuropathy (CIPN).

[0010] CIPN is a dose-limiting side effect of certain common anticancer agents. Despite its prevalence, the underlying molecular mechanisms driving CIPN remain under investigation, and improved options for treatment are needed.

[0011] As such, there remains a need to determine pathogenic factors in CIPN and further develop a reliable therapeutic treatment for preventing or treating CIPN and related axonal disorders.

[0012] SUMMARY

[0013] The disclosed subject matter provides methods and compositions for treating or preventing chemotherapy-induced peripheral neuropathy (CIPN) in a subject receiving an anti-cancer agent. An example method comprises obtaining a first biological sample from the subject prior to administration of the anti-cancer agent; obtaining a second biological sample from the subject after administration of the anti-cancer agent; determining an expression level of delta-2 tubulin in the first biological sample and the second biological sample; comparing the determined delta-2 tubulin expression level in the first biological sample to the determined delta-2 tubulin expression level in the second biological sample to determine whether the delta-2 tubulin expression level has increased; and, when an Docket No:070050.6974_CU18162

[0014] PCT Application

[0015] increase in delta-2 tubulin expression level is determined, and administering to the subject a therapeutically effective amount of a pharmaceutical composition configured to reduce delta-2 tubulin accumulation, thus treating or preventing CIPN. In certain embodiments, the biological sample comprises dorsal root ganglion (DRG).

[0016] In certain embodiments, determining the expression level of delta-2 tubulin comprises detecting delta-2 tubulin by immunofluorescence, immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry. An increase in delta-2 tubulin expression is determined by comparing the second biological sample to the first biological sample and determining that the delta-2 tubulin level in the second biological sample is higher than in the first biological sample by a pre-determined threshold amount.

[0017] In certain embodiments, the pharmaceutical composition comprises an agent configured to reduce formation of delta-2 tubulin by inhibiting one or more cytosolic carboxypeptidases (CCP), wherein the CCP is selected from CCP1 and CCP6. In some embodiments, the agent comprises an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an adeno-associated virus (AAV) vector encoding a Cre recombinase or shRNA sequence that reduces expression of CCP1 or CCP6.

[0018] In alternative embodiments, the pharmaceutical composition comprises an agent configured to promote re-tyrosination of a-tubulin by increasing expression or activity of tubulin tyrosine ligase (TTL), where the agent comprises a small molecule, a polynucleotide encoding TTL, or a viral vector expressing TTL.

[0019] In alternative embodiments, the pharmaceutical composition comprises an agent that upregulates mitochondrial respiration, where the agent comprises mitochondrial electron transport chain enhancers, NAD+precursors, ATP-generating enhancers, or compounds that restore mitochondrial membrane potential.

[0020] In alternative embodiments, the pharmaceutical composition comprises an agent that increases mitochondrial motility by downregulating or inhibiting syntaphilin (SNPH). The agent comprises a small molecule, antisense oligonucleotide, shRNA, or polypeptide antagonist of SNPH.

[0021] In certain embodiments, administering the pharmaceutical composition comprises delivering a viral vector to DRG neurons, wherein the viral vector comprises an adeno-associated virus (AAV). In some embodiments, the AAV comprises an AAV-PHP.S capsid.

[0022] In certain embodiments, the anti-cancer agent is selected from the group consisting of bortezomib, paclitaxel, vincristine, cisplatin, carboplatin, eribulin, thalidomide, and Docket No:070050.6974_CU18162

[0023] PCT Application

[0024] taxane. In some embodiments, the pharmaceutical composition is administered prophylactically prior to onset of CIPN when an increase in delta-2 tubulin is detected.

[0025] In certain embodiments, the method further comprises monitoring delta-2 tubulin levels after administration of the pharmaceutical composition and adjusting a dosing regimen of the pharmaceutical composition based on the monitored delta-2 tubulin levels. In some embodiments, the pharmaceutical composition is formulated for systemic administration, local intrathecal administration, or direct ganglionic delivery.

[0026] In another aspect, the disclosed subject matter further provides a pharmaceutical composition for treating or preventing chemotherapy-induced peripheral neuropathy comprising a therapeutically effective amount of an inhibitor of CCP1 or CCP6 and a pharmaceutically acceptable carrier, wherein the composition is formulated for systemic or neuronal delivery, and wherein the inhibitor of CCP1 or CCP6 is configured to reduce formation of delta-2 tubulin in dorsal root ganglion neurons.

[0027] In certain embodiments, the pharmaceutical composition further comprises a tubulin tyrosine ligase (TTL) activator. In alternative embodiments, the pharmaceutical composition further comprises a mitochondrial enhancer. In some embodiments, the pharmaceutical composition is formulated for systemic or intrathecal administration and includes a pharmaceutically acceptable carrier or excipient.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated and constitute part of this disclosure, illustrate certain embodiments of the disclosed subject matter.

[0030] Figs. 1A-1B illustrate schematic representations of microtubule structure and tubulin post-translational modifications, according to certain embodiments of the disclosed subject matter.

[0031] Fig. 2 provides a schematic diagram of the a-tubulin detyrosination / tyrosination cycle and the enzymatic pathways leading to delta-2 tubulin formation, according to certain embodiments of the disclosed subject matter.

[0032] Figs. 3A-3H show example data of delta-2 tubulin accumulation in sensory neurons of rodents and human biopsies following treatment with the chemotherapeutic agent bortezomib, according to certain embodiments of the disclosed subject matter. Docket No:070050.6974_CU18162

[0033] PCT Application

[0034] Figs.4A-4D show example data showing that accumulation of delta-2 tubulin is both necessary and sufficient to induce axonal degeneration and inhibit mitochondrial motility, as well as bortezomib-induced axonal degeneration and mitochondrial motility impairment.

[0035] Figs. 5A-5C depict somatosensory behavioral perturbations in mice with AAV-mediated TTL downregulation leading to delta-2 tubulin accumulation in DRG neurons, indicating altered mechanical and thermal sensitivity profiles.

[0036] Figs. 6A-6E present data from a bortezomib-induced peripheral neuropathy model, showing heightened tactile sensitivity, impaired motor performance, and reduced epidermal nerve fiber density compared to controls.

[0037] Figs.7A-7B show the example strategy for adeno-associated virus (AAV)-mediated transduction of DRG neurons and the establishment of a preclinical models of CIPN induced by paclitaxel (B) in the mouse.

[0038] Figs. 8A-8I present behavioral and histological analyses from a vincristine-induced peripheral neuropathy model, demonstrating increased mechanical and thermal hypersensitivity, motor impairment, and decreased IENF density.

[0039] Figs.9A-9C present time-course data showing that cisplatin treatment induces delta-2 tubulin accumulation in DRG neurons prior to detectable axonal degeneration.

[0040] Fig. 10 shows a preclinical model of cisplatin-induced peripheral neuropathy, with increased mechanical withdrawal responses and prolonged hypersensitivity following treatment.

[0041] Figs. 11A-11G illustrates that CRE-mediated excision of CCP1 depletes D2 tubulin levels in vivo with no detectable impact on sensory function in CCP1 -depleted mice without Bortezomib treatment (therapeutic potential of this approach).

[0042] Figs. 12A-12E shows that reduction of delta-2 tubulin formation via AAV-mediated excision of CCP1 / 6 ameliorates BIPN (mechanosensory response and IENF density) in mice treated with bortezomib.

[0043] Figs. 13A-13B illustrates comparative mitochondrial respiration analyses and TRP channel activity measurements in DRG neurons expressing delta-2 tubulin.

[0044] Figs. 14A-14I present data showing that depletion of tubulin tyrosine ligase (TTL) to elevate delta-2 tubulin levels in cultured DRG neurons, has profound effects TRP channel activity.

[0045] Figs. 15A-15C present data showing that both bortezomib and delta-2 tubulin overexpression increase SNPH levels in DRG neurons. Docket No:070050.6974_CU18162

[0046] PCT Application

[0047] Fig. 16 illustrates a process flowchart for treating or preventing CIPN in a subject receiving a neurotoxic anti-cancer agent.

[0048] Throughout the Figures and specification, the same reference numerals are used to indicate similar features and / or structures.

[0049] DETAILED DESCRIPTION

[0050] The disclosed subject matter addresses a significant medical problem that can affect cancer patients: nerve damage caused by chemotherapy, known as CIPN. The disclosed subject matter uncovers that this nerve damage is linked to the buildup of a specific modified protein in nerve cells, delta-2 tubulin. When delta-2 tubulin accumulates, it disrupts the cell’s energy-producing structures — mitochondria — and interferes with normal nerve signaling. Further, the disclosed subject matter provides methods to detect this harmful buildup early in patients receiving chemotherapy and to treat or prevent nerve damage by using drugs or genetic tools that block the enzymes responsible for producing A2 -tubulin or boost the enzymes that reverse the modification. By restoring normal mitochondrial function and protecting nerve fibers, these treatments can prevent or lessen the painful, disabling side effects of chemotherapy without interfering with other anti-cancer effects.

[0051] For purposes of the disclosed subject matter, the following terms shall have the meanings ascribed below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of neurobiology, molecular biology, or pharmaceutical sciences.

[0052] “Delta-2 tubulin”, “delta-2 tubulin” or “A2 tubulin” refers to an a-tubulin variant that results from sequential enzymatic removal of the C-terminal tyrosine and the adjacent glutamate residues of a-tubulin.

[0053] “Tubulin tyrosine ligase (TTL)” refers to an enzyme that re-adds tyrosine to the C-terminal end of detyrosinated a-tubulin, thereby regenerating the tyrosinated tubulin pool and maintaining microtubule homeostasis. Decreased TTL activity typically promotes delta-2 tubulin accumulation and neurotoxicity.

[0054] “Cytosolic carboxypeptidase (CCP)” refers to any member of the CCP enzyme family (including CCP1-CCP6) capable of catalyzing the removal of the penultimate glutamate residue from detyrosinated a-tubulin to form delta-2 tubulin. Inhibition of CCP1 or CCP6 can prevent irreversible delta-2 tubulin formation in certain embodiments of the disclosed Docket No:070050.6974_CU18162

[0055] PCT Application

[0056] matter. “CCPl / 6flx / flx” or “CCPl / 6fl / fl” refers to the subject homozygous for floxed alleles of CCP1 and CCP6, allowing Cre-dependent deletion.

[0057] “Mitochondrial respiration” refers to a biochemical process by which mitochondria produce ATP (adenosine triphosphate) through oxidative phosphorylation, measured as oxygen consumption rate (OCR) in certain embodiments of the disclosed subject matter. Impaired mitochondrial respiration in sensory neurons results in reduced energy supply, increased oxidative stress, and neuronal degeneration.

[0058] “Transient receptor potential (TRP) channel” refers to a family of cation channels, including TRPV1, TRPV4, and related isoforms, that mediate sensory transduction in dorsal root ganglion neurons. The term “TRP channel function” encompasses calcium influx, electrical excitability, and nociceptive signaling mediated by these channels.

[0059] "MATCAP" refers to metacaspase-associated truncated catabolic activator protein, a plant enzyme that is a calcium-dependent activator for a type of protease, e.g., metacaspases.

[0060] “Syntaphilin (SNPH)” refers to a neuronal mitochondrial docking protein that anchors mitochondria to microtubules within axons, reducing their motility. “SNPH expression” refers to the production and cellular level of syntaphilin (SNPH) protein in neurons in certain embodiments of the disclosed subject matter. Increased SNPH expression or activity leads to mitochondrial immobilization and impaired axonal energy distribution, whereas decreased levels of the SNPH expression would lead to mitochondria hypermobility.

[0061] “Peripheral neuropathy” refers to a pathological condition characterized by damage or dysfunction of peripheral nerves, resulting in sensory disturbances such as numbness, pain, or hypersensitivity. Peripheral neuropathy encompasses conditions including CIPN, diabetic neuropathy, and toxin-induced neuropathies. CIPN can refer to peripheral nerve injury caused by the administration of chemotherapeutic agents such as bortezomib, paclitaxel, vincristine, or cisplatin in certain embodiments of the disclosed subject matter.

[0062] “php.s-” refers to the AAV-PHP.S capsid variant, an engineered serotype with enhanced tropism for neurons and efficient transduction of the dorsal root ganglia (DRG) and other peripheral neurons following systemic (e.g., retro-orbital) delivery. “Syn-” refers to the human synapsin promoter, which drives neuron-specific expression, ensuring that transgene activity is restricted to neuronal cells. “EGFP-Cre-” refers to a fusion of enhanced green fluorescent protein (EGFP) and Cre recombinase enzyme, allowing simultaneous visualization of infected neurons (via EGFP fluorescence) and site-specific DNA recombination in certain embodiments of the disclosed subject matter. Docket No:070050.6974_CU18162

[0063] PCT Application

[0064] “Sensory neuron” or “dorsal root ganglion (DRG) neuron” refers to a neuron residing in the dorsal root ganglia that conveys sensory information, including pain and temperature signals, from the periphery to the central nervous system.

[0065] “Mitochondrial motility” refers to the movement of mitochondria along axonal microtubules, including anterograde (toward the synapse) and retrograde (toward the soma) transport. Decreased mitochondrial motility indicates impaired energy distribution along the axon.

[0066] “Degeneration index” refers to a quantitative measurement of axonal integrity derived from microscopy or image analysis that reflects the proportion of fragmented axons in a neuronal culture or tissue section.

[0067] “Therapeutically effective amount” refers to the quantity of an active agent that produces a measurable improvement or prevention of a pathological condition, such as restoration of mitochondrial function, reduction of neuropathic pain, or prevention of axonal degeneration, without undue toxicity to the subject in certain embodiments of the disclosed subject matter.

[0068] “Inhibitor to delta-2 tubulin” refers to any molecule, compound, nucleic acid, or biologic that reduces delta-2 tubulin formation or enhances its turnover. Such inhibitors or agents can inhibit CCP1 / 6 activity, increase TTL expression, or indirectly restore microtubule homeostasis for therapeutic purposes in certain embodiments of the disclosed subject matter.

[0069] “Pharmaceutically acceptable carrier” refers to any non-toxic medium, diluent, excipient, or vehicle suitable for the formulation and delivery of an active compound to a subject, including aqueous buffers, liposomes, emulsions, biodegradable polymers, or viral vectors.

[0070] “Subject” includes a human or non-human mammal in need of treatment, prevention, or diagnosis of a neuropathic condition.

[0071] “deTyr / Tyr cycle” refers to the tyrosination and detyrosination of a-tubulin in microtubules. Tyrosinated tubulin (Tyr) contains a C-terminal tyrosine, whereas detyrosinated tubulin (deTyr) has the tyrosine removed. The ratio of deTyr to Tyr reflects microtubule stability and dynamics, with increased detyrosination generally indicating more stable, less dynamic microtubules.

[0072] “AAV:PHP.s.tdTomato” refers to an adeno-associated virus (AAV) vector with a PHP.S capsid variant, engineered for systemic delivery to neurons, carrying a gene encoding Docket No:070050.6974_CU18162

[0073] PCT Application

[0074] the red fluorescent protein tdTomato. Expression of tdTomato allows visualization and tracing of infected neurons in vivo.

[0075] “mito-DsRed” refers to a mitochondrially targeted fluorescent reporter protein derived from the red fluorescent protein DsRed. In certain embodiments of the disclosed subject matter, mito-DsRed can be used as a live-cell imaging marker to visualize and quantify mitochondrial morphology, distribution, and motility within DRG neurons.

[0076] “Diagnostic biomarker” refers to a measurable molecular or cellular feature that correlates with a pathological state. In the context of this invention, delta-2 tubulin accumulation in peripheral neurons or skin biopsy samples serves as a biomarker for neuropathic progression in certain embodiments of the disclosed subject matter.

[0077] “Viral vector” refers to a recombinant viral particle engineered to deliver a therapeutic nucleic acid to a target cell. In certain embodiments, the vector is an adeno-associated virus (AAV), such as AAV-PHP.s, carrying a polynucleotide that modulates CCP1, CCP6, or TTL expression in sensory neurons.

[0078] “Neuronal-specific promoter” means a regulatory sequence that drives gene expression predominantly in neuronal cells, such as synapsin (Syn), CamKIIa, or neurofilament heavy chain promoters, enabling selective targeting of DRG neurons in vivo.

[0079] “Modulate” refers to increasing or decreasing the biological activity, expression, or function of a target molecule (e.g., CCP1, TTL, TRP channel, or syntaphilin) relative to its baseline level.

[0080] “ANOVA”, short for Analysis of Variance, refers to a statistical method used to compare the means of multiple groups to determine whether there are statistically significant differences among them. ANOVA can be applied to assess the effects of different treatments or conditions on a measured variable — such as protein levels, mitochondrial respiration, or behavioral responses. In certain embodiments of the disclosed subject matter, one-way ANOVA is used to compare the effects of delta-2 tubulin overexpression versus controls on mitochondrial respiration, “t-tesf ’ refers to a parametric statistical test used to compare the means of two groups to determine whether they are significantly different from each other. It assumes that the data are normally distributed and that the variances between the groups are similar. In certain embodiments, t-tests can be used to evaluate differences in quantitative parameters such as axonal integrity, calcium response amplitude, or mitochondrial respiration between treated and control groups of neurons. “Mann-Whitney test” refers to a non-parametric statistical test used to compare differences between two independent groups when the data do not necessarily follow a normal distribution. This test Docket No:070050.6974_CU18162

[0081] PCT Application

[0082] ranks all values and evaluates whether one group tends to have higher or lower ranks than the other. In certain embodiments, Mann-Whitney tests can be applied to analyze non-normally distributed datasets, including neuronal degeneration indices, mitochondrial motility fractions, and proportions of TRP channel-responsive cells.

[0083] “GAPDH” refers to glyceraldehyde-3 -phosphate dehydrogenase, a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3 -phosphate to 1,3-bisphosphogly cerate during energy metabolism. In analyses of certain embodiments, GAPDH can be used as a loading control in Western blot (immunoblot) assays because its expression is generally stable across different samples and treatment conditions. In the context of this invention, GAPDH serves as an internal reference to normalize protein levels, ensuring that observed changes in delta-2 tubulin (delta-2 tubulin) reflect true biological variation rather than differences in total protein loading.

[0084] The examples or embodiments herein are provided merely for descriptive purposes, rather than being restrictive or limiting to the disclosed subject matter. The methods and systems provided in the disclosed subject matter and certain exemplary and example data and results thereof can be illustrated and presented below, as shown in the Figures.

[0085] With references to Fig. 1A, the disclosed subject matter presents the dynamic assembly and disassembly of a- and P-tubulin heterodimers into polarized microtubule polymers, demonstrating the structural polarity defined by a P-tubulin-containing “plus” end and an a-tubulin-containing “minus” end. As shown in Fig. 1 A regarding the principle of dynamic instability, the microtubules undergo cycles of polymerization and depolymerization, allowing rapid remodeling required for axonal growth, transport, and plasticity.

[0086] Fig. IB shows representative post-translational modifications (PTMs) that occur on the exposed C-terminal tails of tubulin subunits. These PTMs can include at least one of detyrosination, acetylation, polyglutamylation, polyglycylation, phosphorylation, and irreversible A2 / A3 truncations, and constitute a regulatory “tubulin code” that regulates the interaction of microtubules with molecular motors, microtubule-associated proteins (MAPs), and organelles such as mitochondria. As shown in Fig. IB, the dynamic microtubules are typically less modified, while stable microtubules acquire multiple PTMs, conferring specialized properties such as altered transport efficiency and differential protein binding. This spatial and temporal diversity of PTMs can determine the functional identity of microtubule subsets within neurons. Docket No:070050.6974_CU18162

[0087] PCT Application

[0088] Fig. 2 illustrates the enzymatic pathway governing the tyrosination-detyrosination cycle of a-tubulin and the subsequent formation of delta-2 tubulin in certain embodiments, which is an irreversible modification implicated in neuropathic degeneration. The cycle begins with the removal of the C-terminal tyrosine residue by vasohibin / SVBP (Small Vasohibin-Binding Protein) complexes (VASH1 / 2-SVBP) or by the calcium-dependent enzyme MATCAP, generating detyrosinated tubulin. Such a modification is normally reversible through the action of tubulin tyrosine ligase (TTL), which re-adds the tyrosine residue to soluble a-tubulin, maintaining a dynamic balance between tyrosinated and detyrosinated forms. Notably, a prolonged or excessive detyrosination allows cytosolic carboxypeptidases (CCPs) — particularly CCP1 and CCP6 — to remove the penultimate glutamate residue, producing delta-2 tubulin. Such a modification eliminates the substrate for TTL, rendering a-tubulin permanently unmodifiable by re-tyrosination. As shown schematically in Fig. 2, the conversion to delta-2 tubulin marks long-lived, hyperstable microtubules, reflecting enzymatic processing that occurs over time.

[0089] Thus, through the irreversible accumulation of delta-2 tubulin in adult DRG neurons, the disclosed subject matter disrupts neuronal energy metabolism and intracellular signaling impair and prevent the increased delta-2 tubulin levels from impairing mitochondrial respiration, hindering organelle motility, and altering the dynamic equilibrium of microtubule networks essential for axonal maintenance. These findings also identify delta-2 tubulin as a causal pathogenic factor in neuronal degeneration and form the molecular foundation for the therapeutic approaches disclosed in the disclosed subject matter.

[0090] The disclosed subject matter illustrates the induction and distribution of delta-2 tubulin in DRG neurons and peripheral nerves following exposure to the chemotherapeutic agent bortezomib, demonstrating both example and clinical evidence of A2 accumulation as an effective indicator of CIPN, as shown in Figs. 3 A-3H. Scale bars in Figs. 3 A, 3D, and 3F represent 20 pm; statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0091] Fig. 3 A presents representative immunofluorescence images of DRG sections and sciatic nerves biopsies from untreated control mice and from mice subjected to acute (e.g., less than 24-hour) and chronic bortezomib treatment (e.g., 0.2 mg / kg). In bortezomib-treated subjects, intense delta-2 tubulin immunoreactivity is evident in the neuronal somata and axons, demonstrating a robust and widespread increase in delta-2 tubulin levels compared to controls. Docket No:070050.6974_CU18162

[0092] PCT Application

[0093] Figs. 3B and 3C provide quantitative data of tubulin post-translational modification (PTM) levels measured by immunofluorescence in DRG neurons (35-60 randomly selected) and five sciatic nerve sections per condition. Acute (0.2 mg / kg; 24 hours) and chronic (0.2 mg / kg, three times per week for eight weeks) bortezomib treatments both significantly elevate delta-2 tubulin levels relative to controls, while other PTMs such as tyrosination, acetylation, and polyglutamylation remain unchanged. These data demonstrate that bortezomib selectively increases delta-2 tubulin in a dose- and duration-dependent manner without broadly altering the tubulin modification landscape.

[0094] Fig. 3D presents triple immunostaining for peripherin (a marker for small, unmyelinated C-fibers), NF200 (a marker for medium and large myelinated A-P fibers), and delta-2 tubulin in DRG neurons from acutely treated mice. The data reveal a delta-2 tubulin accumulation in peripherin-positive small sensory neurons, whereas NF200-positive neurons display minimal delta-2 tubulin signal. Fig. 3E quantifies delta-2 tubulin intensity across 35-60 DRG neurons per condition (e.g., 6 mice) and confirms that C-fiber neurons exhibit significantly higher delta-2 tubulin accumulation than A-P fibers following bortezomib treatment. This subtype-specific pattern aligns with the clinical profile of bortezomib-induced peripheral neuropathy, which predominantly affects unmyelinated and thinly myelinated fibers.

[0095] Fig. 3F shows immunofluorescence images of sural nerve biopsies from a subject, e.g., a human patient, diagnosed with bortezomib-induced peripheral neuropathy (BIPN), demonstrating that an increase in delta-2 tubulin staining is relative to pill-tubulin compared to healthy control nerves.

[0096] Fig. 3G provides a ratio analysis of delta-2 tubulin / pill-tubulin levels measured by IF in three to six fixed tissue sections per condition, showing a significant elevation of delta-2 tubulin in the BIPN patient sample compared to three control patients. Fig. 3H presents example results about delta-2 tubulin levels in whole cell lysates from one sural nerve biopsy for a subject treated with Bort displayed with a control subject.

[0097] Collectively, as demonstrated in Figs. 3 A-3H, the disclosed subject matter establishes that bortezomib treatment induces rapid and sustained delta-2 tubulin accumulation in both animal and human sensory neurons, with preferential enrichment in small-diameter C-fiber populations. The observed delta-2 tubulin elevation occurs before any detectable axonal fragmentation or morphological degeneration, identifying delta-2 tubulin accumulation as an early and causative event in the pathogenesis of bortezomib-induced peripheral Docket No:070050.6974_CU18162

[0098] PCT Application

[0099] neuropathy. These findings validate delta-2 tubulin-tubulin as a key biomarker and therapeutic target for preventing chemotherapy-induced neuronal injury.

[0100] Figs. 4A-4D provide degeneration index and mitochondrial dynamic state data, demonstrating that delta-2 tubulin accumulation is both necessary and sufficient to induce axonal degeneration and impair mitochondrial motility in DRG neurons, establishing delta-2 tubulin as a direct pathogenic mediator in chemotherapy -induced neuropathy. Degeneration index refers to a quantitative measure of axonal integrity used to assess the extent of neurite fragmentation or retraction. It can be calculated as the ratio of the area occupied by fragmented axonal segments to the total axonal area within a microscopic field. A higher degeneration index indicates greater axonal breakdown and structural loss. Mitochondrial dynamic state refers to the classification of mitochondria within neuronal axons based on their movement behavior over time, as determined typically by live-cell imaging. Individual mitochondria are categorized as anterograde running (AR), retrograde running (RR), stationary (ST), or in a dynamic pause (DP) state. The relative distribution of these states provides a quantitative assessment of mitochondrial motility and transport efficiency. In delta-2 tubulin overexpressing neurons, the fraction of stationary mitochondria increases markedly, while anterograde and retrograde movement fractions decrease, indicating impaired mitochondrial trafficking. Restoration of these motile states upon CCP1 inhibition demonstrates preservation of mitochondrial transport and axonal health. Fig. 4A shows representative images of DRG neurons (12 days in vitro, DIV) transduced at 5DIV with lentiviral constructs encoding either wild-type (WT) a-tubulin or delta-2 tubulin. Neurons expressing delta-2 tubulin exhibit pronounced axonal fragmentation and neurite retraction, as quantified by an elevated degeneration index compared to WT controls. These findings demonstrate that overexpression of delta-2 tubulin alone, independent of external chemotherapeutic stress, is sufficient to trigger axonopathy. Fig. 4B presents quantification of mitochondrial motility states in DRG neurons (5DIV) coexpressing Mito-DsRed and either WT a-tubulin or delta-2 tubulin prior to detectable axonal degeneration. Compared with WT a-tubulin-expressing neurons, A2-tubulin-expressing neurons exhibit a marked reduction in anterograde and retrograde movement and a significant increase in stationary mitochondria, indicating severe disruption of mitochondrial transport. This motility impairment occurs before visible axonal damage, supporting a primary effect of delta-2 tubulin on organelle trafficking (see Figure 5, Pero et al., Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy, Proc. Docket No:070050.6974_CU18162

[0101] PCT Application

[0102] Natl. Acad. Sci. U.S.A. 118 (4) e2012685118, https: / / doi.org / 10.1073 / pnas.2012685118 (2021).

[0103] Fig. 4C demonstrates the effect of depleting cytosolic carboxypeptidase 1 (CCP1), an enzyme required for delta-2 tubulin formation, on bortezomib-induced neurotoxicity. Adult DRG neurons are transduced at 5DIV with a lentiviral shRNA targeting CCP1 (shCCPl) and subsequently treated with 100 nM bortezomib for 24 hours at 11DIV. Compared with control neurons, CCP1 -silenced neurons display markedly reduced axonal degeneration, indicating that suppression of delta-2 tubulin formation confers resistance to bortezomib-induced axonopathy. Fig. 4D quantifies mitochondrial dynamic states in DRG neurons expressing Mito-DsRed and shCCPl at 1DIV, followed by 24-hour bortezomib treatment at 4DIV. The analysis reveals that CCP1 knockdown restores mitochondrial motility, reducing the proportion of stationary mitochondria and increasing both anterograde and retrograde transport fractions. These results confirm that limiting delta-2 tubulin formation preserves mitochondrial dynamics even under chemotherapeutic stress.

[0104] Data in Figs. 4A and 4C are derived from at least 6-12 microscopic fields per condition (n = 4 experiments), while data in Figs. 4B and 4D is from 9-19 fields across multiple independent experiments. Quantitative results are presented as medians with interquartile ranges and analyzed using the Mann-Whitney U test. Statistical significance is denoted as *p < 0.05, p < 0.01, *p < 0.001, and p < 0.0001*.

[0105] Collectively, the data in Figs. 4A-4D further confirm that delta-2 tubulin accumulation is a causative driver of both axonal degeneration and mitochondrial transport failure. Conversely, inhibition of delta-2 tubulin synthesis through CCP1 (or CCP6) silencing mitigates these effects, directly linking the detyrosination / delta-2 tubulin pathway to neuronal survival and mitochondrial homeostasis. This mechanistic relationship defines delta-2 tubulin as a therapeutic target for preventing or reversing chemotherapy-induced peripheral neuropathy.

[0106] Figs. 5A-5C present behavioral data of TTLFL / FLmice injected retro-orbitally at 5 weeks of age with AAV php.s-Syn-EGFP-Cre (to silence TTL and promote delta-2 tubulin formation) or AAV php.s-Syn-EGFP (control). These data show that depletion of TTL results in accumulation of both detyrosinated tubulin and delta-2 (D2) tubulin and produces measurable alterations in sensory function in mice. TTL-depleted mice exhibit changes in mechanical and thermal responsiveness, supporting the conclusion that increased endogenous levels of detyrosinated / D2 tubulin are detrimental to mechanosensory processing. Three weeks post-injection, when TTL knockdown and delta-2 tubulin Docket No:070050.6974_CU18162

[0107] PCT Application

[0108] accumulation are established, Cre-injected mice exhibited mild mechanical hypersensitivity (slightly elevated responses to 0.16-1 g von Frey filaments), along with prolonged withdrawal latencies to thermal and cold stimuli, indicating heat and cold hyposensitivity. These findings demonstrate that delta-2 tubulin accumulation alone can modulate sensory thresholds, consistent with disrupted mitochondrial and TRP channel function in DRG neurons.

[0109] Figs. 6A-6E presents a model of bortezomib-induced peripheral neuropathy (BTZ-PN). Fig. 6A shows that C57BL / 6J mice (n = 10 per group) treated with bortezomib (subcutaneous injections, twice weekly for six weeks) develop significant and sustained mechanical hypersensitivity, evidenced by increased withdrawal responses to von Frey fibers over seven weeks (p < 0.05, ***p < 0.0001, two-way repeated measures ANOVA). Fig. 6B illustrates increased hindlimb clasping behavior, an indicator of sensorimotor impairment, in BTZ-treated mice relative to vehicle controls (Mann-Whitney U test). Figs.

[0110] 6C-6E depict a significant reduction in IENF density in skin biopsies from BTZ-treated mice, confirming peripheral axonal degeneration at the histological level.

[0111] Figs. 7A-7B show exemplary data to establish a Paclitaxel-IPN preclinical model. Fig. 7A depicts the example images for illustrating adeno-associated virus (AAV)-mediated gene delivery to DRG in a subject, e.g., adult mice, coupled with a preclinical model of paclitaxel (PTX)-induced peripheral neuropathy. In certain embodiments, seven-week-old C57BL / 6J mice receive retro-orbital injections of AAV:PHP.s.tdTomato (l><1012genome copies per animal) and are sacrificed three weeks later. DRG tissues are sectioned (20 pm) and processed for immunohistochemistry (IHC) using antibodies against endogenous pill-tubulin and the reporter tdTomato. The resulting composite image (Fig. 5A) shows efficient in vivo transduction of DRG neurons, with approximately 80% expression efficiency, confirming that the viral system provides a robust tool for targeted genetic manipulation of sensory neurons.

[0112] Fig. 7B (including panels 1-5) provides the neuropathic data in paclitaxel-treated animals. A significant reduction in intraepidermal nerve fiber (IENF) density is displayed (panels 1-2), consistent with peripheral axonopathy. Behavioral data shows a progressive mechanical hypersensitivity to dynamic (brush) and punctate (von Frey) mechanical stimuli (panels 3-5), reflecting tactile allodynia and mechanical hyperalgesia. Statistical analyses confirmed significant differences between paclitaxel-treated and control groups (p < 0.05 to **p < 0.001, by unpaired t-test, Mann-Whitney test, or two-way ANOVA as appropriate). Docket No:070050.6974_CU18162

[0113] PCT Application

[0114] Figs. 8A-8I illustrate a Vincristine-Induced Peripheral Neuropathy Model, wherein the peripheral neuropathy is illustrated via C57BL / 6J mice treated with vincristine (VCR), a microtubule-targeting chemotherapeutic agent known to cause sensory and motor deficits.

[0115] Fig. 8A shows that mice receiving VCR (0.75 mg / kg, intraperitoneally, twice weekly for four weeks) exhibit significantly increased paw withdrawals to von Frey filament stimulation, indicative of mechanical allodynia, which persisted up to five weeks after treatment initiation. Figs. 8B and 8C depict behavioral responses to non-painful (brush) and painful (pinprick) mechanical stimuli. VCR-treated mice show significant withdrawal and grooming responses, confirming the induction of mechanical hypersensitivity and pain sensitization (p < 0.01). Fig. 8D demonstrates thermal hypersensitivity, as VCR-treated mice exhibited reduced latency to cold stimuli compared to vehicle controls. Figs. 8E and 8F reveal motor impairments, with decreased locomotor activity, reduced movement area, and slower beam-walking velocity in treated mice. Figs. 6G-6I show confirmation of axonal degeneration, with significantly decreased IENF density in skin biopsies of VCR-treated animals (scale bar = 100 pm; p < 0.05, *p < 0.01, ***p < 0.0001), compared to controlled samples.

[0116] Figs. 9A-9C show preliminary evidence that cisplatin treatment induces delta-2 tubulin accumulation in DRG neurons, providing a rationale to establish a Cisplatin-IPN preclinical model. Fig. 9A shows representative images of DRG neurons (12DIV) treated with cisplatin (5 or 10 pM) for varying durations, revealing progressive axonal fragmentation in a time- and dose-dependent manner (scale bar = 50 pm). Fig. 9B quantifies this degeneration using an axon degeneration index, confirming significant structural loss with higher doses and prolonged exposure (p < 0.01, *p < 0.001, one-way ANOVA). Fig.

[0117] 9C presents immunoblot (IB) analysis showing increased delta-2 tubulin protein levels following cisplatin treatment, with GAPDH as a loading control.

[0118] Collectively, it can be confirmed that delta-2 tubulin accumulation disrupts neuronal function and integrity both under genetic manipulation (TTL loss) and chemotherapeutic stress (cisplatin exposure), reinforcing delta-2 tubulin as a central mediator and therapeutic target in sensory neuropathy. As noted above, TTL silencing does not fully recapitulate the effects of delta-2 tubulin overexpression on TRP channel activity. This discrepancy can result from at least two factors: (1) TTL silencing increases both detyrosinated tubulin and delta-2 tubulin, rather than selectively elevating D2 alone; and (2) the levels of delta-2 tubulin generated endogenously by TTL silencing is lower than the delta-2 tubulin levels achieved through ectopic overexpression. Therefore, the disclosed subject matter further Docket No:070050.6974_CU18162

[0119] PCT Application

[0120] provides to segregate TTL silencing from delta-2 tubulin accumulation alone by comparing the effects of TTL on live-cell Ca2+imaging in the presence of TRP channel agonists.

[0121] Fig. 10 shows a plot for Cisplatin-Induced Peripheral Neuropathy Model and behavioral data from the pilot evaluating cisplatin-induced peripheral neuropathy in C57BL / 6J mice (n = 10 per group). Mice are treated intraperitoneally with cisplatin (twice weekly for four weeks) and assessed for mechanical sensitivity. As shown in Fig. 10, the Cisplatin-treated animals display significantly increased paw withdrawal responses to von Frey stimulation compared with vehicle-treated controls (p < 0.05, ***p < 0.0001, two-way repeated measures ANOVA). The onset of hypersensitivity can be observed as early as week two and persisted throughout the observation period, consistent with chronic sensory dysfunction.

[0122] Together, the above disclosure demonstrates that distinct chemotherapeutic agents can induce comparable sensory and structural neuropathic phenotypes characterized by IENF loss, hypersensitivity, and motor deficits, and that delta-2 tubulin accumulation serves as a unifying molecular signature of CIPN across drug classes.

[0123] With reference to Figs. 11 A-l 1G, Cre-mediated excision of CCP1 / 6 reduces delta-2 tubulin levels in vivo without producing detectable effects on sensory or motor function in untreated mice. These findings provide proof of principle that in vivo depletion of delta-2 tubulin is biologically well-tolerated and has therapeutic potential for preventing or mitigating chemotherapy-induced peripheral neuropathy.

[0124] Figs. 11A and 11B show immunofluorescence (IF) data of sciatic nerve (SN) axons and DRG neurons from CCPl / 6flx / flxmale mice injected retro-orbitally at five weeks of age with either a control virus (AAVphp.s-hSyn-GFP) or a Cre-expressing virus (AAVphp.s-hSyn-Cre-2A-GFP, 1 x io12genome copies per mouse). Tissue samples collected at different time points post-injection reveal a marked reduction in delta-2 tubulin immunoreactivity in GFP -positive neurons and axons following Cre-mediated excision of CCP 1 / 6, relative to control virus-treated mice. Quantitative data from 29 sciatic nerve axons and 60 DRG cell bodies from two animals per time point can confirm significant delta-2 tubulin reduction.

[0125] Fig. 11C presents immunoblot data from whole sciatic nerve lysates, demonstrating a time-dependent decline in delta-2 tubulin protein levels after AAV-Cre administration. In contrast, polyglutamylated tubulin (polyGlu) levels remained substantially unchanged, indicating that selective deletion of CCP 1 / 6 specifically reduces delta-2 tubulin formation without broadly disrupting other post-translational tubulin modifications. Fig. 1 ID shows Docket No:070050.6974_CU18162

[0126] PCT Application

[0127] behavioral testing results for mechanical sensitivity. CCPl / 6flx / flxmice injected with the Cre virus or control virus are monitored for paw withdrawal responses to von Frey filament stimulation. Across four weeks post-injection, Cre-injected mice showed no increase in withdrawal frequency, indicating absence of mechanical hypersensitivity (two-way repeated measures ANOVA, / ? > 0.05).

[0128] Figs. 1 IE-11G show quantitative data of motor behavior using open-field and beamwalking assays. No significant changes are observed in locomotor velocity, overall mobility (expressed as average percentage of movement area), or meander scores (a measure of gait stability) between Cre- and control -injected groups, confirming that loss of CCP 1 / 6 activity does not impair general motor coordination or sensorimotor performance (unpaired / -test, / ? > 0.05).

[0129] Therefore, the disclosed subject matter demonstrates that a targeted excision of CCP 1 and / or CCP 6 in sensory neurons selectively suppresses delta-2 tubulin accumulation without altering other tubulin modifications or baseline sensory and motor behaviors. These results validate an inhibition of CCP 1 / 6 as a safe and effective strategy to prevent or reverse delta-2 tubulin-mediated axonal pathology and highlight the translational potential of genetic or pharmacologic modulators of the tubulin detyrosination pathway as neuroprotective therapies.

[0130] Figs. 12A-12E demonstrate that in vivo excision of D2 tubulin through AAV-mediated deletion of CCP 1 / 6 ameliorates bortezomib-induced peripheral neuropathy (BIPN), improving mechanosensory responses and preserving intraepidermal nerve fiber (IENF) density in treated mice.

[0131] Fig. 12A outlines an example design. Five-week-old CCPl / 6floxmice are injected systemically with AAV-PHP.S vectors encoding either EGFP (control) or EGFP-Cre (example) under the human synapsin-1 promoter. Two weeks later, both groups received systemic bortezomib (twice weekly for eight weeks), which can induce delta-2 tubulin accumulation as discussed above. Behavioral testing using the von Frey assay is conducted weekly throughout treatment. Fig. 12B shows the time course of 50% paw withdrawal thresholds, where AAV-EGFP-injected mice (control) develop progressive mechanical hypersensitivity, indicative of neuropathic pain. In contrast, AAV-EGFP-Cre-injected mice, in which CCP 1 / 6 are selectively deleted in DRG neurons, maintained significantly higher withdrawal thresholds, demonstrating protection from bortezomib-induced allodynia (p < 0.05, two-way ANOVA). Docket No:070050.6974_CU18162

[0132] PCT Application

[0133] Fig. 12C presents Western blot validation, confirming reduced CCP1 protein levels in DRG tissue from AAV-Cre-injected mice relative to GFP controls at the endpoint of Bort-treatment. Figs. 12D and 12E display representative immunofluorescence images (Scale bar = 100 pm) and quantification of intraepidermal nerve fibers (lENFs) stained for PGP9.5. AAV-Cre-treated mice retain significantly more PGP9.5 and positive fibers than controls, indicating preservation of sensory axons and small fibers.

[0134] Figs. 13A-13B confirm that the delta-2 tubulin overexpression alone impairs mitochondrial respiration and alters TRP channel-mediated calcium signaling in primary DRG neurons. Fig. 13 A presents a quantitative assessment of mitochondrial respiratory function in cultured DRG neurons transduced at 1 day in vitro (1DIV) with lentiviral vectors encoding either wild-type (WT) a-tubulin (pLVX-EGFP-a-tub) or delta-2 tubulin (pLVX-EGFP-A2). After 5 days in vitro (5DIV), oxygen consumption rate (OCR), as a direct indicator of oxidative phosphorylation, is determined using a Seahorse metabolic assay. The results show a significant reduction in basal and maximal mitochondrial respiration in delta-2 tubulin-overexpressing neurons compared to WT controls (p < 0.05, one-way ANOVA, non-parametric test, data normalized to total protein; n = 5 independent experiments). This finding indicates that delta-2 tubulin accumulation compromises mitochondrial efficiency and energy output, consistent with the observed axonal degeneration and motility defects described in earlier Figures. Mechanistically, the impairment of mitochondrial respiration shown in Fig. 12A suggests that delta-2 tubulin disrupts interactions between microtubules and mitochondria, likely through altered anchoring dynamics mediated by syntaphilin and impaired transport along axonal tracks. The resulting decline in ATP availability reduces neuronal resilience to chemotherapeutic stress and contributes to progressive sensory dysfunction.

[0135] Fig. 13B displays the impact of delta-2 tubulin on TRP channel-mediated calcium responses, using Calbryte 590 fluorescence imaging to monitor cytosolic calcium levels (AF / F). DRG neurons (5DIV) expressing WT a-tubulin or delta-2 tubulin are exposed sequentially to GSK1016790A (a TRPV4 agonist), capsaicin (a TRPV1 agonist), and high-potassium depolarization, representing distinct modes of sensory activation. Traces are analyzed, with baseline fluorescence (Fo) defined as the mean intensity over the first 10 seconds prior to agonist application, and responses are classified as positive when the signal increased by >50% relative to baseline. In certain embodiments employing 111 neurons, A2 -tubulin-overexpressing cells exhibit significantly reduced Ca2+response amplitudes to both TRPV4 and TRPV1 agonists (p < 0.01, two-way ANOVA with Sidak’s multiple Docket No:070050.6974_CU18162

[0136] PCT Application

[0137] comparison test), while responses to high-potassium depolarization remained intact. This selective attenuation of TRP-mediated calcium signaling demonstrates that delta-2 tubulin accumulation specifically impairs sensory transduction pathways rather than general membrane excitability. Collectively, these embodiments provide direct mechanistic evidence that delta-2 tubulin accumulation disrupts mitochondrial energy metabolism and sensory receptor signaling, two critical processes for neuronal homeostasis and function. The results underscore the pathogenic cascade whereby A2-tubulin, through microtubule remodeling, alters organelle bioenergetics and ion channel function — culminating in neuronal vulnerability, metabolic stress, and neuropathic dysfunction.

[0138] In certain embodiments, the disclosed subject matter discloses the differential regulation of syntaphilin (SNPH), a mitochondrial anchoring protein, by distinct classes of chemotherapy drugs. Figs. 13A, 13B, and 13C depict representative SNPH immunostaining in cultured DRG neurons following exposure to bortezomib and oxaliplatin, comparatively, and demonstrate that bortezomib -treated neurons exhibit elevated SNPH fluorescence intensity and total protein levels, consistent with increased mitochondrial anchoring. In contrast, oxaliplatin-treated neurons display diminished SNPH signal, correlating with enhanced mitochondrial motility.

[0139] Figs. 14A-14I provide the data illustrating that depletion of tubulin tyrosine ligase (TTL) to elevate delta-2 tubulin levels in cultured DRG neurons alters TRP channel activity and demonstrates that endogenous increases in detyrosinated and delta-2 tubulin resulting from TTL excision modify TRP channel function in primary DRG neurons. In such embodiment, DRGs isolated from adult C57BL / 6J mice are cultured and transduced with two separate lentiviral constructs carrying short hairpin RNAs (shRNAs) targeting TTL. Following five days of infection, neurons were loaded with the calcium indicator Fura-2, and calcium activity was recorded in response to agonists selective for TRPV4 (1 pM GSK1016790A), TRPM8 (500 pM menthol), and TRPV1 (2 pM capsaicin). High potassium depolarization can be used as a control to identify viable neurons.

[0140] Figs. 14A-14C present representative Ca2+response traces from control neurons (A, N=31) and TTL depleted neurons infected with two independent shRNA viruses (B, N=84; C, N=81). Figs. 14D-14F quantify the proportion of neurons responding to each TRP channel agonist. TTL depletion significantly increases the fraction of neurons responsive to TRPV4 and TRPM8 agonists and enhanced the proportion of polymodal neurons, which respond to multiple agonists simultaneously, at the expense of neurons responding solely to TRPV1 or none of the agonists. Docket No:070050.6974_CU18162

[0141] PCT Application

[0142] Figs. 14G-14I present data summarizing the area under the curve (AUC) for calcium responses to each agonist. Compared to control, TTL-depleted neurons exhibit an increased response amplitude to the TRPV4 agonist GSK1016790A (Fig. 14G), whereas capsaicin-induced TRPV1 responses remain unchanged (Fig. 14H). Notably, TTL depletion can induce a stronger calcium response to high potassium depolarization (Fig. 141), suggesting heightened overall neuronal excitability. Statistical analysis is performed using the Kruskal-Wallis test.

[0143] These results presented as Figs. 14A-14I demonstrate that reduced TTL expression, which increases delta-2 tubulin accumulation, alters sensory neuron responsiveness by dysregulating TRP channel signaling. The enhanced activation of TRPV4 and TRPM8 channels and the expansion of polymodal neuronal populations indicate that delta-2 tubulin accumulation modulates ion channel sensitivity and neuronal excitability, potentially contributing to sensory abnormalities observed in CIPN. Together with previous data showing mitochondrial dysfunction and axonal degeneration under delta-2 tubulin accumulating conditions, these data support a model in which TTL depletion and subsequent delta-2 tubulin accumulation disrupt both metabolic and electrophysiological homeostasis in DRG neurons, linking cytoskeletal dysregulation to altered pain and sensory signaling pathways.

[0144] Mechanistically, the observed alteration in TRP channel function upon TTL depletion suggests that delta-2 tubulin accumulation sensitizes sensory neurons by disrupting microtubule-membrane signaling interfaces. The excessive delta-2 tubulin modification alters cytoskeletal organization near the plasma membrane, where TRP VI, TRPV4, and TRPM8 channels reside, leading to aberrant calcium influx and neuronal hyperexcitability. This dysregulation enhances pain transduction and contributes to the mechanical and thermal hypersensitivity characteristic of CIPN. Thus, maintaining proper TTL activity and preventing delta-2 tubulin buildup are critical for preserving normal TRP channel function and neuronal sensory balance.

[0145] Thus, the disclosed subject matter demonstrates that a suppression of delta-2 tubulin formation through CCP1 / 6 deletion confers both behavioral and structural neuroprotection against bortezomib-induced damage. These data confirm that delta-2 tubulin-tubulin accumulation is a primary pathogenic driver of CIPN and that targeting enzymes in the tubulin detyrosination pathway (e.g., CCP1 / 6 inhibition or TTL activation) represents a viable therapeutic approach to prevent or treat chemotherapy-induced neuropathy. Docket No:070050.6974_CU18162

[0146] PCT Application

[0147] In certain embodiments, the disclosed subject matter provides a method 1600 for treating or preventing CIPN in a subject receiving a neurotoxic anti-cancer agent, as illustrated in Fig. 16, comprising obtaining a first biological sample from a subject prior to administration of the anti-cancer agent at 1601, obtaining a second biological sample from the subject after administration of the anti-cancer agent at 1602, determining an expression level of delta-2 tubulin at 1603 in the first biological sample and the second biological sample, comparing the delta-2 tubulin expression level in the first biological sample to the determined delta-2 tubulin expression level in the second biological sample to further determine whether the delta-2 tubulin expression level has increased at 1604, and upon the increase of delta-2 tubulin, a composition is administered to the subject for pharmaceutical purposes. The pharmaceutical composition is configured to reduce delta-2 tubulin accumulation, thereby treating or preventing CIPN, as discussed above.

[0148] The biological sample used for delta-2 tubulin determination can include a tissue sample, biopsy, or cell preparation obtained from the DRG, peripheral nerve, or other sensory neuron-enriched region, delta-2 tubulin expression levels can be measured using immunofluorescence (IF), immunohistochemistry (IHC), Western blot (immunoblotting), enzyme-linked immunosorbent assay (ELISA), or quantitative mass spectrometry. An increase in the delta-2 tubulin expression can be defined relative to baseline by a predetermined threshold (e.g., a 1.2-fold or greater increase) or by statistical significance using paired analysis between pre- and post-treatment samples.

[0149] When an elevation of delta-2 tubulin is observed, a pharmaceutical composition targeting the enzymatic regulators of delta-2 tubulin formation or degradation is administered to the subject. In certain embodiments, the composition includes an inhibitor of cytosolic carboxypeptidases (CCPs), particularly CCP1 or CCP6. The inhibitor can comprise an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an adeno-associated virus (AAV) vector encoding a Cre recombinase or shRNA sequence that reduces expression of CCP1 or CCP6.

[0150] In certain embodiments, the pharmaceutical composition can include an agent that enhances re-tyrosination of a-tubulin through upregulation of tubulin tyrosine ligase (TTL). TTL activators or TTL gene delivery vectors can restore the reversible tyrosination cycle, thereby counteracting irreversible delta-2 tubulin accumulation. Exemplary TTL-activating compositions can include small molecules, nucleic acids encoding TTL, or adeno-associated virus (AAV) vectors expressing TTL under neuron-specific promoters. Docket No:070050.6974_CU18162

[0151] PCT Application

[0152] In further embodiments, the composition comprises an agent that enhances mitochondrial bioenergetics, such as NAD+precursors (nicotinamide riboside, nicotinamide mononucleotide), coenzyme Q10, or ATP-generating enhancers that restore oxidative phosphorylation and membrane potential disrupted by delta-2 tubulin accumulation. In some embodiments, the pharmaceutical composition includes an agent that inhibits syntaphilin (SNPH) expression or activity, thereby increasing mitochondrial motility and promoting redistribution of mitochondria along neuronal axons. Such SNPH inhibitors can be antisense oligonucleotides, shRNAs, or small-molecule antagonists that disrupt SNPH-mitochondria binding. In additional embodiments, the pharmaceutical composition includes a transient receptor potential (TRP) channel agonist, such as capsaicin (a TRPV1 agonist) or GSK1016790A (a TRPV4 agonist), to normalize calcium signaling and improve sensory neuron function.

[0153] In certain embodiments, the pharmaceutical composition is delivered via a viral vector, such as an AAV carrying a php.S capsid and a neuron-specific promoter (e.g., Synapsin or hSyn). The vector can encode Cre recombinase for conditional deletion of CCP1 or CCP6 alleles, or express TTL or SNPH-targeting shRNAs.

[0154] The chemotherapeutic agent inducing delta-2 tubulin accumulation can be selected from bortezomib, paclitaxel, vincristine, cisplatin, carboplatin, eribulin, thalidomide, or other microtubule- or proteasome-targeting drugs known to induce peripheral neuropathy. The method can be further implemented prophylactically by monitoring delta-2 tubulin prior to the onset of symptoms and initiating treatment upon detecting an early increase in delta-2 tubulin, thereby preventing irreversible axonal degeneration. In certain embodiments, delta-2 tubulin levels are periodically monitored after therapy initiation, and the dosage or frequency of the pharmaceutical composition is adjusted according to delta-2 tubulin response kinetics.

[0155] The compositions disclosed herein can be administered by systemic routes (intravenous, intraperitoneal, or oral) or local routes (intrathecal, epidural, or direct ganglionic injection). Dosage regimens can be determined based on delta-2 tubulin biomarker monitoring, body weight, and the chemotherapeutic regimen employed.

[0156] In another aspect, the disclosed subject matter provides pharmaceutical compositions comprising a therapeutically effective amount of a CCP1 or CCP6 inhibitor, optionally in combination with a TTL activator or mitochondrial enhancer, and a pharmaceutically acceptable carrier or excipient. The compositions can be formulated for systemic delivery (e.g., intravenous, oral) or for localized neuronal administration (e.g., intrathecal or Docket No:070050.6974_CU18162

[0157] PCT Application

[0158] perineural injection). The CCP inhibitor can be provided as a small molecule or genesilencing construct that reduces enzymatic conversion of detyrosinated a-tubulin to delta-2 tubulin. The TTL activator component can include small molecules or nucleic acid-based vectors that increase TTL expression. In some embodiments, mitochondrial enhancers, such as coenzyme Q10 or NAD+precursors, are included to restore oxidative capacity.

[0159] Suitable pharmaceutically acceptable carriers include saline, phosphate-buffered saline, mannitol, glycerol, trehalose, or liposomal formulations. For gene therapy compositions, stabilizing excipients such as pluronic surfactants or polysorbate can be included to maintain viral particle integrity.

[0160] The pharmaceutical compositions disclosed herein can be administered alone or in combination with chemotherapeutic agents, either sequentially or concurrently, to reduce or prevent chemotherapy-induced peripheral neuropathy by mitigating delta-2 tubulin accumulation and preserving neuronal mitochondrial function.

[0161] It will be understood that the foregoing is only illustrative of the principles described herein, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosed subject matter. For example, the system and methods described herein are used for monitoring delta-2 tubulin level of a subject to reduce CIPN. It is understood that that techniques described herein are useful for other peripheral neuropathies. Further, the techniques described have been performed on mice in certain embodiments, but it is understood the techniques are applicable to other subjects, such as humans. Moreover, the features in embodiments described herein can be combined and / or rearranged to create new embodiments. For instance, the type of microtubules, the expression and regulation same thereof, are adjustable and expandable within the knowledge of person skilled in the art.

[0162] Exemplary Embodiments

[0163] The following embodiments are provided to illustrate, but not limit, the scope of the disclosed subject matter. It will be understood that variations and modifications can be made without departing from the spirit or scope of the disclosed subject matter as defined by the exemplary embodiments.

[0164] Embodiment 1.

[0165] A method for treating or preventing chemotherapy-induced peripheral neuropathy (CIPN) in a subject receiving an anti-cancer agent, the method comprising: obtaining a first biological sample from the subject prior to administration of the anti-cancer agent; obtaining Docket No:070050.6974_CU18162

[0166] PCT Application

[0167] a second biological sample from the subject after administration of the anti-cancer agent; determining an expression level of delta-2 tubulin in the first and second biological samples; comparing the determined delta-2 tubulin expression levels to determine whether the delta-2 tubulin expression level has increased; and when such increase is determined, administering to the subject a therapeutically effective amount of a pharmaceutical composition configured to reduce delta-2 tubulin accumulation, thereby treating or preventing CIPN.

[0168] Embodiment 2.

[0169] The method of Embodiment 1, wherein the biological sample comprises dorsal root ganglion (DRG) tissue or cells.

[0170] Embodiment 3.

[0171] The method of any of the preceding embodiments, wherein determining the expression level of delta-2 tubulin comprises detecting delta-2 tubulin by immunofluorescence, immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry.

[0172] Embodiment 4.

[0173] The method of any of the preceding embodiments, wherein an increase in delta-2 tubulin expression is determined by comparing the second biological sample to the first biological sample and determining that the delta-2 tubulin level in the second biological sample is higher by a predetermined threshold amount.

[0174] Embodiment 5.

[0175] The method of any of the preceding embodiments, wherein the pharmaceutical composition comprises an agent configured to reduce formation of delta-2 tubulin by inhibiting one or more cytosolic carboxypeptidases (CCPs), wherein the CCP is selected from CCP1 and CCP6.

[0176] Embodiment 6.

[0177] The method of Embodiment 5, wherein the agent comprises an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an adeno-associated virus (AAV) vector encoding a Cre recombinase or shRNA sequence that reduces expression of CCP1 or CCP6.

[0178] Embodiment 7.

[0179] The method of any of the preceding embodiments, wherein the pharmaceutical composition comprises an agent configured to promote re-tyrosination of a-tubulin by increasing expression or activity of tubulin tyrosine ligase (TTL), wherein the agent Docket No:070050.6974_CU18162

[0180] PCT Application

[0181] comprises a small molecule, a polynucleotide encoding TTL, or a viral vector expressing TTL.

[0182] Embodiment 8.

[0183] The method of any of the preceding embodiments, wherein the pharmaceutical composition comprises an agent that upregulates mitochondrial respiration, wherein the agent comprises mitochondrial electron transport chain enhancers, NAD+precursors, ATP-generating enhancers, or compounds that restore mitochondrial membrane potential.

[0184] Embodiment 9.

[0185] The method of any of the preceding embodiments, wherein the pharmaceutical composition comprises an agent that increases mitochondrial motility by downregulating or inhibiting syntaphilin (SNPH).

[0186] Embodiment 10.

[0187] The method of any of the preceding embodiments, wherein the agent comprises a small molecule, antisense oligonucleotide, shRNA, or polypeptide antagonist of SNPH.

[0188] Embodiment 11.

[0189] The method of any of the preceding embodiments, wherein administering the pharmaceutical composition comprises delivering a viral vector to DRG neurons, wherein the viral vector comprises an adeno-associated virus (AAV).

[0190] Embodiment 12.

[0191] The method of Embodiment 11, wherein the AAV comprises an AAV-PHP.S capsid. Embodiment 13.

[0192] The method of any of the preceding embodiments, wherein the anti-cancer agent is selected from the group consisting of bortezomib, paclitaxel, vincristine, cisplatin, carboplatin, eribulin, thalidomide, and taxane.

[0193] Embodiment 14.

[0194] The method of any of the preceding embodiments, wherein the pharmaceutical composition is administered prophylactically prior to onset of CIPN when an increase in delta-2 tubulin is detected.

[0195] Embodiment 15.

[0196] The method of any of the preceding embodiments, further comprising monitoring delta-2 tubulin levels after administration of the pharmaceutical composition and adjusting a dosing regimen of the pharmaceutical composition based on the monitored delta-2 tubulin levels.

[0197] Embodiment 16. Docket No:070050.6974_CU18162

[0198] PCT Application

[0199] The method of any of the preceding embodiments, wherein the pharmaceutical composition is formulated for systemic administration, local intrathecal administration, or direct ganglionic delivery.

[0200] Embodiment 17.

[0201] A pharmaceutical composition for treating or preventing chemotherapy-induced peripheral neuropathy comprising a therapeutically effective amount of an inhibitor of CCP1 or CCP6 and a pharmaceutically acceptable carrier, wherein the composition is formulated for systemic or neuronal delivery, and wherein the inhibitor of CCP1 or CCP6 is configured to reduce formation of delta-2 tubulin in dorsal root ganglion neurons.

[0202] Embodiment 18.

[0203] The pharmaceutical composition of Embodiment 17, further comprising a tubulin tyrosine ligase (TTL) activator.

[0204] Embodiment 19.

[0205] The pharmaceutical composition of Embodiment 17, further comprising a mitochondrial enhancer.

[0206] Embodiment 20.

[0207] The pharmaceutical composition of any of Embodiments 17-19, wherein the composition is formulated for systemic or intrathecal administration and includes a pharmaceutically acceptable carrier or excipient.

[0208] EXAMPLES

[0209] Example 1

[0210] Figs. 13A-13B confirm that overexpression of delta-2 tubulin in DRG neurons disrupts both mitochondrial bioenergetic capacity and transient receptor potential (TRP) channel-mediated calcium signaling. Fig. 13 A presents a quantitative assessment of mitochondrial respiratory function in cultured DRG neurons transduced at 1 day in vitro (1DIV) with lentiviral vectors encoding either wild-type (WT) a-tubulin (pLVX-EGFP-a-tub) or delta-2 tubulin (pLVX-EGFP-A2). After 5 days in vitro (5DIV), oxygen consumption rate (OCR), as a direct indicator of oxidative phosphorylation, is determined using a Seahorse metabolic assay. The results show a significant reduction in basal and maximal mitochondrial respiration in delta-2 tubulin-overexpressing neurons compared to WT controls (p < 0.05, one-way ANOVA, non-parametric test, data normalized to total protein; n = 5 independent experiments). This finding indicates that delta-2 tubulin accumulation compromises mitochondrial efficiency and energy output, consistent with the Docket No:070050.6974_CU18162

[0211] PCT Application

[0212] observed axonal degeneration and motility defects described in earlier Figures. Mechanistically, the impairment of mitochondrial respiration shown in Fig. 13 A suggests that delta-2 tubulin disrupts interactions between microtubules and mitochondria, likely through altered anchoring dynamics mediated by syntaphilin and impaired transport along axonal tracks. The resulting decline in ATP availability reduces neuronal resilience to chemotherapeutic stress and contributes to progressive sensory dysfunction.

[0213] Fig. 13B displays the impact of delta-2 tubulin on TRP channel-mediated calcium responses, using Calbryte 590 fluorescence imaging to monitor cytosolic calcium levels (AF / F). DRG neurons (5DIV) expressing WT a-tubulin or delta-2 tubulin are exposed sequentially to GSK1016790A (a TRPV4 agonist), capsaicin (a TRPV1 agonist), and high-potassium depolarization, representing distinct modes of sensory activation. Traces are analyzed, with baseline fluorescence (Fo) defined as the mean intensity over the first 10 seconds prior to agonist application, and responses are classified as positive when the signal increased by >50% relative to baseline. In certain embodiments employing 111 neurons, A2-tubulin-overexpressing cells exhibit significantly reduced Ca2+response amplitudes to both TRPV4 and TRPV1 agonists (p < 0.01, two-way ANOVA with Sidak’s multiple comparison test), while responses to high-potassium depolarization remained intact. This selective attenuation of TRP-mediated calcium signaling demonstrates that delta-2 tubulin accumulation specifically impairs sensory transduction pathways rather than general membrane excitability. Collectively, these embodiments provide direct mechanistic evidence that delta-2 tubulin accumulation disrupts mitochondrial energy metabolism and sensory receptor signaling, two critical processes for neuronal homeostasis and function. The results underscore the pathogenic cascade whereby A2-tubulin, through microtubule remodeling, alters organelle bioenergetics and ion channel function — culminating in neuronal vulnerability, metabolic stress, and neuropathic dysfunction.

[0214] Example 2

[0215] Syntaphilin (SNPH), a mitochondrial-anchoring protein that restricts organelle mobility along axons, is regulated by distinct classes of chemotherapy drugs. In this example, the disclosed subject matter recognizes a mechanistic link between delta-2 tubulin accumulation and impaired mitochondrial transport in sensory neurons. As shown in the experimental data, both bortezomib treatment (which induces endogenous generation of delta-2 tubulin, shown in Fig. 15 A), and direct overexpression of delta-2 tubulin in cultured DRG neurons (shown Figs. 15B-15C) lead to increased levels of syntaphilin (SNPH),. The observed elevation of SNPH under both conditions indicates that delta-2 tubulin-driven Docket No:070050.6974_CU18162

[0216] PCT Application

[0217] cytoskeletal alterations promote mitochondrial immobilization by enhancing SNPH expression, thereby providing a unifying mechanism through which chemotherapeutic exposure or forced delta-2 tubulin expression can inhibit mitochondrial transport and contribute to axonal dysfunction.

[0218] Example 3

[0219] Figs. 12A-12E provide in vivo confirmation that therapeutically reducing delta-2 tubulin formation via AAV-mediated excision of CCP1 / 6 in sensory neurons mitigates both behavioral and structural symptoms of bortezomib-induced peripheral neuropathy (BTZ-PN).

[0220] Fig. 12A outlines the example design. Five-week-old CCPl / 6floxmice are injected systemically with AAV-PHP.S vectors encoding either EGFP (control) or EGFP-Cre (example) under the human synapsin-1 promoter. Two weeks later, both groups received systemic bortezomib (twice weekly for eight weeks), which can induce delta-2 tubulin accumulation as discussed above. Behavioral testing using the von Frey assay is conducted weekly throughout treatment. Fig. 12B shows the time course of 50% paw withdrawal thresholds, where AAV-EGFP-injected mice (control) develop progressive mechanical hypersensitivity, indicative of neuropathic pain. In contrast, AAV-EGFP-Cre-injected mice, in which CCP1 / 6 are selectively deleted in DRG neurons, maintained significantly higher withdrawal thresholds, demonstrating protection from bortezomib-induced allodynia (p < 0.05, two-way ANOVA).

[0221] Fig. 12C presents Western blot validation, confirming reduced CCP1 protein levels in DRG tissue from AAV-Cre-injected mice relative to GFP controls at the endpoint of Bort-treatment. Figs. 12D and 12E display representative immunofluorescence images (Scale bar = 100 pm) and quantification of intraepidermal nerve fibers (lENFs) stained for PGP9.5. AAV-Cre-treated mice retain significantly more PGP9.5 and positive fibers than controls, indicating preservation of sensory axons and small fibers.

[0222] Thus, the disclosed subject matter demonstrates that a suppression of delta-2 tubulin formation through CCP1 / 6 deletion confers both behavioral and structural neuroprotection against bortezomib-induced damage. These data confirm that delta-2 tubulin-tubulin accumulation is a primary pathogenic driver of CIPN and that targeting enzymes in the tubulin detyrosination pathway (e.g., CCP1 / 6 inhibition or TTL activation) represents a viable therapeutic approach to prevent or treat chemotherapy-induced neuropathy.

[0223] Example 4 Docket No:070050.6974_CU18162

[0224] PCT Application

[0225] The method 1600 provides a treatment for preventing CIPN in a subject receiving a neurotoxic anti-cancer agent, as illustrated in Fig. 16. A first biological sample from the subject is obtained prior to administration of the anti-cancer agent at 1601. Following 1601, a second biological sample from the subject is obtained after administration of the anticancer agent at 1602. The first and second biological samples can be DRG tissue.

[0226] Then, an expression level of delta-2 tubulin is determined at 1603 in the first biological sample and the second biological sample. Upon the determination, the delta-2 tubulin expression level is compared in the first biological sample to the determined delta-2 tubulin expression level in the second biological sample to further determine whether the delta-2 tubulin expression level has increased at 1604, and upon the increase of delta-2 tubulin, a composition is administered to the subject for pharmaceutical purposes. The pharmaceutical composition is configured to reduce delta-2 tubulin accumulation, thereby treating or preventing CIPN, as discussed above.

[0227] The biological sample used for delta-2 tubulin determination is a tissue sample, biopsy, or cell preparation obtained from the DRG, peripheral nerve, or other sensory neuron-enriched region, delta-2 tubulin expression levels can be measured using immunofluorescence (IF), immunohistochemistry (IHC), Western blot (immunoblotting), enzyme-linked immunosorbent assay (ELISA), or quantitative mass spectrometry. An increase in the delta-2 tubulin expression can be defined relative to baseline by a predetermined threshold (e.g., a 1.2-fold or greater increase) or by statistical significance using paired analysis between pre- and post-treatment samples.

[0228] When an elevation of delta-2 tubulin is observed, a pharmaceutical composition targeting the enzymatic regulators of delta-2 tubulin formation or degradation is administered to the subject. In certain embodiments, the composition includes an inhibitor of cytosolic carboxypeptidases (CCPs), particularly CCP1 or CCP6. The inhibitor can comprise an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an adeno-associated virus (AAV) vector encoding a Cre recombinase or shRNA sequence that reduces expression of CCP1 or CCP6.

[0229] The pharmaceutical composition can include an agent that enhances re-tyrosination of a-tubulin through upregulation of tubulin tyrosine ligase (TTL). TTL activators or TTL gene delivery vectors can restore the reversible tyrosination cycle, thereby counteracting irreversible delta-2 tubulin accumulation. Exemplary TTL-activating compositions can include small molecules, nucleic acids encoding TTL, or adeno-associated virus (AAV) vectors expressing TTL under neuron-specific promoters. Docket No:070050.6974_CU18162

[0230] PCT Application

[0231] The composition comprises an agent that enhances mitochondrial bioenergetics, such asNAD+precursors (nicotinamide riboside, nicotinamide mononucleotide), coenzyme Q10, or ATP-generating enhancers that restore oxidative phosphorylation and membrane potential disrupted by delta-2 tubulin accumulation. The pharmaceutical composition can include an agent that inhibits syntaphilin (SNPH) expression or activity, thereby increasing mitochondrial motility and promoting redistribution of mitochondria along neuronal axons. Such SNPH inhibitors can be antisense oligonucleotides, shRNAs, or small-molecule antagonists that disrupt SNPH-mitochondria binding. In additional embodiments, the pharmaceutical composition includes a transient receptor potential (TRP) channel agonist, such as capsaicin (a TRPV1 agonist) or GSK1016790A (a TRPV4 agonist), to normalize calcium signaling and improve sensory neuron function.

[0232] The pharmaceutical composition can be delivered via a viral vector, such as an AAV carrying a php.S capsid and a neuron-specific promoter (e.g., Synapsin or hSyn). The vector can encode Cre recombinase for conditional deletion of CCP1 or CCP6 alleles, or express TTL or SNPH-targeting shRNAs.

[0233] The chemotherapeutic agent inducing delta-2 tubulin accumulation can be selected from bortezomib, paclitaxel, vincristine, cisplatin, carboplatin, eribulin, thalidomide, or other microtubule- or proteasome-targeting drugs known to induce peripheral neuropathy. The method can be further implemented prophylactically by monitoring delta-2 tubulin prior to the onset of symptoms and initiating treatment upon detecting an early increase in delta-2 tubulin, thereby preventing irreversible axonal degeneration. In certain embodiments, delta-2 tubulin levels are periodically monitored after therapy initiation, and the dosage or frequency of the pharmaceutical composition is adjusted according to delta-2 tubulin response kinetics.

[0234] The compositions disclosed herein can be administered by systemic routes (intravenous, intraperitoneal, or oral) or local routes (intrathecal, epidural, or direct ganglionic injection). Dosage regimens can be determined based on delta-2 tubulin biomarker monitoring, body weight, and the chemotherapeutic regimen employed.

[0235] A pharmaceutical composition comprising a therapeutically effective amount of a CCP1 or CCP6 inhibitor can also be used, optionally in combination with a TTL activator or mitochondrial enhancer, and a pharmaceutically acceptable carrier or excipient. The compositions can be formulated for systemic delivery (e.g., intravenous, oral) or for localized neuronal administration (e.g., intrathecal or perineural injection). The CCP inhibitor can be provided as a small molecule or gene-silencing construct that reduces Docket No:070050.6974_CU18162

[0236] PCT Application

[0237] enzymatic conversion of detyrosinated a-tubulin to delta-2 tubulin. The TTL activator component can include small molecules or nucleic acid-based vectors that increase TTL expression. In some embodiments, mitochondrial enhancers, such as coenzyme Q10 or NAD+precursors, are included to restore oxidative capacity.

[0238] Suitable pharmaceutically acceptable carriers include saline, phosphate-buffered saline, mannitol, glycerol, trehalose, or liposomal formulations. For gene therapy compositions, stabilizing excipients such as pluronic surfactants or polysorbate can be included to maintain viral particle integrity.

[0239] The pharmaceutical compositions disclosed herein can be administered alone or in combination with chemotherapeutic agents, either sequentially or concurrently, to reduce or prevent chemotherapy-induced peripheral neuropathy by mitigating delta-2 tubulin accumulation and preserving neuronal mitochondrial function.

[0240] It will be understood that the foregoing is only illustrative of the principles described herein, and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosed subject matter. For example, the system and methods described herein are used for monitoring delta-2 tubulin level of a subject to reduce CIPN. It is understood that that techniques described herein are useful for other peripheral neuropathies. Further, the techniques described have been performed on mice in certain embodiments, but it is understood the techniques are applicable to other subjects, such as humans. Moreover, the features in embodiments described herein can be combined and / or rearranged to create new embodiments. For instance, the type of microtubules, the expression and regulation same thereof, are adjustable and expandable within the knowledge of person skilled in the art.

Claims

Docket No:070050.6974_CU18162PCT ApplicationWhat is claimed is,1. A method for treating or preventing chemotherapy-induced peripheral neuropathy (CIPN) in a subject receiving an anti-cancer agent, the method comprising: obtaining a first biological sample from the subject prior to administration of the anti-cancer agent;obtaining a second biological sample from the subject after administration of the anti-cancer agent;determining an expression level of delta-2 tubulin in the first biological sample and the second biological sample;comparing the determined delta-2 tubulin expression level in the first biological sample to the determined delta-2 tubulin expression level in the second biological sample to determine whether the delta-2 tubulin expression level has increased; andwhen an increase in delta-2 tubulin expression level is determined, administering to the subject a therapeutically effective amount of a pharmaceutical composition configured to reduce delta-2 tubulin accumulation, thereby treating or preventing CIPN.

2. The method of claim 1, wherein the biological sample comprises dorsal root ganglion (DRG).

3. The method of claim 1, wherein determining the expression level of delta-2 tubulin comprises detecting delta-2 tubulin by immunofluorescence, immunohistochemistry, immunoblotting, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry.

4. The method of claim 1, wherein an increase in delta-2 tubulin expression is determined by at least one of comparing the second biological sample to the first biological sample or determining that the delta-2 tubulin level in the second biological sample is higher than in the first biological sample by a pre-determined threshold amount.

5. The method of claim 1, wherein the pharmaceutical composition comprises an agent configured to reduce formation of delta-2 tubulin by inhibiting one or more cytosolic carboxypeptidases (CCP), wherein the CCP is selected from CCP1 and CCP6.Docket No:070050.6974_CU18162PCT Application6. The method of claim 5, wherein the agent comprises an antisense oligonucleotide, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an adeno-associated virus (AAV) vector encoding a Cre recombinase or shRNA sequence that reduces expression of CCP1 or CCP6.

7. The method of claim 1, wherein the pharmaceutical composition comprises an agent configured to promote re-tyrosination of a-tubulin by increasing expression or activity of tubulin tyrosine ligase (TTL), wherein the agent comprises a small molecule, a polynucleotide encoding TTL, or a viral vector expressing TTL.

8. The method of claim 1, wherein the pharmaceutical composition comprises an agent that upregulates mitochondrial respiration, wherein the agent comprises mitochondrial electron transport chain enhancers, NAD+ precursors, ATP-generating enhancers, or compounds that restore mitochondrial membrane potential.

9. The method of claim 1, wherein the pharmaceutical composition comprises an agent that increases mitochondrial motility by downregulating or inhibiting syntaphilin (SNPH).

10. The method of claim 9, wherein the agent comprises a small molecule, antisense oligonucleotide, shRNA, or polypeptide antagonist of SNPH.

11. The method of claim 1, wherein administering the pharmaceutical composition comprises delivering a viral vector to DRG neurons, wherein the viral vector comprises an adeno-associated virus (AAV).

12. The method of claim 11, wherein the AAV comprises an AAV-PHP.S capsid.

13. The method of claim 1, wherein the anti-cancer agent is selected from the group consisting of bortezomib, paclitaxel, vincristine, cisplatin, carboplatin, eribulin, thalidomide, and taxane.Docket No:070050.6974_CU18162PCT Application14. The method of claim 1, wherein the pharmaceutical composition is administered prophylactically prior to onset of CIPN when an increase in delta-2 tubulin is detected.

15. The method of claim 1, further comprising monitoring delta-2 tubulin levels after administration of the pharmaceutical composition and adjusting a dosing regimen of the pharmaceutical composition based on the monitored delta-2 tubulin levels.

16. The method of claim 1, wherein the pharmaceutical composition is formulated for systemic administration, local intrathecal administration, or direct ganglionic delivery.

17. A pharmaceutical composition for treating or preventing chemotherapy-induced peripheral neuropathy comprising a therapeutically effective amount of an inhibitor of CCP1 or CCP6 and a pharmaceutically acceptable carrier, wherein the composition is formulated for systemic or neuronal delivery, where the inhibitor of CCP1 or CCP6 is configured to reduce formation of delta-2 tubulin in dorsal root ganglion neurons.

18. The pharmaceutical composition of claim 17, further comprising a tubulin tyrosine ligase (TTL) activator.

19. The pharmaceutical composition of claim 17, further comprising a mitochondrial enhancer.

20. The pharmaceutical composition of claim 17, wherein the composition is formulated for systemic or intrathecal administration and includes a pharmaceutically acceptable carrier or excipient.

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