A dose-escalation and safety study of gene therapy for CMT4C neuropathy

A dose-escalation and safety study of gene therapy for CMT4C neuropathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A dose-escalation and safety study of gene therapy for CMT4C neuropathy Elena Georgiou, Alexia Kagiava, Andreas Hentschel, Irene Sargiannidou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8334328/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 May, 2026 Read the published version in Gene Therapy → Version 1 posted 11 You are reading this latest preprint version Abstract Charcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the SH3TC2 gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage vector with a minimal human MPZ promoter driving expression of SH3TC2 . Groups of 1-month old Sh3tc2 −/− mice were treated with 3 different doses of AAV9- hMPZmini.SH3TC2.SV40pA or the formulation buffer by lumbar intrathecal injection. Outcomes were compared 8 weeks post injection by behavioral, electrophysiological, proteomics, morphological analysis and evaluation of tissue integrity and inflammatory responses. Vector biodistribution to the peripheral nerves and high rates of cell-specific therapeutic gene expression in Schwann cells resulted in significant therapeutic benefits in the CMT4C model. Treated mice showed improved motor performance in grip strength, rotarod testing and motor nerve conduction velocities. Morphological analysis revealed significant improvement in g-ratios, myelin thickness and ratios of demyelinated fibers in lumbar roots and femoral nerves of treated mice. Proteomic profiles showed correction of muscle denervation associated pathobiochemical processes in treated mice. Not observed tissue toxicity or immune reactions in neural tissues or peripheral organs. This study provides proof of principle for dose-dependent effectiveness and safety of intrathecal AAV9-mediated gene replacement paving the way for clinical translation. Biological sciences/Neuroscience/Peripheral nervous system Biological sciences/Biological techniques/Gene delivery/Genetic vectors CMT4C gene therapy SH3TC2 Schwann cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Charcot-Marie-Tooth disease type 4C (CMT4C) is the most common autosomal recessively inherited demyelinating neuropathy (1-3) Patients with CMT4C usually present in the first decade of life with delayed walking, progressive distal muscle atrophy and weakness, areflexia and sensory loss. Almost all patients develop foot deformities and early onset spinal deformities with scoliosis, often requiring surgery (4-6). Cranial nerve involvement with hearing loss, slow pupillary light reflexes, and lingual fasciculations is also characteristic (7) (8) (9). A combination of proprioceptive loss and vestibular neuropathy may cause disabling imbalance early in disease evolution (10). Electrophysiological studies in CMT4C patients confirm the demyelinating process with mean median motor nerve conduction velocity (MNCV) of 22.6 m/s. Rare cases with upper limb MNCVs >38 m/s have been reported (9). Nerve biopsies are characterized by an increase of basement membranes around myelinated, demyelinated, and unmyelinated axons, relatively few onion bulbs, and, most typically, large cytoplasmic extensions of Schwann cells (1, 4, 5). The clinical characteristics and severity of CMT4C are quite variable along with phenotypic variations within families (11) Clear relationships between pathogenic variants and the spectrum of disease manifestations are to date lacking, although recent studies in large patient series suggest the possibility of a milder phenotype in individuals with one or two non-protein-truncating variants (11). CMT4C is caused by bi-allelic, loss of function variants in the SH3 domain and tetratricopeptide repeats 2 ( SH3TC2 ) gene (1, 12). At least 28 different SH3TC2 mutations have been described to date, mostly truncating but also missense, with higher frequency among certain ethnic groups (13) likely due to founder effects (7). The most prevalent pathogenic SH3TC2 variants are Arg954Ter (especially North African, Dutch and French-Canadian populations) and Arg1109Ter (commonly found in the Spanish Romani population), likely due to founder effects (6, 14, 15). SH3TC2 is well conserved among vertebrate species, whereas no non-vertebrate orthologs were identified. SH3TC2 is exclusively expressed in myelinating Schwann cells and affects receptor dynamics by regulating endosome recycling (16, 17). The protein is localized to the plasma membrane and perinuclear recycling compartment. In vitro studies demonstrated that SH3TC2 interacts with Rab11, a small GTPase that regulates the recycling of membranes and receptors to the cell surface. Current hypotheses suggest that recycling endosomes play a central role in protein sorting and trafficking, both during plasma membrane recycling and as an intermediate step during cargo transport from the trans-Golgi network to the plasma membrane (18). SH3TC2 could be transported from endosomal storage sites to the plasma membrane when needed for myelin formation, as described recently for proteolipid protein (PLP) in oligodendrocytes (19). Otherwise, SH3TC2 could be involved in the regulation of cargo transport through the recycling endosome. In vitro studies have shown that both the more common protein truncating nonsense and frameshift variants ending in premature stop codon, as well as rarer missense variants, all lead to loss of the SH3TC2 protein function in endosomes and abate protein–protein interactions between SH3TC2 and RAB7 that disturbs the endocytic and membrane recycling pathway (20). Thus, the endocytic and membrane trafficking pathway may be involved in the pathogenesis of CMT4C disease, and SH 3TC2 missense mutations could impair the communication between the Schwann cell and the axon causing abnormal myelin formation. Sh3tc2 -/- mice represent a well characterized model of CMT4C developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. Ultrastructural analysis of myelinated fibers in this model showed abnormal organization of the node of Ranvier, a phenotype that was also confirmed in nerve biopsies from CMT4C patients, further supporting the crucial role of SH3TC2 in myelination and in the integrity of the node of Ranvier (16). Thus, Sh3tc2 -/- mice recapitulate all major features of CMT4C disease and provide a relevant model to test therapies. Since loss of SH3TC2 function in myelinating cells appears to be the cause of CMT4C, we have developed a gene replacement therapy using intrathecally injected adeno-associated viral (AAV) vector 9 (21-25) that provides Schwann cell-specific expression of SH3TC2 when driven by a minimal version of the rat myelin-specific myelin protein zero (Mpz) promoter (miniMpz) (26). This vector provides high expression levels, while its episomal persistence without integration into the host genome increases the safety of in vivo delivery (27, 28). The AAV9 serotype is already approved for clinical application in patients with spinal muscular atrophy (SMA) after extensive safety evaluation in pre-clinical (29) (30) and in Phase 1 (NCT02122952) and Phase 3 (NCT03461289) clinical trials, as well as in a Phase 1/2 clinical trial for the treatment of giant axonal neuropathy (GAN) (NCT02362438). Furthermore, AAV9 has shown effective Schwann cell targeting in our preclinical studies in other models for gene replacement in CMT1X (24) and gene silencing in CMT1A (31) demyelinating neuropathies. In our recent proof of concept study in Sh3tc2 -/- mice, treatment with AAV9-miniMpz. SH3TC2 .SV40pA resulted in significant functional and morphological improvements when delivered at early as well as at late stages of the neuropathy (26). To further advance this approach towards clinical translation, we used in the current study a modified AAV9 viral vector, with a human minimal MPZ promoter driving the expression of SH3TC2 with modification of polyA sequence for enhanced expression, and performed for the first time a dose escalation and safety study in the CMT4C mouse model. Lumbar intrathecal administration of modified AAV9- hMPZmini.SH3TC2.SV40pA resulted in Schwann cell specific expression of SH3TC2 and in statistically significant improvement in nerve conduction velocities and in various motor behavioural tests. No adverse effects were observed, as demonstrated by extensive histopathological examination and analysis of possible inflammatory reaction in neural and peripheral tissues. These results indicate effective targeting of Schwann cells and a clear dose-response up to the highest dose tested that is still in clinically feasible range, supporting the clinical translation of this approach for the treatment of CMT4C. MATERIALS AND METHODS Cloning of a clinical stage hMPZmin . SH3TC2.SV40pA expression cassette In our previous work (26) a minimal version of the rat myelin protein zero (miniMpz/P0) promoter was generated and used to drive expression of myc-tagged SH3TC2 in Schwann cells. As a humanized promoter would be preferable for further clinical translation and for the final testing process beyond the mouse model (toxicity, NHP), the rat miniMPz/P0 was replaced with a human minimal MPZ promoter (designated hMPZmini ). The human hMPZmini promoter was PCR amplified from a construct that contained the full sequence of human MPZ promoter introducing also the Kpn I restriction site on each end. The 429bp DNA was ligated to the previously used AAV transfer plasmid that carried the human SH3TC2 open reading frame (ORF). In addition, the myc tag at the end of the SH3TC2 coding sequence and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were removed. Finally, the polyA tail was replaced by an SV40pA sequence. For this modification, the hMPZmini.SH3TC2 insert was cloned into the Virovek single-stranded AAV backbone V445-pFB-GFP using restriction site enzyme/site Spel and BspEI/AgeI. Moreover, a mock vector that contained the human mini MPZ/P0 promoter to drive the expression of EGFP was generated. The hMPZmini.EGFP-WPRE-bGHpA insert was cloned into the Virovek single- stranded AAV backbone V445-pFB-GFP using restriction site enzymes/sites SpeI and BspEI. Lumbar intrathecal injections Vector delivery was performed under general anaesthesia by slow intrathecal injection into the L5–L6 intervertebral space of 1- or 2-month-old mice as previously described (32–35). Briefly, following a small skin incision along the lower lumbar spine level to visualize the spine, the AAV9 vector was injected into the L5-L6 intervertebral space. A 50-µL Hamilton syringe connected to a 26-gauge needle was used to inject 20 µL of the vector stock with appropriate dilutions in formulation buffer PBS (PBS with 0.001% Pluronic F68) containing different total amounts ranging between 4e10 vg (low dose), 1.2e11 vg (mid dose) and 3.5e11 vg (high dose). A flick of the tail was considered indicative of successful intrathecal administration. Treatment trial design The aim of this study was to determine whether a dose-escalation gene addition therapy can improve the manifestations of peripheral neuropathy in the mouse model of CMT4C following intrathecal injection at the L5-L6 intervertebral space. Sh3tc2 −/− mice were treated at the age of 1 month. A total volume of 20 µL containing three different vector doses ranging from 4e10 vg (low dose), 1.2e11 vg (mid dose) and 3.5e11 vg (high dose) was injected intrathecally as described above. Littermate mice were randomized to either receiving the therapeutic (full) vector AAV9- hMPZmini.SH3TC2 vector (treatment groups, n = 10 animals per dose) or equal volume of the formulation buffer (serving as the control group) and were assigned a coding number for further identification. Randomization was based on animal numbering after tailing. To assess the level of phenotype correction, groups of age-matched WT mice were similarly evaluated as controls in all outcome measures. All animals were evaluated at 1 month of age (at baseline) by behavioral testing before treatment, and again at the age of 3 months, 2 months after treatment. At the end of the treatment trial, 3-month-old mice were evaluated by electrophysiology (n = 10 per dose) and then sacrificed for quantitative morphometric analysis of myelination (n = 10 per dose) as outlined below. All therapeutic outcome evaluations including motor performance, electrophysiology, as well as myelin morphology were performed by examiners blinded to the treatment condition. Morphometric analysis of myelination For quantitative analysis of myelination, we obtained toluidine blue-stained transverse semithin sections (1 µm) of PNS tissues following perfusion with 2.5% glutaraldehyde and resin embedding, as previously described (26, 36). We calculated the g-ratios for all myelinated fibers in all treatment groups using the Image Pro software and a custom-made macro, which detects the axons and their myelin sheath according to color. This macro calculates g-ratio by dividing the average inner perimeter of the axon by the average outer perimeter of the axon, as well as the average myelin thickness for each myelinated fibre. Because more pronounced myelination deficits were observed in fibres > 4 µm in diameter in the original characterization of this model (16), we also analyzed the subset of fibres > 4 µm in diameter separately along with the analysis of all fibres > 1 µm, as previously described (36) (26). In addition, completely demyelinated fibres (defined as axons > 1 µm in diameter devoid of myelin sheath) in anterior lumbar roots and mid-sciatic nerves were manually counted and compared to the total fiber numbers, to obtain the ratio of demyelinated fibers. Evaluation of possible toxicity and SH3TC2 expression in the PNS and peripheral organs of Sh3tc2 −/− mice injected with different therapeutic vector doses. Immunohistochemistry To assess potential toxicity associated with intrathecal AAV9- hMPZmini.SH3TC2.SV40pA vector injection and gene delivery to Schwann cells as well as SH3TC2 expression was evaluated. After deparaffinisation, antigen retrieval was achieved by heating the slides for 30min in the pressure cooker in 0.1M citric acid monohydrate buffer (pH 6). Unspecific binding sites were blocked with 5% bovine serum albumin human at room temperature (RT) for 1h and slides were then incubated overnight with specific antibodies against SH3TC2 (anti-rabbit 1:100; Abcam, Cambridge, UK), CD45 (anti-rat 1:100; Abcam, Cambridge, UK), CD3 (anti-rabbit 1:100; Abcam, Cambridge, UK), CD68 (anti-rabbit 1:100; Abcam, Cambridge, UK), and CD20 (anti-goat 1:100; Santa-Cruz, USA) followed by appropriate secondary antibodies at room temperature. Slides were then washed in PBS and incubated with mouse cross-affinity fluorescein-conjugated (1:3000; Invitrogen, A21202), rabbit fluorescein (FITC)-conjugated (1:1000; Jackson ImmunoResearch, 111-486-003), rabbit cross-affinity purified rhodamine-conjugated (1:3000; Jackson ImmunoResearch, 111-026-003), mouse cross-affinity purified rhodamine-conjugated (1:1000; Jackson ImmunoResearch,115-026-068) and goat fluorescein (FITC)-conjugated (1:700; Jackson ImmunoResearch, 111-486-003). Slides were mounted with fluorescent mounting medium (DAKO) and images photographed under a fluorescence microscope (Nikon Eclipse Nἱ) with a digital camera (DS-Qi2) using NIS-Elements software. We first assessed inflammatory changes in tissues at low magnification (x10) and specific areas were further assessed at a higher magnification (x20 & x40). To assess possible inflammatory responses, paraffin sections were stained for the following markers: CD3 (T lymphocytes), CD68 (macrophages), CD45 (leukocytes) and CD20 (B-lymphocytes). For neural tissues, images of whole transverse nerve sections, femoral nerve and multiple anterior lumbar spinal roots per mouse were obtained at x20 magnification. The number of CD-positive cells were counted in all images and normalized to the total tissue area analyzed. For peripheral non-neural tissues, a total of 3 images were obtained per tissue section, per mouse, at x20 magnification. Muscle proteomic analysis Sample Preparation Murine TA muscle was snap-frozen in liquid nitrogen and stored at -80°C until further processing. Tissue lysis was performed using 200 µL of lysis buffer containing 50 mM TEAB (pH 7.8), 5% SDS, and cOmplete ULTRA protease inhibitor (Roche). The samples were homogenized with a Bioruptor® (Diagenode) for 10 minutes (30-second cycles of sonication and rest) at 4°C. To ensure complete lysis, an additional sonication step using an ultrasonic probe (30 seconds, 1-second pulse on/off, 40% amplitude) was conducted, followed by centrifugation at 20,000 g for 15 minutes at 4°C. The protein concentration of the resulting supernatant was measured using the BCA assay according to the manufacturer’s protocol. Disulfide bonds were reduced with 10 mM TCEP at 37°C for 30 minutes, and free sulfhydryl groups were alkylated with 15 mM IAA in the dark at room temperature for 30 minutes. Proteolysis was performed using the S-Trap protocol (Protifi) with 100 µg of protein per sample and a trypsin-to-protein ratio of 1:20. The samples were incubated with trypsin at 42°C for 2 hours, and proteolysis was terminated by acidifying the samples to pH < 3.0 with formic acid (FA). Assessment of Digestion Efficiency Proteolytic digests were desalted and analyzed for completeness using monolithic column separation (PepSwift monolithic PS-DVB PL-CAP200-PM, Dionex) on an Ultimate 3000 HPLC system (Dionex, Germering, Germany). For quality control, 0.5 µg of each sample was injected. Chromatographic separation was performed using a binary gradient with solvent A (0.1% TFA) and solvent B (0.08% TFA, 84% acetonitrile), with a gradient from 5–12% solvent B over 5 minutes, followed by 12–50% solvent B over 15 minutes, at a flow rate of 2.2 µL/min and 60°C. UV detection was conducted at 214 nm as described (37). LC-MS/MS Analysis Peptide samples (1 µg per injection) were analyzed using an UltiMate 3000 RSLC nano UHPLC system coupled to a Q Exactive HF mass spectrometer. Peptides were first loaded onto a 75 µm × 2 cm, 100 Å, C18 precolumn at a flow rate of 10 µL/min for 20 minutes, followed by separation on a 75 µm × 50 cm, 100 Å, C18 analytical column at 250 nL/min. A linear gradient of solution A (99.9% water, 0.1% formic acid) and solution B (84% acetonitrile, 15.9% water, 0.1% formic acid) was applied, progressing from 3% to 45% solution B over 120 minutes, with washing steps at 95% solution B and equilibration of the system between runs. Data were acquired in data-independent acquisition (DIA) mode spiked with iRT standards (Biognosys). Full MS scans were performed from 300–1100 m/z at a resolution of 60,000 (Orbitrap) using a lock mass of 445.12002 m/z (polysiloxane ion). The AGC target was set to 3 × 10⁶, with a maximum injection time of 20 ms. DIA windows were set to cover 400–1100 m/z in 23 variable windows of 28 m/z width with 1 m/z overlap, collected at a resolution of 30,000 (Orbitrap), an AGC target of 3 × 10⁶, and a normalized collision energy (nCE) of 27. Data Analysis DIA data were analyzed using Spectronaut software (Biognosys, v18.7) and analyzed with a direct-DIA based search with default search and extraction settings (BGS Factory settings). The mouse proteome reference database (UniProt) was employed. For label-free quantification, only proteins identified with at least two unique peptides were considered. Average normalized protein abundances were calculated, and log2 ratios were determined for comparisons between the different conditions. RESULTS Generation and in vivo validation of a human minimal MPZ promoter To first validate the newly cloned minimal fraction of the human MPZ promoter, the AAV9- hMPZmini.EGFP vector ( Supplementary Fig. 1A ) was delivered by lumbar intrathecal injection into 2-month-old WT mice. Vector biodistribution and EGFP expression were analyzed 6 weeks after injection. Vector genome copy numbers (VGCNs) were detected in DNA extracted from peripheral nervous system (PNS) tissues with a gradient from injection site toward the distal nerves, reaching 1.97 ± 0.5 in lumbar spinal roots, 0.31 ± 0.27 in sciatic nerves, and 0.04 ± 0.02 in femoral nerves ( Supplementary Fig. 1B, n = 3 mice; one-way ANOVA F (3,8) = 10,54, p = 0.0037). Examination of fixed lumbar spinal root sections attached to the spinal cord, and of bilateral sciatic nerve sections revealed widespread expression of the EGFP reporter gene in the perinuclear cytoplasm restricted to myelinating Schwann cells in all PNS tissues of the injected mice, in contrast to non-injected controls ( Supplementary Fig. 1C–1F ). The percentage of EGFP-expressing cells reached an average of 53.67 ± 5.56% in anterior lumbar roots and 71.14 ± 2.66% in sciatic nerves ( Supplementary Fig. 1G, n = 3 mice; Mann-Whitney U test p = 0.1). AAV9- hMPZmini.SH3TC2.SV40pA therapeutic vector cloning and expression in Sh3tc2 –/– mice The novel hMPZmini.SH3TC2.SV40pA therapeutic expression cassette ( Supplementary Fig. 1H ) was successfully packaged into the AAV9 capsid with vector production achieving titers of 1.75x10^13vg/ml. A total of 3.5x10^11 vg in a volume of 20 µl was delivered by lumbar intrathecal injection into 2-month-old Sh3tc2 −/− mice (n = 3 mice). Vector biodistribution and expression was examined 6 weeks after injection. DNA extracted from spinal roots and sciatic nerves of injected mice showed high levels of vector biodistribution throughout the PNS with VGCNs reaching 1.17 ± 1.10 in spinal roots, 0.392 ± 0.173 in sciatic nerves and 0.03 ± 0.012 in femoral nerves ( Supplementary Fig. 1I, n = 3 mice; one-way ANOVA F (3,8) = 0.8706, p = 0.4953). Dose-dependent vector biodistribution was also detected in CNS and peripheral organ tissues ( Supplementary Fig. 2A-G ). Immunostaining for human SH3TC2 in lumbar spinal root sections, as well as in sciatic nerve sections and teased fibers confirmed the presence of virally expressed SH3TC2 in the perinuclear Schwann cell cytoplasm in all PNS tissues, in a characteristic perinuclear granular appearance, and occasionally along the entire length of the Schwann cell, while it was absent from tissues of non-injected Sh3tc2 −/− mice stained as negative controls ( Supplementary Fig. 1J-M ). Quantification of the percentage of SH3TC2-immunoreactive Schwann cells showed average expression rates of 45.51 ± 7.60% in spinal roots and 54.57 ± 11.15% in sciatic nerves ( Supplementary Fig. 1N, n = 3 mice; Mann-Whitney U test p = 0.7). Thus, we were able to achieve adequate therapeutic vector biodistribution and high SH3TC2 expression rates in Schwann cells of the CMT4C model. Dose-dependent Schwann cell targeting and SH3TC2 expression in the PNS of the CMT4C model After confirming the high expression efficiency of the novel therapeutic vector AAV9- hMPZmini.SH3TC2.SV40pA , we performed a randomized, controlled treatment trial at 1 month of age. The level of phenotypic rescue in treated animals was compared with littermates injected with the formulation buffer alone by motor behavioral, electrophysiological, and morphological studies 2 months after treatment. For the dose escalation gene therapy trial, three dose groups (4e10 vg, 1.2e11 vg, and 3.5e11 vg/animal; n = 10 per dose group) of Sh3tc2 −/− mice along with age-matched WT mice (n = 5–6) were assessed by behavioral testing at the age of 1 (before injection) and at 3 months followed by electrophysiological evaluation at 3 months of age (2 months after injection), and then sacrificed for quantitative morphometric analysis. Additional groups of mice (n = 4 per dose) were injected with the therapeutic vector at the low, mid and high vector doses, in which we evaluated PNS biodistribution and expression rates of SH3TC2 in myelinating Schwann cells in lumbar roots, sciatic and femoral nerves (Fig. 1 A). SH3TC2 expression was detected in all PNS tissues examined (Fig. 1 B). VGCNs determined 8 weeks after injection reached 0.34 ± 0.3 (low dose), 0.89 ± 0.4 (mid dose), 1.4 ± 0.8 (high dose) in lumbar roots (n = 4 mice, one-way ANOVA F(2,9) = 0.9184, p = 0.4336); 0.003 ± 0.003 (low dose), 0.19 ± 0.1 (mid dose), 0.16 ± 0.1 (high dose) in the sciatic nerves (n = 3–4 mice, one-way ANOVA F(2,7) = 4.525, p = 0.0548); and 0.02 ± 0.01 (low dose), 0.05 ± 0.04 (mid dose), 0.41 ± 0.3 (high dose) in the femoral nerves (n = 4; one-way ANOVA F(2,9) = 1.609, p = 0.2527) (Fig. 1 C-E). The percentage of SH3TC2-expressing cells reached an average of 16.38 ± 2.11% (low dose), 42.70 ± 3.78% (mid dose) and 59.57 ± 4.60% (high dose) in anterior lumbar roots (one-way ANOVA F(2,9) = 0.9184, p = 0.4336); 18.29 ± 2.47% (low dose), 38.03 ± 3.84% (mid dose) and 48.18 ± 2.41% (high dose) in the sciatic nerves (one-way ANOVA F(2,9) = 26, p = 0.0002); and 17.38 ± 3.05% (low dose), 37.31 ± 2.90% (mid dose) and 37.78 ± 3.52% (high dose) in the femoral nerves (one-way ANOVA F(2,9) = 13,55, p = 0.0019) (n = 4 mice; Fig. 1 F-H). Functional improvements following dose escalation treatment in the CMT4C model We have focused our behavioral analysis of treated, buffer-treated and WT animals on testing motor strength and coordination. Rotarod tests at low and high speeds showed at baseline, before treatment, significantly worse motor performance with less time staying on the rotarod in all Sh3tc2 −/− mouse groups compared to WT mice (Fig. 2 B, one-way ANOVA F(4,41) = 3.572, p = 0.0137), (Fig. 2 E, one-way ANOVA F(4,41) = 3.562, p = 0.0139). At 2 months after treatment with AAV9- hMPZmini.SH3TC2, Sh3tc2 −/− mice showed improvement of rotarod performance approaching the performance of age-matched WT mice at both speeds tested, whereas formulation buffer-treated Sh3tc2 −/− mice showed deterioration over time (Fig. 2 C, one-way ANOVA F(4,41) = 2.011, p = 0.1109), (Fig. 2 F, one-way ANOVA F(4,41) = 2.181, p = 0.0881), (Fig. 2 A-F). Likewise, foot grip strength at age 1 month, at baseline before starting treatment, showed lower strength generated by Sh3tc2 −/− mouse groups compared to WT mice (Fig. 2 H, one-way ANOVA F(4,41) = 11.53, p < 0.0001) whereas at age 3 months (2 months post-injection) muscle strength improved in mid- and high vector dose treated Sh3tc2 −/− mice compared with the low dose and buffer group and reached WT levels (Fig. 2 I, one-way ANOVA F(4,41) = 8.164, p < 0.0001). Longitudinal comparison also demonstrated that the strength produced by the hindlimbs improved with time in mid- and high vector dose treated mice in contrast to buffer and low dose treated mice (Fig. 2 G-I). Nerve conduction studies in 3-month-old mice (2 months after treatment) revealed significantly higher sciatic nerve MNCVs in AAV9- hMPZmini.SH3TC2.SV40pA -treated Sh3tc2 −/− mice reaching 37.78 ± 1.16 m/s (low dose), 35.07 ± 1.07 m/s (mid dose), 32.07 ± 1.35 m/s (high) compared with the buffer-treated group (23.51 ± 1.07 m/s), although not reaching those of age-matched WT animals (41.47 ± 1.04 m/s) (one-way ANOVA F(4,39) = 42.13, p < 0.0001). There was also a trend for higher compound muscle action potential (CMAP) amplitude in treated mice reaching 3.53 ± 0.41 mV (low dose group), 3.95 ± 0.34 mV (mid), and 3.97 ± 0.60 mV (high), compared to the buffer group (3.36 ± 0.28 mV), but without reaching statistical significance and also remaining below WT levels (4.46 ± 0.42 mV) (one-way ANOVA F (4,41) = 1.865, p = 0.1350) (Fig. 2 J-K). Since axonal loss and neuromuscular junction (NMJ) denervation leading to muscle atrophy is a common pathological process correlating with clinical weakness in all patients with peripheral nerve diseases including CMT4C (Cipriani et al 2018), we further evaluated the degree of proximal and distal hind limb muscle atrophy in our Sh3tc2 −/− treatment groups. At 3 months of age, 2 months post injection, the quadriceps and tibialis anterior muscles showed significantly increased muscle weight in treated mice injected with the mid and the high dose compared to buffer treated controls or to mice injected with the low dose (Fig. 2 L, one-way ANOVA F(4,33) = 8.478, p < 0.0001), (Fig. 2 M, one-way ANOVA F(4,32) = 2.680, p = 0.0493) (Fig. 2 L-M). Finally, in addition to these functional evaluations, we also observed an apparent improvement in the phenotype of abnormal clenching of toes and clasping of hind limbs upon suspension by the tail, which was previously described in Sh3tc2 −/− mice (Arnaud et al., 2009). Treated animals were able to stretch their legs and toes much more compared to buffer-treated littermates, and did not differ from WT mice. This phenotype was quantified by measuring the hind limb angle as an indicator of limb opening ability, which confirmed significant improvements in treated mice (Fig. 2 O, one-way ANOVA F(4,41) = 10.12, p < 0.0001)(Fig. 2 N-O). Dose-dependent improvement of PNS myelination in treated CMT4C mice Morphological examination was carried out in transverse toluidine stained semithin sections of anterior lumbar motor roots and femoral motor nerves of 3-month-old Sh3tc2 −/− mice injected with the therapeutic vector or with formulation buffer at age 1 month (n = 10 per dose group) and in tissues of WT mice (n = 5). In all PNS tissues evaluated, dose-dependent improvement of myelination in treated as opposed to buffer-treated Sh3tc2 −/− mice was observed but without reaching WT levels. In lumbar roots (Fig. 3 A), average g-ratios of myelinated fibers > 1 µm in diameter in the treated groups were 0.70 ± 0.01 (low dose), 0.71 ± 0.01 (mid dose), and 0.68 ± 0.01 (high), compared with 0.74 ± 0.01 in the buffer-treated group and 0.65 ± 0.01 in WT mice (Fig. 3 B, one-way ANOVA F(4,40) = 10.81, p 4 µm in diameter, which are more affected by demyelination in this model (Arnaud et al., 2009), average g-ratios reached 0.79 ± 0.01 (low dose), 0.80 ± 0.01 (mid dose), and 0.77 ± 0.01 (high dose) in treated mice compared with 0.83 ± 0.01 in the buffer group and 0.71 ± 0.004 in WT (Fig. 3 C, one-way ANOVA F(4,40) = 25.53,p < 0.0001). (Fig. 3 B-C and Supplementary Fig. 3A-B ). Corresponding with the g-ratio reductions, average myelin thickness increased in treated mice reaching 0.52 ± 0.03 µm (low dose), 0.50 ± 0.02 µm (mid dose) and 0.57 ± 0.02 µm (high dose) compared with the buffer group (0.43 ± 0.01 µm), with the high dose mice approaching the WT group (0.64 ± 0.01 µm) (Fig. 3 D, one-way ANOVA F(4,40) = 8.365, p 4 µm, myelin thickness in treated mice increased to 0.56 ± 0.03 µm (low dose), 0.54 ± 0.02 µm (mid dose) and 0.63 ± 0.02 µm (high dose), compared with the buffer group (0.47 ± 0.02 µm), and almost reached the WT group 0.70 ± 0.004 µm (Fig. 3 E, one-way ANOVA F(4,40) = 11.44. p < 0.0001) (Fig. 3 D-E). Furthermore, the percentage of completely demyelinated fibers was significantly reduced in treated mice to 0.02 ± 0.003 (low dose), 0.02 ± 0.001 (mid dose), 0.02 ± 0.002 (high dose), compared with 0.03 ± 0.01 in the buffer group and 0.0003 ± 0.0003 in the WT group (one-way ANOVA F(4,40) = 9.057, p < 0.0001), (Fig. 3 F). Furthermore, axonal profiling analysis revealed a shift of axonal diameter distribution toward the population of smaller axons (< 5 µm) in Sh3tc2 −/− compared to WT lumbar roots, without significant correction after treatment (two-way ANOVA F(4,327) = 1.088, p = 0.3624).( Supplementary Fig. 3C ). The observed profile changes, likely reflecting hypomyelination, did not lead to significant axonal loss in either treated or buffer-treated animals compared with WT, as determined by similar total number of axons per lumbar root in all groups (two-way ANOVA F(4,41) = 1.469, p = 0.2293) ( Supplementary Fig. 3D ). In femoral motor nerves (Fig. 3 G) a similar dose-dependent improvement of myelination was observed in treated CMT4C mice with g-ratios reaching 0.67 ± 0.01 (low dose), 0.63 ± 0.02 (mid) and 0.64 ± 0.01 (high dose) in treated compared to 0.66 ± 0.01 in buffer-treated mice and 0.65 ± 0.01 in WT mice (Fig. 3 H, one-way ANOVA F(4,42) = 1.557, p = 0.2035). In the subset of fibers > 4 µm in diameter the corresponding g-ratios also showed a dose-dependent reduction reaching 0.77 ± 0.01 (low), 0.76 ± 0.01 (mid), and 0.74 ± 0.01 (high dose) in treated compared to 0.77 ± 0.01 in buffer treated Sh3tc2 −/− mice and 0.71 ± 0.01 in the WT group (Fig. 3 I, one-way ANOVA F(4,40) = 12.18, p < 0.0001). (Fig. 3 H-I and Supplementary Fig. 4A-B ). Average myelin thickness increased in the treated groups to 0.52 ± 0.02 µm (low), 0.61 ± 0.04 µm (mid) and 0.58 ± 0.02 µm (high) compared to 0.55 ± 0.02 µm in the buffer group and 0.71 ± 0.02 µm in WT mice (Fig. 3 J, one-way ANOVA, F(4,40) = 8.208, p 4 µm in diameter, again a dose-dependent increase in myelin thickness was found reaching 0.60 ± 0.02 µm in low dose, 0.67 ± 0.05 µm in mid dose, and 0.69 ± 0.02 µm in high dose treated group, compared to 0.63 ± 0.02 mm in the buffer group and 0.95 ± 0.03 in WT group (Fig. 3 K, one-way ANOVA F(4,40) = 21.45, p < 0.0001). (Fig. 3 J-K). As in the lumbar roots, the ratio of demyelinated fibers in femoral nerves decreased to 0.02 ± 0.004 (low), 0.02 ± 0.003 (mid) and 0.01 ± 0.003 (high) in treated compared with 0.03 ± 0.003 in the buffer treated group, while no demyelinated fibers were found in the WT group (Fig. 3 L one-way ANOVA F(4,40) = 8.946, p < 0.0001). Axonal profiling analysis revealed a shift of axonal diameter distribution toward the population of smaller axons (< 2 mm) in all Sh3tc2 −/− femoral nerves compared with WT mice, without significant correction in treated animals ( Supplementary Fig. 4C , two-way ANOVA F(4,336) = 3.303, p = 0.0113). As in the lumbar roots, no significant axonal loss was found in Sh3tc2 −/− femoral nerves compared with WT mice ( Supplementary Fig. 4D ). Analysis of inflammatory response and tissue integrity in treated CMT4C mice To assess for potential toxicity in neural and peripheral organ tissues, Sh3tc2 −/− mice were injected intrathecally with the AAV9- hMPZmini.SH3TC2.SV40pA vector at the low (4e10 vg), mid (1.2e11 vg) and high (3.5e11 vg) doses (n = 4 mice per dose) and sacrificed 8 weeks post injection, at which time point the maximum of possible immune response is expected (38). Buffer injected littermate Sh3tc2 −/− mice (n = 4) were used as controls, as well as additional groups of WT mice and untreated Sh3tc2 −/− mice of the same age. Assessment of inflammatory markers in neural tissues (Fig. 4 B-D) revealed only mildly elevated numbers of CD68 + macrophages in all PNS tissues of Sh3tc2 −/− mice compared to WT mice, without any difference between untreated, buffer- or vector-injected groups, indicating that this difference likely reflects a feature of CMT4C PNS pathology and not a reaction to vector injection. Interestingly, treated mice showed reduced macrophage numbers in the femoral nerve compared to buffer injected controls, suggesting an improvement of nerve pathology and secondary inflammation. No significant elevation of CD68 + macrophages or any of the other cell types (CD3+, CD20 + or CD45 + cells) was found in any of the peripheral organ tissues examined from vector injected compared to buffer control Sh3tc2 −/− or WT mice ( Supplementary Fig. 5A-F ). To evaluate overall tissue integrity in treated mice, paraffin sections were stained with H&E and visualized under the light microscope. This study revealed no evidence of impaired tissue integrity in neural tissues ( Supplementary Fig. 6A) or in peripheral organs ( Supplementary Fig. 6B, 7 ). Improvement of muscle proteomic profiles in treated CMT4C mice We performed proteomic profiling on tibialis anterior muscle (total of n = 26 samples) to investigate the effect of the different treatment regimens on reinnervation-based restoration of proteostasis (Fig. 5 A). Our label-free proteomic profiling approach allowed the robust quantification of a total of 18115 unique peptides referring to 1487 proteins (1199 quantifiable proteins) spanning 7 orders of magnitude across the different quantification approaches (Fig. 5 B). To visualize protein changes underlying de- and re-innervation, we generated heat maps on one hand covering all proteins identified across the different quantification approaches ( Supplementary Fig. 8 ) and on the other hand, covering “only” proteins displaying a statistically significant dysregulation across these group quantifications. The latter approach included 58 proteins (Fig. 5 C) covering different biological processes including translation at pre- and post-synapse, mitochondrial-based ATP production, intermediate filament organization and muscle development. Affected subcellular compartments and structures include synapses, myelin sheath, microfibrils, ribonucleoprotein complexes, nuclei (with chromatin remodelling) and mitochondria as illustrated by our GO-term-based in silico analysis of proteomic findings (Fig. 5 D). The most robust and consistent differences were noticed between Sh3tc2 −/− and WT animals for proteins involved in transcription including histones (H1-0, H1-1, H1-2, H1-3, H1-4 & H1-5), Elongin-C, Splicing factor U2AF 65 kDa subunit, in translation (Rpl15 & 29 & Ubiquitin-like FUBI-ribosomal protein eS30 fusion protein), for Serpins (Serpin3k & Serpinb6), for mitochondrial proteins (Cytochrome c oxidase subunit 5B & NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3) and proteins modulating oxidative stress burden (Thioredoxin domain-containing protein 17 & Carboxic anhydrase 3), Chaperones (Heat shock protein beta-6 &7), Isochorismatase domain-containing protein 2A and Dual specificity phosphatase 29 (DUSP29). Out of these proteins, our therapeutic intervention associated with re-innervation, in particular corrected dysregulations of proteins involved and transcription and translation whereby low dosages seem to have the most beneficial effect (Fig. 5 C). Of note, Kynurenine–oxoglutarate transaminase 1 is increased only in the group of treated animals. DISCUSSION In this study, we have further optimized and validated a gene therapy approach to treat CMT4C, the most prevalent recessively inherited demyelinating neuropathy, arising from loss-of-function mutations in the SH3TC2 gene. By using a minimal version of the myelin-specific human MPZ promoter ( hMPZmini ) to drive human SH3TC2 gene expression restricted to Schwann cells, and an SV40pA sequence to enhance the expression, we completed for the first time a dose escalation and safety study in the Sh3tc2 − /− mouse model of the disease. This is a significant advance compared to our earlier using a non-clinical stage expression cassette based on a minimal version of the myelin specific rat Mpz promoter ( miniMpz ) to drive human SH3TC2 gene tested in a single AAV9 vector dose at early and late stages of the neuropathy in the CMT4C model (Georgiou et al, 2023). We also build on our initial proof-of-concept studies using a lentiviral vector system injected intrathecally to express SH3TC2 under the control of the full-length Mpz promoter, showing expression restricted to Schwann cells and a therapeutic benefit (36). However, the limitations of in vivo lentiviral vector delivery (28) motivated us to explore as a clinically more translatable approach the possibility of delivering the therapeutic SH3TC2 gene using the AAV9 vector that has shown better biodistribution, improved safety profile, and higher expression levels in the PNS. (24, 32). In the current study, we tested 3 different therapeutic vector doses injected intrathecally in an effort to clarify the minimum effective as well as safe dose in the model of the disease. We demonstrate that beginning with the mid, and even more consistently with the high dose of AAV9- hMPZmini.SH3TC2.SV40pA vector leads to adequate biodistribution to the peripheral nervous system and to high rates of therapeutic gene expression specifically in myelinating Schwann cells. The high dose also showed significantly higher expression rates in most PNS tissues. The therapeutic efficacy of this gene therapy approach was demonstrated using multiple functional and pathological outcome measures that are relevant not only for the phenotype of this model (Arnaud 2009) but also for the human disease CMT4C. (1, 4, 5) Our results confirm that a minimal version of the human MPZ promoter can provide strong gene expression specifically in myelinating Schwann cells. The minimal promoters, or core promoters, are short sequences that allow for the accurate formation of the initiation complex. These sequences play a critical role in the synthetic promoter properties, from its background expression, or leakiness, to its maximum potential induction and can maximize the transgene capacity of recombinant AAV vector genomes (39, 40) (41). Previous work has indicated that most of the functional regulatory elements and crucial transcription factor binding sites of the MPZ promoter, including that of SOX10 and EGR2, are located within this distal promoter fragment downstream of the AvrII site (42). Thus, we PCR amplified this 412-bp-long sequence and validated its efficacy and fidelity initially through evaluation of the reporter gene expression. Similar to the full-length Mpz promoter tested previously (24, 32, 33, 36) we showed that a high percentage of myelinating Schwann cells was expressing EGFP in the PNS. Our resent work (26) showed that in the CNS, less than 2% of neurons in the lumbar spinal cord and a very low percentage of oligodendrocytes were EGFP positive, confirming the largely preserved specificity of this reduced promoter version to restrict expression in Schwann cells. Thus, we provide strong evidence for the usefulness of this minimal human PNS myelin-specific promoter to facilitate packaging of larger coding sequences into the AAV expression cassette that can be utilized not only for CMT4C treatment, but also for various gene therapy approaches to treat other demyelinating CMT neuropathies. AAV vectors are leading gene therapy vehicles (43), as they offer unique advantages, such as tissue tropism, specificity in transduction, a relatively low immunogenicity, no integration into the host chromosome, and long-lasting robust transgene expression in post-mitotic cells. In addition to application in many ongoing clinical trials, different AAV serotypes have already been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA) for the treatment of rare genetic disorders, including Leber’s congenital amaurosis (AAV2), SMA (AAV9) and Duchenne Muscular Dystrophy (AAVrh74) (44, 45). While different AAV serotypes have demonstrated varied tropism for various cells and tissues, AAV9 was found to have the highest tropism for the CNS with efficient targeting of motor neurons for SMN gene replacement to treat SMA patients (46). Importantly, AAV9 has been shown to target efficiently the PNS via intrathecal (24, 26, 31, 32), intravenous (32), or intraneural administration (47) with high degree of tropism for myelinating Schwann cells, leading to therapeutic GJB1 gene expression controlled by the Schwann cell-specific Mpz promoter to treat CMT1X neuropathy in various model of the disease (24, 32) (48), or to PMP22 gene silencing by shRNA (47) or microRNA (31) under ubiquitous promoters. In this work, we delivered by AAV9 a minimal version of the myelin-specific human MPZ promoter ( hMPZmini ) to drive human SH3TC2 gene expression restricted to Schwann cells combined with an SV40pA sequence to enhance the expression level at 3 different doses via lumbar intrathecal injection. The mid (1.2x10^11 vg/animal) and even better the high (3.5x10^11 vg) doses of AAV9- hminiMpz-SH3TC2 .SV40pA vector led to adequate biodistribution to the PNS and to high rates of cell-specific therapeutic gene expression in myelinating Schwann cells, with the high dose showing significantly higher expression rates in most tissues. These results reproduce our previous studies using a single IT dose of 2x10^11 vg. Both the analysis of VGCNs biodistributed to the PNS tissues, as well as the percentage of transduced Schwann cells expressing the reporter or the therapeutic gene in lumbar roots and in sciatic nerves indicates a gradient of biodistribution from the site of injection, as in our previous studies (24, 32). Lumbar intrathecal delivery could be the safest and most effective route of administration to target both CNS and PNS, as it has been shown to result in widespread expression of AAV9 (49). The intravenous injection has significant disadvantages including high vector amount needed for injection in order to achieve expression in the CNS (50, 51) (21, 52, 53) with higher risk for toxicity and immune reactions, while the direct intraneural injection is more invasive and does not provide widespread expression (47, 54). We demonstrated here that virally delivered SH3TC2 localizes correctly to the perinuclear cytoplasm of myelinating Schwann cells of the PNS, as also shown in our previous studies using both lentiviral and AAV9-mediated SH3TC2 gene replacement, and colocalization with its interacting molecule Rab11 (26, 36). Although the overall VGCNs and SH3TC2 expression rates were not very high even in the high dose in the sciatic nerves, they were still sufficient to produce significant therapeutic effects in the CMT4C model. However, we acknowledge that phenotype rescue was not complete, similar to the results obtained previously in the CMT4C, CMT1X, and CMT1A neuropathy models (24, 36) (26, 31, 33, 34). Both in the functional and electrophysiological outcome measures, as well as in the morphological analysis of myelination, Sh3tc2 −/− mice treated with either of the three vector doses mostly failed to reach the levels of age-matched WT control mice. This may reflect the severity and early onset of the pathology in this model, as well as the fact that only a subset of myelinating Schwann cells was transduced. In addition, we observed that the phenotype of abnormal clenching of toes and clasping of hind limbs upon tail suspension reflecting the PNS dysfunction in this neuropathy model (16, 55) improved significantly in treated Sh3tc2 −/− mice compared to controls. Moreover, the muscle weight of the quadricep and tibialis anterior muscles increased in treated mice and the high vector dose group reached the levels of the WT mice in contrast to buffer and low dose mice groups which had lower muscle weight, reflecting atrophy. This model of CMT4C shows neuromuscular junction (NMJ) alterations on a structural and transcriptional level that could contribute to the pathomechanisms of the disease and the resulting phenotype. Increased axon branching at the NMJ of Sh3tc2 −/− mice has been shown hypothesized to result from adapting to the pathology, probably mediated by an increase in neurotrophic factors produced by muscle and proteomic changes in the sciatic nerve (55). These findings are also relevant for clinical translation, as muscle atrophy and intramuscular fat accumulation (IMFA) are characteristic manifestations of CMT neuropathies and can be monitored by MRI as a reliable biomarker to reflect disease severity (56–58), and as a possible surrogate marker of treatment response (59). Our study further supports the use of muscle atrophy and IMFA as a biomarker in CMT neuropathies which, depending on the stage of the neuropathy, may show an early response to treatment. Sh3tc2 −/− mice develop progressive peripheral neuropathy confirmed by decreased motor and sensory nerve conduction velocity and hypomyelination (16). We used this mouse model because it reproduces the main phenotypic features of the human disease while preserving fertility and normal lifespan. To assess functional improvement after treatment, we applied foot grip strength and rotarod analysis, which were shown to be responsive to treatment in our previous experiments using this mouse model (26, 36). Although foot grip strength testing verified significant improvement of mid- and high-dose treated mice compared with buffer-treated mice, reaching WT levels, rotarod test improvement did not reach statistical significance, likely due to increase variability within groups. Importantly, sciatic MNCV increased significantly in all doses-groups of treated Sh3tc2 −/− mice, albeit not reaching WT levels. This improvement reflects not only enhanced myelination shown here, but also remodeling of the elongated nodes of Ranvier, demonstrated in our previous studies in this model (26, 36). In contrast, no significant CMAP improvement was achieved in this study with early treatment, in line with previous results showing significant CMAP increase only in the late-treated and older Sh3tc2 −/− mice (26), likely because at this age secondary axonal pathology and muscle denervation is more pronounced than in 3-month-old CMT4C mice, allowing for a clearer treatment effect on axonal pathology to be obtained. The morphometric evaluation of lumbar motor roots and femoral motor nerves focused on assessment of the hypomyelination phenotype of Sh3tc2 −/− mice and revealed dose-dependent improvement in g-ratios as well as in the average myelin thickness, in addition to reduced ratios of completely demyelinated fibers. Sh3tc2 −/− mice present with hypomyelination at 1 month of age, with slowly progressive demyelination ensuing overtime (16). Thus, a combination of developmental and progressive abnormalities in myelination generate the neuropathy in this model. Here we demonstrate significant but only partial improvement in treated mice in most myelination parameters when compared to mock treated and WT controls. As in our previous treatment studies (26, 36), these improvements were more significant in the subset of myelinated fibers > 4 µm in diameter, which are known to be more affected in this CMT4C model (16). Since safety and immunological reaction to vector injections for gene therapy remain an important consideration before moving to clinical translation (60), we further analysed the possible inflammatory and toxic responses caused by IT administration of the AAV9- hminiMpz-SH3TC2 .SV40pA vector. Our data show that overall numbers of immune cells in relevant tissues of Sh3tc2 −/− mice show no elevation compared to the non-injected animals. Despite the high VGCNs, no toxic or inflammatory effects were observed in the liver, likely due to the Schwann cell-specific expression of the transgene driven by the myelin-specific MPZ promoter. The same reason may also account for lack of DRG toxicity with this intrathecal approach, since DRG neurons do not express the transgene, in contrast to previous applications with reported DRG toxicity in primates with the neuronally overexpressed SMN transgene (61). This is also supported by our H&E staining that revealed no evidence of impaired tissue integrity in neural tissues or in relevant peripheral organs. Taken together, our results indicate that this AAV9 vector is overall safe and expected to neither cause any significant capsid-related toxicity when delivered intrathecally, as has already been demonstrated in the GAN clinical trial (62) nor any payload-related toxicity due to the cell-targeted expression. No or mild toxicity was also observed in a toxicology study after intra-cisterna magna injection in non-human primates (60) and high doses of AAV9 remained non-toxic (63). However, further studies and appropriate preventive immunosuppression protocols that have been established in other similar clinical stage gene therapy programs (62), are needed to ensure patient safety in future clinical applications for CMT4C. In particular, due to high percentage of CMT4C patients with biallelic premature stop codon variants likely resulting in lack of any SH3TC2 protein expression, stronger immunosuppressive protocols may be needed to avoid immune reactions to the virally expressed SH3TC3 protein itself. To address the beneficial effect of gene therapeutically based intervention in our CMT4C mouse model, we performed unbiased proteomic profiling on WT and Sh3tc2 −/− mice whereby for the latter group, untreated, mock treated and vector (different dosages) treated animals were included. Grouped protein analysis and heatmap-based visualization of proteomic findings enabled the determination of 58 relevant proteins covering different biological functions and thus cellular processes. Hereby, transcription and translation as well as mitochondrial function and associated oxidative stress as well as protein folding seem to represent the most vulnerable processes underlying denervation. Of note, these processes have already been linked to muscle denervation in studies of human musculature (64). Interestingly, our therapeutic intervention in particular corrected dysregulations of proteins involved in transcriptional and translational processes whereby the low dosage seems to have the most pronounced effect. One might speculate that this reflects the correction of the production of proteins associated with denervation such as “atrogenes” (65) or other proteins involved in atrophic remodelling of skeletal musculature. Along this line, DUSP29 showed a correction of increased abundance upon low-dosage treatment. DUSP29 is known to affect MAP kinase signalling though modulation of the MAPK1/2 cascade in skeletal muscle promoting muscle cell atrophy (66). Hence, our proteomic findings support the concept that re-innervation promoted by gene therapy corrected the pathobiochemical processes associated with muscle atrophy. These data are also in keeping with our findings showing reversal of muscular atrophy and improved muscle function in treated Sh3tc2 −/− mice DATA AVAILABILITY All data, analytical methods, and study materials are available from the corresponding author on request. Declarations ETHICAL APROVAL All experimental procedures were conducted in accordance with animal care protocols approved by the Cyprus Government’s Chief Veterinary Officer (project license CY/EXP/PR. L11/2022) according to national law and European guidelines (EC Directive 86/609/EEC). COMPETING INTERESTS The study is part of a PCT/EP2020/065312 application in which AK, IS and KAK are co-inventors. FUNDING This research was funded by Neurogene Inc and by Charcot-Marie-Tooth Association (CMTA Grant 2019-21 to KAK). AUTHOR CONTRIBUTIONS E.G. performed the experiments, acquired the data, analyzed the data, and wrote the manuscript. A.K. performed electrophysiology experiments. I.S. conducted cloning and mice PCR screening. R.P. performed paraffin embedding and sectioning. M.S. performed evaluations of control animal groups. C.T. and J.R. performed and analyzed VGCNs. A.H and A.R. performed and analyzed the proteomic analysis. K.A.K. conceptualization, supervision, project administration, writing-original draft, writing-review and editing. All authors critically reviewed and approved the final manuscript. ACKNOWLEDGEMENTS We thank Dr.Kyriaki Michaelidou and Dr. Maria Zanti for their invaluable assistance with the statistical analysis of the data. 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1","display":"","copyAsset":false,"role":"figure","size":14309050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of the dose escalation treatment trial and dose-dependent vector biodistribution and SH3TC2 expression. A:\u003c/strong\u003e Dose escalation treatment trial design and outcome measures used. \u003cstrong\u003eB:\u003c/strong\u003e Expression of the AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e vector injected at different doses as indicated in paraffin sections\u003cstrong\u003e \u003c/strong\u003eof lumbar roots, sciatic and femoral nerves 6 weeks after intrathecal injection in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. Immunostaining for SH3TC2 (red) shows expression of virally-delivered SH3TC2 in a subset of Schwann cells, similar to WT mice (positive control), while it is absent in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e tissues from buffer-injected littermates. \u003cstrong\u003eC-E:\u003c/strong\u003e Quantification of vector genome copy numbers confirms dose-dependent biodistribution of AAV9 in anterior lumbar roots as well as in sciatic more than femoral nerves. \u003cstrong\u003eF-H:\u003c/strong\u003e Quantification of SH3TC2 expression rates confirms a dose-dependent increase with high-dose injected animals showing the highest expression rates. Values represent mean ±SEM (n=3-4 mice/group; one-way ANOVA and Tukey’s post hoc test, *: p\u0026lt;0.05; **:p\u0026lt;0.01, ***:p\u0026lt;0.001). Scale bars:20 μm.\u003c/p\u003e","description":"","filename":"Figure1GEORGIOUCING.png","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/c17757c3cf6e9982e03b989b.png"},{"id":100369898,"identity":"0f75ec3f-f94d-4920-9922-681ae0928413","added_by":"auto","created_at":"2026-01-16 07:59:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9166369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose-dependent functional improvements after treatment in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSh3tc2\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e–/– \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice. \u003c/strong\u003eAnalysis of functional outcomes of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice at baseline before injection at 1 month of age and 2 months after treatment at 3 months of age, with either the formulation buffer or with the AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e vector at the low (4.0e\u003csup\u003e10\u003c/sup\u003e vg, vector genomes), standard (1.2e\u003csup\u003e11\u003c/sup\u003e vg) or high (3.5e\u003csup\u003e11\u003c/sup\u003e vg) doses, as indicated. \u003cstrong\u003eA-F:\u003c/strong\u003e Trend for improved rotarod performance of treated 3-month-old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice compared with the buffer injected group, approaching WT levels, including time spent on rotarod at 20 (\u003cstrong\u003eA-C)\u003c/strong\u003e and 32 \u003cstrong\u003e(D-F\u003c/strong\u003e) rotations per minute (rpm), but without reaching statistical significance. \u003cstrong\u003eG-I:\u003c/strong\u003e Significant improvement of hindlimb foot grip strength in mid- and high-dose treated animals compared to littermate controls. \u003cstrong\u003eJ-K:\u003c/strong\u003e Electrophysiological analysis in 3-month-old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice injected at age 1 month shows improved sciatic nerve motor conduction velocities (MNCV) (\u003cstrong\u003eJ\u003c/strong\u003e) and a trend for improved compound muscle action potential (CMAP) amplitudes (\u003cstrong\u003eK\u003c/strong\u003e) in treated compared with mock-treated mice (n=10/dose group), although not reaching WT levels (n=6).\u003cstrong\u003e L-M: \u003c/strong\u003eResults of the quadriceps and tibialis anterior muscle weight evaluation shows a significant increase in the weight of both muscles in fully-treated mice injected with the mid or the high dose compared to buffer treated controls or mice injected with the low dose. \u003cstrong\u003eN-O:\u003c/strong\u003e Improvement of abnormal clenching of toes and clasping of hind limbs\u003cstrong\u003e \u003c/strong\u003ewhen suspended by the tail in treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice compared to buffer-treated littermates. \u003cstrong\u003eO:\u003c/strong\u003e Quantification of the hind limb opening angle from all animals in each group confirm this improvement with a wider angle in all treatment groups. Values represent the mean±SEM. (One-way ANOVA with Tukey’s multiple comparison test, *: p\u0026lt;0.05; **:p\u0026lt;0.01, ***:p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure2GEORGIOUCING.png","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/0f419df74a27722e4d6ba8c3.png"},{"id":100369658,"identity":"1f7e10e9-aaf0-4441-8be8-edc890ec5817","added_by":"auto","created_at":"2026-01-16 07:59:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36862900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose-dependent improvement of PNS myelination in treated CMT4C mice. A:\u003c/strong\u003e Representative low (upper panels) and higher (lower panels) magnification images of toluidine blue-stained semithin sections from anterior lumbar motor roots attached to the spinal cord (SC) of 3-mo old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice injected at 1 mo of age either with buffer, or with the therapeutic vector at low, mid, and high doses, as indicated, as well as age-matched WT mice as control. Buffer-injected mice show thin myelin sheaths in most of the axons as well as several completely demyelinated fibres (black asterisks), whereas therapeutic vector-injected mice show improvement of this myelin pathology. \u003cstrong\u003eB-E:\u003c/strong\u003e Comparison of g-ratios from all fibres \u0026gt;1 μm in diameter (\u003cstrong\u003eB\u003c/strong\u003e), as well as from the subset of fibres \u0026gt;4 μm (\u003cstrong\u003eC\u003c/strong\u003e) shows significant g-ratio reduction in treated (n=10 mice per group) compared to buffer treated mice (n=10), while the corresponding myelin thickness is increased in treated compared to buffer treated mice, both when comparing all fibres \u0026gt;1 μm (\u003cstrong\u003eD\u003c/strong\u003e), as well as the subset of fibres \u0026gt;4 μm (\u003cstrong\u003eE\u003c/strong\u003e). \u003cstrong\u003eF: \u003c/strong\u003eThe ratio of completely demyelinated fibres is also significantly reduced in mid-and high dose treated compared to mock-treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003eG:\u003c/strong\u003e Representative low (upper panels) and higher (lower panels) magnification images of toluidine blue-stained semithin sections from femoral motor nerves of 3-mo old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/- \u003c/sup\u003emice injected at 1 mo of age either with buffer, or with the therapeutic vector at low, mid, and high doses, as indicated, as well as age-matched WT mice as control. Buffer-injected mice show thin myelin sheaths as well as several completely demyelinated fibres (black asterisks), whereas therapeutic vector-injected mice show improvement of this myelin pathology. \u003cstrong\u003eH-K:\u003c/strong\u003e Comparison of g-ratios from all fibres \u0026gt;1 μm in diameter (\u003cstrong\u003eH\u003c/strong\u003e), as well as from the subset of fibres \u0026gt;4 μm (\u003cstrong\u003eI\u003c/strong\u003e) shows significant g-ratio reduction in treated (n=10 mice per group) compared to buffer treated mice (n=10), while the corresponding myelin thickness is increased in treated compared to buffer treated mice, both when comparing all fibres \u0026gt;1 μm (\u003cstrong\u003eJ\u003c/strong\u003e), as well as the subset of fibres \u0026gt;4 μm (\u003cstrong\u003eK\u003c/strong\u003e). \u003cstrong\u003eL: \u003c/strong\u003eThe ratio of completely demyelinated fibres in femoral nerves is also significantly reduced in mid-and high dose treated compared to mock-treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice. (One-way ANOVA with Tukey’s multiple comparison test, *: p\u0026lt;0.05; **:p\u0026lt;0.01, ***:p\u0026lt;0.001). Scale bars: 25 μm in low (upper panels) and 50μm in higher (lower panels) magnification images of A and B.\u003c/p\u003e","description":"","filename":"Figure3GEORGIOUCING.png","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/ad45697ae4c36dad38b1b781.png"},{"id":100178239,"identity":"99ae59d6-753a-46e1-b3fe-2ea965448c89","added_by":"auto","created_at":"2026-01-13 18:34:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20684608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of possible inflammation in sciatic nerve sections from treated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSh3tc2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-/- mice 8 weeks post injection. A: \u003c/strong\u003eRepresentative images of sciatic nerve tissues from \u003cem\u003eSh3tc2\u003c/em\u003e-/- mice treated with low, mid or high vector doses or buffer, and from WT mice, immunostained for inflammatory cell markers CD3 (red), CD68 (red), or CD45 (red) and CD20 (green), as indicated. CD+ cells are pointed by a white arrowhead. Note increased numbers of mostly CD68+ macrophages in nerves from \u003cem\u003eSh3tc2\u003c/em\u003e-/- mice regardless of treatment condition, including buffer, as opposed to WT nerves, while other CD+ cell types appear similar in all groups\u003cstrong\u003e. B-D: \u003c/strong\u003eCounts of different inflammatory cells per area visualized in PNS tissue sections (n=4 mice per group) as indicated, show no significant changes across groups for CD20+ and CD45+ cells, while CD68+ macrophage numbers are significantly elevated in all neural tissues of \u003cem\u003eSh3tc2\u003c/em\u003e-/- mice compared to WT, without differences between vector and buffer injected animals, except for decreased numbers in femoral nerves of some treated compared to buffer injected animals. There is also a trend for increased CD3+ T cell numbers in \u003cem\u003eSh3tc2\u003c/em\u003e-/- mice compared to WT. (One-way ANOVA and Tukey’s post hoc test, *: p\u0026lt;0.05; **:p\u0026lt;0.01, ***:p\u0026lt;0.001). Scale bars: 20 μm.\u003c/p\u003e","description":"","filename":"Figure4GEORGIOUCING.png","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/78e144c4dac5d25ae610c44f.png"},{"id":100178236,"identity":"6ea1b6c1-0908-4dc8-a967-ebd78d22f5cf","added_by":"auto","created_at":"2026-01-13 18:34:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10194959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of proteomic profiling on TA muscle derived from WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSh3tc2\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice without and with gene therapeutic intervention\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the applied analytical approach. (\u003cstrong\u003eB\u003c/strong\u003e) Abundance plots showing the dynamic range of all proteins identified in protein extracts of TA muscles via liquid chromatography coupled to tandem mass spectrometry based on their relative quantification using the 3 highest abundant peptides for each protein and allowing protein comparison within an experiment. All identified proteins of the WT mice (black) are sorted with decreasing abundance while the different conditions of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice\u003cstrong\u003e \u003c/strong\u003e(red) were respectively plotted in the same order to directly compare the different abundances. All identified proteins cover a dynamic range of seven orders of magnitude. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap illustrating the respective of the statistically significantly dysregulated 58 proteins across the different subgroups. (\u003cstrong\u003eD\u003c/strong\u003e) GO-term based \u003cem\u003ein silico\u003c/em\u003e analysis of proteomic findings depicting biological processes and cellular compartments affected by the dysregulation of the 58 proteins.\u003c/p\u003e","description":"","filename":"Figure5GEORGIOUCING.png","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/f7408f572a96a8d73cfcbe81.png"},{"id":109054051,"identity":"79e2660f-664a-4aad-ac82-4d5e4ef6636e","added_by":"auto","created_at":"2026-05-12 07:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":82624357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/75d73221-3253-40c3-aa97-98d7cc0bcd0b.pdf"},{"id":100178231,"identity":"5715f9ee-0e0c-4ab4-acf7-406874363db6","added_by":"auto","created_at":"2026-01-13 18:34:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1732878,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8334328/v1/8109b9f247bb0022ae11674f.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential conflict of interest.\nThe study is part of a PCT/EP2020/065312 application in which AK, IS and KAK are co-inventors.","formattedTitle":"A dose-escalation and safety study of gene therapy for CMT4C neuropathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCharcot-Marie-Tooth disease type 4C (CMT4C) is the most common autosomal recessively inherited demyelinating neuropathy (1-3) Patients with CMT4C usually present in the first decade of life with delayed walking, progressive distal muscle atrophy and weakness, areflexia and sensory loss. Almost all patients develop foot deformities and early onset spinal deformities with scoliosis, often requiring surgery (4-6). Cranial nerve involvement with hearing loss, slow pupillary light reflexes, and lingual fasciculations is also characteristic (7) (8) (9). A combination of proprioceptive loss and vestibular neuropathy may cause disabling imbalance early in disease evolution (10). Electrophysiological studies in CMT4C patients confirm the demyelinating process with mean median motor nerve conduction velocity (MNCV) of 22.6 m/s. Rare cases with upper limb MNCVs \u0026gt;38 m/s have been reported (9). Nerve biopsies are characterized by an increase of basement membranes around myelinated, demyelinated, and unmyelinated axons, relatively few onion bulbs, and, most typically, large cytoplasmic extensions of Schwann cells (1, 4, 5). The clinical characteristics and severity of CMT4C are quite variable along with phenotypic variations within families (11) Clear relationships between pathogenic variants and the spectrum of disease manifestations are to date lacking, although recent studies in large patient series suggest the possibility of a milder phenotype in individuals with one or two non-protein-truncating variants (11).\u003c/p\u003e\n\u003cp\u003eCMT4C is caused by bi-allelic, loss of function variants in the SH3 domain and tetratricopeptide repeats 2 (\u003cem\u003eSH3TC2\u003c/em\u003e) gene\u0026nbsp;(1, 12). At least 28 different \u003cem\u003eSH3TC2\u003c/em\u003e mutations have been described to date, mostly truncating but also missense, with higher frequency among certain ethnic groups (13) likely due to founder effects (7). The most prevalent pathogenic \u003cem\u003eSH3TC2\u003c/em\u003e variants are Arg954Ter (especially North African, Dutch and French-Canadian populations) and Arg1109Ter (commonly found in the Spanish Romani population), likely due to founder effects (6, 14, 15). SH3TC2 is well conserved among vertebrate species, whereas no non-vertebrate orthologs were identified. \u003cem\u003eSH3TC2\u003c/em\u003e is exclusively expressed in myelinating Schwann cells and affects receptor dynamics by regulating endosome recycling (16, 17). The protein is localized to the plasma membrane and perinuclear recycling compartment. \u003cem\u003eIn\u003c/em\u003e \u003cem\u003evitro\u0026nbsp;\u003c/em\u003estudies demonstrated that SH3TC2 interacts with Rab11, a small GTPase that regulates the recycling of membranes and receptors to the cell surface. Current hypotheses suggest that recycling endosomes play a central role in protein sorting and trafficking, both during plasma membrane recycling and as an intermediate step during cargo transport from the trans-Golgi network to the plasma membrane (18). SH3TC2 could be transported from endosomal storage sites to the plasma membrane when needed for myelin formation, as described recently for proteolipid protein (PLP) in oligodendrocytes (19). Otherwise, SH3TC2 could be involved in the regulation of cargo transport through the recycling endosome.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003estudies have shown that both the more common protein truncating nonsense and frameshift variants ending in premature stop codon, as well as rarer missense variants, all lead to loss of the \u003cem\u003eSH3TC2\u0026nbsp;\u003c/em\u003eprotein function in endosomes and abate protein–protein interactions between SH3TC2 and RAB7 that disturbs the endocytic and membrane recycling pathway (20). Thus, the endocytic and membrane trafficking pathway may be involved in the pathogenesis of CMT4C disease, and SH\u003cem\u003e3TC2\u003c/em\u003e missense mutations could impair the communication between the Schwann cell and the axon causing abnormal myelin formation. \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice represent a well characterized model of CMT4C developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. Ultrastructural analysis of myelinated fibers in this model showed abnormal organization of the node of Ranvier, a phenotype that was also confirmed in nerve biopsies from CMT4C patients, further supporting the crucial role of SH3TC2 in myelination and in the integrity of the node of Ranvier (16). Thus, \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice recapitulate all major features of CMT4C disease and provide a relevant model to test therapies.\u003c/p\u003e\n\u003cp\u003eSince loss of\u0026nbsp;SH3TC2 function in myelinating cells appears to be the cause of CMT4C, we have developed a gene replacement therapy using intrathecally injected adeno-associated viral (AAV) vector 9 (21-25) that provides Schwann cell-specific expression of SH3TC2 when driven by a minimal version of the rat myelin-specific myelin protein zero (Mpz) promoter (miniMpz) (26). This vector provides high expression levels, while its episomal persistence without integration into the host genome increases the safety of \u003cem\u003ein vivo\u003c/em\u003e delivery (27, 28). The AAV9 serotype is already approved for clinical application in patients with spinal muscular atrophy (SMA) after extensive safety evaluation in pre-clinical (29) (30) and in Phase 1 (NCT02122952) and Phase 3 (NCT03461289) clinical trials, as well as in a Phase 1/2 clinical trial for the treatment of giant axonal neuropathy (GAN) (NCT02362438). Furthermore, AAV9 has shown effective Schwann cell targeting in our preclinical studies in other models for gene replacement in CMT1X (24) and gene silencing in CMT1A (31) demyelinating neuropathies.\u003c/p\u003e\n\u003cp\u003eIn our recent proof of concept study in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, treatment with AAV9-miniMpz.\u003cem\u003eSH3TC2\u003c/em\u003e.SV40pA resulted in significant functional and morphological improvements when delivered at early as well as at late stages of the neuropathy (26). To further advance this approach towards clinical translation, we used in the current study a modified AAV9 viral vector, with a human minimal \u003cem\u003eMPZ\u003c/em\u003e promoter driving the expression of \u003cem\u003eSH3TC2\u003c/em\u003e with modification of polyA sequence for enhanced expression, and performed for the first time a dose escalation and safety study in the CMT4C mouse model. Lumbar intrathecal administration of modified AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e resulted in Schwann cell specific expression of SH3TC2 and in statistically significant improvement in nerve conduction velocities and in various motor behavioural tests. No adverse effects were observed, as demonstrated by extensive histopathological examination and analysis of possible inflammatory reaction in neural and peripheral tissues. These results indicate effective targeting of Schwann cells and a clear dose-response up to the highest dose tested that is still in clinically feasible range, supporting the clinical translation of this approach for the treatment of CMT4C.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eCloning of a clinical stage\u003c/b\u003e \u003cb\u003ehMPZmin\u003c/b\u003e.\u003cb\u003eSH3TC2.SV40pA\u003c/b\u003e \u003cb\u003eexpression cassette\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn our previous work (26) a minimal version of the rat myelin protein zero (miniMpz/P0) promoter was generated and used to drive expression of myc-tagged SH3TC2 in Schwann cells. As a humanized promoter would be preferable for further clinical translation and for the final testing process beyond the mouse model (toxicity, NHP), the rat miniMPz/P0 was replaced with a human minimal \u003cem\u003eMPZ\u003c/em\u003e promoter (designated \u003cem\u003ehMPZmini\u003c/em\u003e). The human \u003cem\u003ehMPZmini\u003c/em\u003e promoter was PCR amplified from a construct that contained the full sequence of human \u003cem\u003eMPZ\u003c/em\u003e promoter introducing also the \u003cem\u003eKpn\u003c/em\u003eI restriction site on each end. The 429bp DNA was ligated to the previously used AAV transfer plasmid that carried the human \u003cem\u003eSH3TC2\u003c/em\u003e open reading frame (ORF). In addition, the myc tag at the end of the SH3TC2 coding sequence and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were removed. Finally, the polyA tail was replaced by an SV40pA sequence. For this modification, the \u003cem\u003ehMPZmini.SH3TC2\u003c/em\u003e insert was cloned into the Virovek single-stranded AAV backbone V445-pFB-GFP using restriction site enzyme/site Spel and BspEI/AgeI. Moreover, a mock vector that contained the human mini MPZ/P0 promoter to drive the expression of EGFP was generated. The \u003cem\u003ehMPZmini.EGFP-WPRE-bGHpA\u003c/em\u003e insert was cloned into the Virovek single- stranded AAV backbone V445-pFB-GFP using restriction site enzymes/sites SpeI and BspEI.\u003c/p\u003e\n\u003ch3\u003eLumbar intrathecal injections\u003c/h3\u003e\n\u003cp\u003eVector delivery was performed under general anaesthesia by slow intrathecal injection into the L5\u0026ndash;L6 intervertebral space of 1- or 2-month-old mice as previously described (32\u0026ndash;35). Briefly, following a small skin incision along the lower lumbar spine level to visualize the spine, the AAV9 vector was injected into the L5-L6 intervertebral space. A 50-\u0026micro;L Hamilton syringe connected to a 26-gauge needle was used to inject 20 \u0026micro;L of the vector stock with appropriate dilutions in formulation buffer PBS (PBS with 0.001% Pluronic F68) containing different total amounts ranging between 4e10 vg (low dose), 1.2e11 vg (mid dose) and 3.5e11 vg (high dose). A flick of the tail was considered indicative of successful intrathecal administration.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTreatment trial design\u003c/h2\u003e \u003cp\u003eThe aim of this study was to determine whether a dose-escalation gene addition therapy can improve the manifestations of peripheral neuropathy in the mouse model of CMT4C following intrathecal injection at the L5-L6 intervertebral space. \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were treated at the age of 1 month. A total volume of 20 \u0026micro;L containing three different vector doses ranging from 4e10 vg (low dose), 1.2e11 vg (mid dose) and 3.5e11 vg (high dose) was injected intrathecally as described above. Littermate mice were randomized to either receiving the therapeutic (full) vector AAV9-\u003cem\u003ehMPZmini.SH3TC2\u003c/em\u003e vector (treatment groups, n\u0026thinsp;=\u0026thinsp;10 animals per dose) or equal volume of the formulation buffer (serving as the control group) and were assigned a coding number for further identification. Randomization was based on animal numbering after tailing. To assess the level of phenotype correction, groups of age-matched WT mice were similarly evaluated as controls in all outcome measures.\u003c/p\u003e \u003cp\u003eAll animals were evaluated at 1 month of age (at baseline) by behavioral testing before treatment, and again at the age of 3 months, 2 months after treatment. At the end of the treatment trial, 3-month-old mice were evaluated by electrophysiology (n\u0026thinsp;=\u0026thinsp;10 per dose) and then sacrificed for quantitative morphometric analysis of myelination (n\u0026thinsp;=\u0026thinsp;10 per dose) as outlined below. All therapeutic outcome evaluations including motor performance, electrophysiology, as well as myelin morphology were performed by examiners blinded to the treatment condition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMorphometric analysis of myelination\u003c/h3\u003e\n\u003cp\u003eFor quantitative analysis of myelination, we obtained toluidine blue-stained transverse semithin sections (1 \u0026micro;m) of PNS tissues following perfusion with 2.5% glutaraldehyde and resin embedding, as previously described (26, 36). We calculated the g-ratios for all myelinated fibers in all treatment groups using the Image Pro software and a custom-made macro, which detects the axons and their myelin sheath according to color. This macro calculates g-ratio by dividing the average inner perimeter of the axon by the average outer perimeter of the axon, as well as the average myelin thickness for each myelinated fibre. Because more pronounced myelination deficits were observed in fibres\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter in the original characterization of this model (16), we also analyzed the subset of fibres\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter separately along with the analysis of all fibres\u0026thinsp;\u0026gt;\u0026thinsp;1 \u0026micro;m, as previously described (36) (26). In addition, completely demyelinated fibres (defined as axons\u0026thinsp;\u0026gt;\u0026thinsp;1 \u0026micro;m in diameter devoid of myelin sheath) in anterior lumbar roots and mid-sciatic nerves were manually counted and compared to the total fiber numbers, to obtain the ratio of demyelinated fibers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of possible toxicity and SH3TC2 expression in the PNS and peripheral organs of\u003c/b\u003e \u003cb\u003eSh3tc2\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice injected with different therapeutic vector doses.\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo assess potential toxicity associated with intrathecal AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e vector injection and gene delivery to Schwann cells as well as SH3TC2 expression was evaluated. After deparaffinisation, antigen retrieval was achieved by heating the slides for 30min in the pressure cooker in 0.1M citric acid monohydrate buffer (pH 6). Unspecific binding sites were blocked with 5% bovine serum albumin human at room temperature (RT) for 1h and slides were then incubated overnight with specific antibodies against SH3TC2 (anti-rabbit 1:100; Abcam, Cambridge, UK), CD45 (anti-rat 1:100; Abcam, Cambridge, UK), CD3 (anti-rabbit 1:100; Abcam, Cambridge, UK), CD68 (anti-rabbit 1:100; Abcam, Cambridge, UK), and CD20 (anti-goat 1:100; Santa-Cruz, USA) followed by appropriate secondary antibodies at room temperature. Slides were then washed in PBS and incubated with mouse cross-affinity fluorescein-conjugated (1:3000; Invitrogen, A21202), rabbit fluorescein (FITC)-conjugated (1:1000; Jackson ImmunoResearch, 111-486-003), rabbit cross-affinity purified rhodamine-conjugated (1:3000; Jackson ImmunoResearch, 111-026-003), mouse cross-affinity purified rhodamine-conjugated (1:1000; Jackson ImmunoResearch,115-026-068) and goat fluorescein (FITC)-conjugated (1:700; Jackson ImmunoResearch, 111-486-003). Slides were mounted with fluorescent mounting medium (DAKO) and images photographed under a fluorescence microscope (Nikon Eclipse Nἱ) with a digital camera (DS-Qi2) using NIS-Elements software.\u003c/p\u003e \u003cp\u003eWe first assessed inflammatory changes in tissues at low magnification (x10) and specific areas were further assessed at a higher magnification (x20 \u0026amp; x40). To assess possible inflammatory responses, paraffin sections were stained for the following markers: CD3 (T lymphocytes), CD68 (macrophages), CD45 (leukocytes) and CD20 (B-lymphocytes). For neural tissues, images of whole transverse nerve sections, femoral nerve and multiple anterior lumbar spinal roots per mouse were obtained at x20 magnification. The number of CD-positive cells were counted in all images and normalized to the total tissue area analyzed. For peripheral non-neural tissues, a total of 3 images were obtained per tissue section, per mouse, at x20 magnification.\u003c/p\u003e\n\u003ch3\u003eMuscle proteomic analysis\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eSample Preparation\u003c/strong\u003e \u003cp\u003eMurine TA muscle was snap-frozen in liquid nitrogen and stored at -80\u0026deg;C until further processing. Tissue lysis was performed using 200 \u0026micro;L of lysis buffer containing 50 mM TEAB (pH 7.8), 5% SDS, and cOmplete ULTRA protease inhibitor (Roche). The samples were homogenized with a Bioruptor\u0026reg; (Diagenode) for 10 minutes (30-second cycles of sonication and rest) at 4\u0026deg;C. To ensure complete lysis, an additional sonication step using an ultrasonic probe (30 seconds, 1-second pulse on/off, 40% amplitude) was conducted, followed by centrifugation at 20,000 g for 15 minutes at 4\u0026deg;C. The protein concentration of the resulting supernatant was measured using the BCA assay according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eDisulfide bonds were reduced with 10 mM TCEP at 37\u0026deg;C for 30 minutes, and free sulfhydryl groups were alkylated with 15 mM IAA in the dark at room temperature for 30 minutes. Proteolysis was performed using the S-Trap protocol (Protifi) with 100 \u0026micro;g of protein per sample and a trypsin-to-protein ratio of 1:20. The samples were incubated with trypsin at 42\u0026deg;C for 2 hours, and proteolysis was terminated by acidifying the samples to pH\u0026thinsp;\u0026lt;\u0026thinsp;3.0 with formic acid (FA).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAssessment of Digestion Efficiency\u003c/strong\u003e \u003cp\u003eProteolytic digests were desalted and analyzed for completeness using monolithic column separation (PepSwift monolithic PS-DVB PL-CAP200-PM, Dionex) on an Ultimate 3000 HPLC system (Dionex, Germering, Germany). For quality control, 0.5 \u0026micro;g of each sample was injected. Chromatographic separation was performed using a binary gradient with solvent A (0.1% TFA) and solvent B (0.08% TFA, 84% acetonitrile), with a gradient from 5\u0026ndash;12% solvent B over 5 minutes, followed by 12\u0026ndash;50% solvent B over 15 minutes, at a flow rate of 2.2 \u0026micro;L/min and 60\u0026deg;C. UV detection was conducted at 214 nm as described (37).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLC-MS/MS Analysis\u003c/strong\u003e \u003cp\u003ePeptide samples (1 \u0026micro;g per injection) were analyzed using an UltiMate 3000 RSLC nano UHPLC system coupled to a Q Exactive HF mass spectrometer. Peptides were first loaded onto a 75 \u0026micro;m \u0026times; 2 cm, 100 \u0026Aring;, C18 precolumn at a flow rate of 10 \u0026micro;L/min for 20 minutes, followed by separation on a 75 \u0026micro;m \u0026times; 50 cm, 100 \u0026Aring;, C18 analytical column at 250 nL/min. A linear gradient of solution A (99.9% water, 0.1% formic acid) and solution B (84% acetonitrile, 15.9% water, 0.1% formic acid) was applied, progressing from 3% to 45% solution B over 120 minutes, with washing steps at 95% solution B and equilibration of the system between runs.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eData were acquired in data-independent acquisition (DIA) mode spiked with iRT standards (Biognosys). Full MS scans were performed from 300\u0026ndash;1100 m/z at a resolution of 60,000 (Orbitrap) using a lock mass of 445.12002 m/z (polysiloxane ion). The AGC target was set to 3 \u0026times; 10⁶, with a maximum injection time of 20 ms. DIA windows were set to cover 400\u0026ndash;1100 m/z in 23 variable windows of 28 m/z width with 1 m/z overlap, collected at a resolution of 30,000 (Orbitrap), an AGC target of 3 \u0026times; 10⁶, and a normalized collision energy (nCE) of 27.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData Analysis\u003c/strong\u003e \u003cp\u003eDIA data were analyzed using Spectronaut software (Biognosys, v18.7) and analyzed with a direct-DIA based search with default search and extraction settings (BGS Factory settings). The mouse proteome reference database (UniProt) was employed. For label-free quantification, only proteins identified with at least two unique peptides were considered. Average normalized protein abundances were calculated, and log2 ratios were determined for comparisons between the different conditions.\u003c/p\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eGeneration and\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003evalidation of a human minimal MPZ promoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo first validate the newly cloned minimal fraction of the human MPZ promoter, the AAV9-\u003cem\u003ehMPZmini.EGFP\u003c/em\u003e vector (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e) was delivered by lumbar intrathecal injection into 2-month-old WT mice. Vector biodistribution and EGFP expression were analyzed 6 weeks after injection. Vector genome copy numbers (VGCNs) were detected in DNA extracted from peripheral nervous system (PNS) tissues with a gradient from injection site toward the distal nerves, reaching 1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 in lumbar spinal roots, 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 in sciatic nerves, and 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 in femoral nerves (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B, n\u003c/b\u003e\u0026thinsp;=\u0026thinsp;3 mice; one-way ANOVA F (3,8)\u0026thinsp;=\u0026thinsp;10,54, p\u0026thinsp;=\u0026thinsp;0.0037). Examination of fixed lumbar spinal root sections attached to the spinal cord, and of bilateral sciatic nerve sections revealed widespread expression of the EGFP reporter gene in the perinuclear cytoplasm restricted to myelinating Schwann cells in all PNS tissues of the injected mice, in contrast to non-injected controls (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u0026ndash;1F\u003c/b\u003e). The percentage of EGFP-expressing cells reached an average of 53.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56% in anterior lumbar roots and 71.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66% in sciatic nerves (\u003cb\u003eSupplementary Fig.\u0026nbsp;1G, n\u003c/b\u003e\u0026thinsp;=\u0026thinsp;3 mice; Mann-Whitney U test p\u0026thinsp;=\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAAV9-\u003c/b\u003e \u003cb\u003ehMPZmini.SH3TC2.SV40pA\u003c/b\u003e \u003cb\u003etherapeutic vector cloning and expression in\u003c/b\u003e \u003cb\u003eSh3tc2\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026ndash;/\u0026ndash;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe novel \u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e therapeutic expression cassette (\u003cb\u003eSupplementary Fig.\u0026nbsp;1H\u003c/b\u003e) was successfully packaged into the AAV9 capsid with vector production achieving titers of 1.75x10^13vg/ml. A total of 3.5x10^11 vg in a volume of 20 \u0026micro;l was delivered by lumbar intrathecal injection into 2-month-old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;3 mice). Vector biodistribution and expression was examined 6 weeks after injection. DNA extracted from spinal roots and sciatic nerves of injected mice showed high levels of vector biodistribution throughout the PNS with VGCNs reaching 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 in spinal roots, 0.392\u0026thinsp;\u0026plusmn;\u0026thinsp;0.173 in sciatic nerves and 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 in femoral nerves (\u003cb\u003eSupplementary Fig.\u0026nbsp;1I, n\u003c/b\u003e\u0026thinsp;=\u0026thinsp;3 mice; one-way ANOVA F (3,8)\u0026thinsp;=\u0026thinsp;0.8706, p\u0026thinsp;=\u0026thinsp;0.4953). Dose-dependent vector biodistribution was also detected in CNS and peripheral organ tissues (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A-G\u003c/b\u003e). Immunostaining for human SH3TC2 in lumbar spinal root sections, as well as in sciatic nerve sections and teased fibers confirmed the presence of virally expressed SH3TC2 in the perinuclear Schwann cell cytoplasm in all PNS tissues, in a characteristic perinuclear granular appearance, and occasionally along the entire length of the Schwann cell, while it was absent from tissues of non-injected \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice stained as negative controls (\u003cb\u003eSupplementary Fig.\u0026nbsp;1J-M\u003c/b\u003e). Quantification of the percentage of SH3TC2-immunoreactive Schwann cells showed average expression rates of 45.51\u0026thinsp;\u0026plusmn;\u0026thinsp;7.60% in spinal roots and 54.57\u0026thinsp;\u0026plusmn;\u0026thinsp;11.15% in sciatic nerves (\u003cb\u003eSupplementary Fig.\u0026nbsp;1N, n\u003c/b\u003e\u0026thinsp;=\u0026thinsp;3 mice; Mann-Whitney U test p\u0026thinsp;=\u0026thinsp;0.7). Thus, we were able to achieve adequate therapeutic vector biodistribution and high SH3TC2 expression rates in Schwann cells of the CMT4C model.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDose-dependent Schwann cell targeting and SH3TC2 expression in the PNS of the CMT4C model\u003c/h2\u003e \u003cp\u003eAfter confirming the high expression efficiency of the novel therapeutic vector AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e, we performed a randomized, controlled treatment trial at 1 month of age. The level of phenotypic rescue in treated animals was compared with littermates injected with the formulation buffer alone by motor behavioral, electrophysiological, and morphological studies 2 months after treatment. For the dose escalation gene therapy trial, three dose groups (4e10 vg, 1.2e11 vg, and 3.5e11 vg/animal; n\u0026thinsp;=\u0026thinsp;10 per dose group) of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice along with age-matched WT mice (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;6) were assessed by behavioral testing at the age of 1 (before injection) and at 3 months followed by electrophysiological evaluation at 3 months of age (2 months after injection), and then sacrificed for quantitative morphometric analysis. Additional groups of mice (n\u0026thinsp;=\u0026thinsp;4 per dose) were injected with the therapeutic vector at the low, mid and high vector doses, in which we evaluated PNS biodistribution and expression rates of SH3TC2 in myelinating Schwann cells in lumbar roots, sciatic and femoral nerves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). SH3TC2 expression was detected in all PNS tissues examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). VGCNs determined 8 weeks after injection reached 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 (low dose), 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 (mid dose), 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 (high dose) in lumbar roots (n\u0026thinsp;=\u0026thinsp;4 mice, one-way ANOVA F(2,9)\u0026thinsp;=\u0026thinsp;0.9184, p\u0026thinsp;=\u0026thinsp;0.4336); 0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 (low dose), 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (mid dose), 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (high dose) in the sciatic nerves (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 mice, one-way ANOVA F(2,7)\u0026thinsp;=\u0026thinsp;4.525, p\u0026thinsp;=\u0026thinsp;0.0548); and 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (low dose), 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (mid dose), 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 (high dose) in the femoral nerves (n\u0026thinsp;=\u0026thinsp;4; one-way ANOVA F(2,9)\u0026thinsp;=\u0026thinsp;1.609, p\u0026thinsp;=\u0026thinsp;0.2527) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). The percentage of SH3TC2-expressing cells reached an average of 16.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11% (low dose), 42.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78% (mid dose) and 59.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.60% (high dose) in anterior lumbar roots (one-way ANOVA F(2,9)\u0026thinsp;=\u0026thinsp;0.9184, p\u0026thinsp;=\u0026thinsp;0.4336); 18.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47% (low dose), 38.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84% (mid dose) and 48.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41% (high dose) in the sciatic nerves (one-way ANOVA F(2,9)\u0026thinsp;=\u0026thinsp;26, p\u0026thinsp;=\u0026thinsp;0.0002); and 17.38\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05% (low dose), 37.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90% (mid dose) and 37.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.52% (high dose) in the femoral nerves (one-way ANOVA F(2,9)\u0026thinsp;=\u0026thinsp;13,55, p\u0026thinsp;=\u0026thinsp;0.0019) (n\u0026thinsp;=\u0026thinsp;4 mice; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFunctional improvements following dose escalation treatment in the CMT4C model\u003c/h3\u003e\n\u003cp\u003eWe have focused our behavioral analysis of treated, buffer-treated and WT animals on testing motor strength and coordination. Rotarod tests at low and high speeds showed at baseline, before treatment, significantly worse motor performance with less time staying on the rotarod in all \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse groups compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;3.572, p\u0026thinsp;=\u0026thinsp;0.0137), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;3.562, p\u0026thinsp;=\u0026thinsp;0.0139). At 2 months after treatment with AAV9-\u003cem\u003ehMPZmini.SH3TC2, Sh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003emice showed improvement of rotarod performance approaching the performance of age-matched WT mice at both speeds tested, whereas formulation buffer-treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed deterioration over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;2.011, p\u0026thinsp;=\u0026thinsp;0.1109), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;2.181, p\u0026thinsp;=\u0026thinsp;0.0881), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F). Likewise, foot grip strength at age 1 month, at baseline before starting treatment, showed lower strength generated by \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse groups compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;11.53, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) whereas at age 3 months (2 months post-injection) muscle strength improved in mid- and high vector dose treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared with the low dose and buffer group and reached WT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;8.164, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Longitudinal comparison also demonstrated that the strength produced by the hindlimbs improved with time in mid- and high vector dose treated mice in contrast to buffer and low dose treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNerve conduction studies in 3-month-old mice (2 months after treatment) revealed significantly higher sciatic nerve MNCVs in AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e-treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice reaching 37.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 m/s (low dose), 35.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 m/s (mid dose), 32.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 m/s (high) compared with the buffer-treated group (23.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 m/s), although not reaching those of age-matched WT animals (41.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 m/s) (one-way ANOVA F(4,39)\u0026thinsp;=\u0026thinsp;42.13, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). There was also a trend for higher compound muscle action potential (CMAP) amplitude in treated mice reaching 3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mV (low dose group), 3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 mV (mid), and 3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 mV (high), compared to the buffer group (3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 mV), but without reaching statistical significance and also remaining below WT levels (4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 mV) (one-way ANOVA F (4,41)\u0026thinsp;=\u0026thinsp;1.865, p\u0026thinsp;=\u0026thinsp;0.1350) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003eSince axonal loss and neuromuscular junction (NMJ) denervation leading to muscle atrophy is a common pathological process correlating with clinical weakness in all patients with peripheral nerve diseases including CMT4C (Cipriani et al 2018), we further evaluated the degree of proximal and distal hind limb muscle atrophy in our \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e treatment groups. At 3 months of age, 2 months post injection, the quadriceps and tibialis anterior muscles showed significantly increased muscle weight in treated mice injected with the mid and the high dose compared to buffer treated controls or to mice injected with the low dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, one-way ANOVA F(4,33)\u0026thinsp;=\u0026thinsp;8.478, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM, one-way ANOVA F(4,32)\u0026thinsp;=\u0026thinsp;2.680, p\u0026thinsp;=\u0026thinsp;0.0493) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-M).\u003c/p\u003e \u003cp\u003eFinally, in addition to these functional evaluations, we also observed an apparent improvement in the phenotype of abnormal clenching of toes and clasping of hind limbs upon suspension by the tail, which was previously described in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Arnaud et al., 2009). Treated animals were able to stretch their legs and toes much more compared to buffer-treated littermates, and did not differ from WT mice. This phenotype was quantified by measuring the hind limb angle as an indicator of limb opening ability, which confirmed significant improvements in treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO, one-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;10.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-O).\u003c/p\u003e\n\u003ch3\u003eDose-dependent improvement of PNS myelination in treated CMT4C mice\u003c/h3\u003e\n\u003cp\u003eMorphological examination was carried out in transverse toluidine stained semithin sections of anterior lumbar motor roots and femoral motor nerves of 3-month-old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice injected with the therapeutic vector or with formulation buffer at age 1 month (n\u0026thinsp;=\u0026thinsp;10 per dose group) and in tissues of WT mice (n\u0026thinsp;=\u0026thinsp;5). In all PNS tissues evaluated, dose-dependent improvement of myelination in treated as opposed to buffer-treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was observed but without reaching WT levels. In lumbar roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), average g-ratios of myelinated fibers\u0026thinsp;\u0026gt;\u0026thinsp;1 \u0026micro;m in diameter in the treated groups were 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (low dose), 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (mid dose), and 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (high), compared with 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the buffer-treated group and 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;10.81, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). When considering only the subset of fibers\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter, which are more affected by demyelination in this model (Arnaud et al., 2009), average g-ratios reached 0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (low dose), 0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (mid dose), and 0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (high dose) in treated mice compared with 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the buffer group and 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;25.53,p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C \u003cb\u003eand Supplementary Fig.\u0026nbsp;3A-B\u003c/b\u003e). Corresponding with the g-ratio reductions, average myelin thickness increased in treated mice reaching 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;m (low dose), 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (mid dose) and 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (high dose) compared with the buffer group (0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;m), with the high dose mice approaching the WT group (0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;8.365, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In the subset of fibers\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m, myelin thickness in treated mice increased to 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;m (low dose), 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (mid dose) and 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (high dose), compared with the buffer group (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m), and almost reached the WT group 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;11.44. p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Furthermore, the percentage of completely demyelinated fibers was significantly reduced in treated mice to 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 (low dose), 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 (mid dose), 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 (high dose), compared with 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the buffer group and 0.0003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0003 in the WT group (one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;9.057, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Furthermore, axonal profiling analysis revealed a shift of axonal diameter distribution toward the population of smaller axons (\u0026lt;\u0026thinsp;5 \u0026micro;m) in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to WT lumbar roots, without significant correction after treatment (two-way ANOVA F(4,327)\u0026thinsp;=\u0026thinsp;1.088, p\u0026thinsp;=\u0026thinsp;0.3624).(\u003cb\u003eSupplementary Fig.\u0026nbsp;3C\u003c/b\u003e). The observed profile changes, likely reflecting hypomyelination, did not lead to significant axonal loss in either treated or buffer-treated animals compared with WT, as determined by similar total number of axons per lumbar root in all groups (two-way ANOVA F(4,41)\u0026thinsp;=\u0026thinsp;1.469, p\u0026thinsp;=\u0026thinsp;0.2293) (\u003cb\u003eSupplementary Fig.\u0026nbsp;3D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn femoral motor nerves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) a similar dose-dependent improvement of myelination was observed in treated CMT4C mice with g-ratios reaching 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (low dose), 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (mid) and 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (high dose) in treated compared to 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in buffer-treated mice and 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, one-way ANOVA F(4,42)\u0026thinsp;=\u0026thinsp;1.557, p\u0026thinsp;=\u0026thinsp;0.2035). In the subset of fibers\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter the corresponding g-ratios also showed a dose-dependent reduction reaching 0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (low), 0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (mid), and 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (high dose) in treated compared to 0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in buffer treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;12.18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I \u003cb\u003eand Supplementary Fig.\u0026nbsp;4A-B\u003c/b\u003e). Average myelin thickness increased in the treated groups to 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (low), 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;m (mid) and 0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m (high) compared to 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m in the buffer group and 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, one-way ANOVA, F(4,40)\u0026thinsp;=\u0026thinsp;8.208, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In fibers\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter, again a dose-dependent increase in myelin thickness was found reaching 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m in low dose, 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;m in mid dose, and 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;m in high dose treated group, compared to 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mm in the buffer group and 0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 in WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;21.45, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-K). As in the lumbar roots, the ratio of demyelinated fibers in femoral nerves decreased to 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 (low), 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 (mid) and 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 (high) in treated compared with 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 in the buffer treated group, while no demyelinated fibers were found in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL one-way ANOVA F(4,40)\u0026thinsp;=\u0026thinsp;8.946, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Axonal profiling analysis revealed a shift of axonal diameter distribution toward the population of smaller axons (\u0026lt;\u0026thinsp;2 mm) in all \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e femoral nerves compared with WT mice, without significant correction in treated animals (\u003cb\u003eSupplementary Fig.\u0026nbsp;4C\u003c/b\u003e, two-way ANOVA F(4,336)\u0026thinsp;=\u0026thinsp;3.303, p\u0026thinsp;=\u0026thinsp;0.0113). As in the lumbar roots, no significant axonal loss was found in \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e femoral nerves compared with WT mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;4D\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of inflammatory response and tissue integrity in treated CMT4C mice\u003c/h2\u003e \u003cp\u003eTo assess for potential toxicity in neural and peripheral organ tissues, \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were injected intrathecally with the AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e vector at the low (4e10 vg), mid (1.2e11 vg) and high (3.5e11 vg) doses (n\u0026thinsp;=\u0026thinsp;4 mice per dose) and sacrificed 8 weeks post injection, at which time point the maximum of possible immune response is expected (38). Buffer injected littermate \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;4) were used as controls, as well as additional groups of WT mice and untreated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice of the same age. Assessment of inflammatory markers in neural tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D) revealed only mildly elevated numbers of CD68\u0026thinsp;+\u0026thinsp;macrophages in all PNS tissues of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to WT mice, without any difference between untreated, buffer- or vector-injected groups, indicating that this difference likely reflects a feature of CMT4C PNS pathology and not a reaction to vector injection. Interestingly, treated mice showed reduced macrophage numbers in the femoral nerve compared to buffer injected controls, suggesting an improvement of nerve pathology and secondary inflammation. No significant elevation of CD68\u0026thinsp;+\u0026thinsp;macrophages or any of the other cell types (CD3+, CD20\u0026thinsp;+\u0026thinsp;or CD45\u0026thinsp;+\u0026thinsp;cells) was found in any of the peripheral organ tissues examined from vector injected compared to buffer control \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or WT mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A-F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate overall tissue integrity in treated mice, paraffin sections were stained with H\u0026amp;E and visualized under the light microscope. This study revealed no evidence of impaired tissue integrity in neural tissues (\u003cb\u003eSupplementary Fig.\u0026nbsp;6A)\u003c/b\u003e or in peripheral organs (\u003cb\u003eSupplementary Fig.\u0026nbsp;6B, 7\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImprovement of muscle proteomic profiles in treated CMT4C mice\u003c/h2\u003e \u003cp\u003eWe performed proteomic profiling on tibialis anterior muscle (total of n\u0026thinsp;=\u0026thinsp;26 samples) to investigate the effect of the different treatment regimens on reinnervation-based restoration of proteostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our label-free proteomic profiling approach allowed the robust quantification of a total of 18115 unique peptides referring to 1487 proteins (1199 quantifiable proteins) spanning 7 orders of magnitude across the different quantification approaches (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To visualize protein changes underlying de- and re-innervation, we generated heat maps on one hand covering all proteins identified across the different quantification approaches (\u003cb\u003eSupplementary Fig.\u0026nbsp;8\u003c/b\u003e) and on the other hand, covering \u0026ldquo;only\u0026rdquo; proteins displaying a statistically significant dysregulation across these group quantifications. The latter approach included 58 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) covering different biological processes including translation at pre- and post-synapse, mitochondrial-based ATP production, intermediate filament organization and muscle development. Affected subcellular compartments and structures include synapses, myelin sheath, microfibrils, ribonucleoprotein complexes, nuclei (with chromatin remodelling) and mitochondria as illustrated by our GO-term-based \u003cem\u003ein silico\u003c/em\u003e analysis of proteomic findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The most robust and consistent differences were noticed between \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and WT animals for proteins involved in transcription including histones (H1-0, H1-1, H1-2, H1-3, H1-4 \u0026amp; H1-5), Elongin-C, Splicing factor U2AF 65 kDa subunit, in translation (Rpl15 \u0026amp; 29 \u0026amp; Ubiquitin-like FUBI-ribosomal protein eS30 fusion protein), for Serpins (Serpin3k \u0026amp; Serpinb6), for mitochondrial proteins (Cytochrome c oxidase subunit 5B \u0026amp; NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3) and proteins modulating oxidative stress burden (Thioredoxin domain-containing protein 17 \u0026amp; Carboxic anhydrase 3), Chaperones (Heat shock protein beta-6 \u0026amp;7), Isochorismatase domain-containing protein 2A and Dual specificity phosphatase 29 (DUSP29). Out of these proteins, our therapeutic intervention associated with re-innervation, in particular corrected dysregulations of proteins involved and transcription and translation whereby low dosages seem to have the most beneficial effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Of note, Kynurenine\u0026ndash;oxoglutarate transaminase 1 is increased only in the group of treated animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we have further optimized and validated a gene therapy approach to treat CMT4C, the most prevalent recessively inherited demyelinating neuropathy, arising from loss-of-function mutations in the \u003cem\u003eSH3TC2\u003c/em\u003e gene. By using a minimal version of the myelin-specific human \u003cem\u003eMPZ\u003c/em\u003e promoter (\u003cem\u003ehMPZmini\u003c/em\u003e) to drive human SH3TC2 gene expression restricted to Schwann cells, and an SV40pA sequence to enhance the expression, we completed for the first time a dose escalation and safety study in the \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e/\u0026minus;\u003c/sup\u003emouse model of the disease. This is a significant advance compared to our earlier using a non-clinical stage expression cassette based on a minimal version of the myelin specific rat \u003cem\u003eMpz\u003c/em\u003e promoter (\u003cem\u003eminiMpz\u003c/em\u003e) to drive human SH3TC2 gene tested in a single AAV9 vector dose at early and late stages of the neuropathy in the CMT4C model (Georgiou et al, 2023). We also build on our initial proof-of-concept studies using a lentiviral vector system injected intrathecally to express \u003cem\u003eSH3TC2\u003c/em\u003e under the control of the full-length Mpz promoter, showing expression restricted to Schwann cells and a therapeutic benefit (36). However, the limitations of in vivo lentiviral vector delivery (28) motivated us to explore as a clinically more translatable approach the possibility of delivering the therapeutic SH3TC2 gene using the AAV9 vector that has shown better biodistribution, improved safety profile, and higher expression levels in the PNS. (24, 32).\u003c/p\u003e \u003cp\u003eIn the current study, we tested 3 different therapeutic vector doses injected intrathecally in an effort to clarify the minimum effective as well as safe dose in the model of the disease. We demonstrate that beginning with the mid, and even more consistently with the high dose of AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e vector leads to adequate biodistribution to the peripheral nervous system and to high rates of therapeutic gene expression specifically in myelinating Schwann cells. The high dose also showed significantly higher expression rates in most PNS tissues. The therapeutic efficacy of this gene therapy approach was demonstrated using multiple functional and pathological outcome measures that are relevant not only for the phenotype of this model (Arnaud 2009) but also for the human disease CMT4C. (1, 4, 5)\u003c/p\u003e \u003cp\u003eOur results confirm that a minimal version of the human \u003cem\u003eMPZ\u003c/em\u003e promoter can provide strong gene expression specifically in myelinating Schwann cells. The minimal promoters, or core promoters, are short sequences that allow for the accurate formation of the initiation complex. These sequences play a critical role in the synthetic promoter properties, from its background expression, or leakiness, to its maximum potential induction and can maximize the transgene capacity of recombinant AAV vector genomes (39, 40) (41). Previous work has indicated that most of the functional regulatory elements and crucial transcription factor binding sites of the \u003cem\u003eMPZ\u003c/em\u003e promoter, including that of SOX10 and EGR2, are located within this distal promoter fragment downstream of the AvrII site (42). Thus, we PCR amplified this 412-bp-long sequence and validated its efficacy and fidelity initially through evaluation of the reporter gene expression. Similar to the full-length \u003cem\u003eMpz\u003c/em\u003e promoter tested previously (24, 32, 33, 36) we showed that a high percentage of myelinating Schwann cells was expressing EGFP in the PNS. Our resent work (26) showed that in the CNS, less than 2% of neurons in the lumbar spinal cord and a very low percentage of oligodendrocytes were EGFP positive, confirming the largely preserved specificity of this reduced promoter version to restrict expression in Schwann cells. Thus, we provide strong evidence for the usefulness of this minimal human PNS myelin-specific promoter to facilitate packaging of larger coding sequences into the AAV expression cassette that can be utilized not only for CMT4C treatment, but also for various gene therapy approaches to treat other demyelinating CMT neuropathies.\u003c/p\u003e \u003cp\u003eAAV vectors are leading gene therapy vehicles (43), as they offer unique advantages, such as tissue tropism, specificity in transduction, a relatively low immunogenicity, no integration into the host chromosome, and long-lasting robust transgene expression in post-mitotic cells. In addition to application in many ongoing clinical trials, different AAV serotypes have already been approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA) for the treatment of rare genetic disorders, including Leber\u0026rsquo;s congenital amaurosis (AAV2), SMA (AAV9) and Duchenne Muscular Dystrophy (AAVrh74) (44, 45). While different AAV serotypes have demonstrated varied tropism for various cells and tissues, AAV9 was found to have the highest tropism for the CNS with efficient targeting of motor neurons for \u003cem\u003eSMN\u003c/em\u003e gene replacement to treat SMA patients (46). Importantly, AAV9 has been shown to target efficiently the PNS via intrathecal (24, 26, 31, 32), intravenous (32), or intraneural administration (47) with high degree of tropism for myelinating Schwann cells, leading to therapeutic \u003cem\u003eGJB1\u003c/em\u003e gene expression controlled by the Schwann cell-specific \u003cem\u003eMpz\u003c/em\u003e promoter to treat CMT1X neuropathy in various model of the disease (24, 32) (48), or to \u003cem\u003ePMP22\u003c/em\u003e gene silencing by shRNA (47) or microRNA (31) under ubiquitous promoters.\u003c/p\u003e \u003cp\u003eIn this work, we delivered by AAV9 a minimal version of the myelin-specific human \u003cem\u003eMPZ\u003c/em\u003e promoter (\u003cem\u003ehMPZmini\u003c/em\u003e) to drive human \u003cem\u003eSH3TC2\u003c/em\u003e gene expression restricted to Schwann cells combined with an SV40pA sequence to enhance the expression level at 3 different doses via lumbar intrathecal injection. The mid (1.2x10^11 vg/animal) and even better the high (3.5x10^11 vg) doses of AAV9-\u003cem\u003ehminiMpz-SH3TC2\u003c/em\u003e.SV40pA vector led to adequate biodistribution to the PNS and to high rates of cell-specific therapeutic gene expression in myelinating Schwann cells, with the high dose showing significantly higher expression rates in most tissues. These results reproduce our previous studies using a single IT dose of 2x10^11 vg. Both the analysis of VGCNs biodistributed to the PNS tissues, as well as the percentage of transduced Schwann cells expressing the reporter or the therapeutic gene in lumbar roots and in sciatic nerves indicates a gradient of biodistribution from the site of injection, as in our previous studies (24, 32). Lumbar intrathecal delivery could be the safest and most effective route of administration to target both CNS and PNS, as it has been shown to result in widespread expression of AAV9 (49). The intravenous injection has significant disadvantages including high vector amount needed for injection in order to achieve expression in the CNS (50, 51) (21, 52, 53) with higher risk for toxicity and immune reactions, while the direct intraneural injection is more invasive and does not provide widespread expression (47, 54).\u003c/p\u003e \u003cp\u003eWe demonstrated here that virally delivered SH3TC2 localizes correctly to the perinuclear cytoplasm of myelinating Schwann cells of the PNS, as also shown in our previous studies using both lentiviral and AAV9-mediated \u003cem\u003eSH3TC2\u003c/em\u003e gene replacement, and colocalization with its interacting molecule Rab11 (26, 36). Although the overall VGCNs and SH3TC2 expression rates were not very high even in the high dose in the sciatic nerves, they were still sufficient to produce significant therapeutic effects in the CMT4C model. However, we acknowledge that phenotype rescue was not complete, similar to the results obtained previously in the CMT4C, CMT1X, and CMT1A neuropathy models (24, 36) (26, 31, 33, 34). Both in the functional and electrophysiological outcome measures, as well as in the morphological analysis of myelination, \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice treated with either of the three vector doses mostly failed to reach the levels of age-matched WT control mice. This may reflect the severity and early onset of the pathology in this model, as well as the fact that only a subset of myelinating Schwann cells was transduced.\u003c/p\u003e \u003cp\u003eIn addition, we observed that the phenotype of abnormal clenching of toes and clasping of hind limbs upon tail suspension reflecting the PNS dysfunction in this neuropathy model (16, 55) improved significantly in treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to controls. Moreover, the muscle weight of the quadricep and tibialis anterior muscles increased in treated mice and the high vector dose group reached the levels of the WT mice in contrast to buffer and low dose mice groups which had lower muscle weight, reflecting atrophy. This model of CMT4C shows neuromuscular junction (NMJ) alterations on a structural and transcriptional level that could contribute to the pathomechanisms of the disease and the resulting phenotype. Increased axon branching at the NMJ of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice has been shown hypothesized to result from adapting to the pathology, probably mediated by an increase in neurotrophic factors produced by muscle and proteomic changes in the sciatic nerve (55). These findings are also relevant for clinical translation, as muscle atrophy and intramuscular fat accumulation (IMFA) are characteristic manifestations of CMT neuropathies and can be monitored by MRI as a reliable biomarker to reflect disease severity (56\u0026ndash;58), and as a possible surrogate marker of treatment response (59). Our study further supports the use of muscle atrophy and IMFA as a biomarker in CMT neuropathies which, depending on the stage of the neuropathy, may show an early response to treatment.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSh3tc2\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice develop progressive peripheral neuropathy confirmed by decreased motor and sensory nerve conduction velocity and hypomyelination (16). We used this mouse model because it reproduces the main phenotypic features of the human disease while preserving fertility and normal lifespan. To assess functional improvement after treatment, we applied foot grip strength and rotarod analysis, which were shown to be responsive to treatment in our previous experiments using this mouse model (26, 36). Although foot grip strength testing verified significant improvement of mid- and high-dose treated mice compared with buffer-treated mice, reaching WT levels, rotarod test improvement did not reach statistical significance, likely due to increase variability within groups. Importantly, sciatic MNCV increased significantly in all doses-groups of treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, albeit not reaching WT levels. This improvement reflects not only enhanced myelination shown here, but also remodeling of the elongated nodes of Ranvier, demonstrated in our previous studies in this model (26, 36). In contrast, no significant CMAP improvement was achieved in this study with early treatment, in line with previous results showing significant CMAP increase only in the late-treated and older \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003emice (26), likely because at this age secondary axonal pathology and muscle denervation is more pronounced than in 3-month-old CMT4C mice, allowing for a clearer treatment effect on axonal pathology to be obtained.\u003c/p\u003e \u003cp\u003eThe morphometric evaluation of lumbar motor roots and femoral motor nerves focused on assessment of the hypomyelination phenotype of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and revealed dose-dependent improvement in g-ratios as well as in the average myelin thickness, in addition to reduced ratios of completely demyelinated fibers. \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice present with hypomyelination at 1 month of age, with slowly progressive demyelination ensuing overtime (16). Thus, a combination of developmental and progressive abnormalities in myelination generate the neuropathy in this model. Here we demonstrate significant but only partial improvement in treated mice in most myelination parameters when compared to mock treated and WT controls. As in our previous treatment studies (26, 36), these improvements were more significant in the subset of myelinated fibers\u0026thinsp;\u0026gt;\u0026thinsp;4 \u0026micro;m in diameter, which are known to be more affected in this CMT4C model (16).\u003c/p\u003e \u003cp\u003eSince safety and immunological reaction to vector injections for gene therapy remain an important consideration before moving to clinical translation (60), we further analysed the possible inflammatory and toxic responses caused by IT administration of the AAV9-\u003cem\u003ehminiMpz-SH3TC2\u003c/em\u003e.SV40pA vector. Our data show that overall numbers of immune cells in relevant tissues of \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice show no elevation compared to the non-injected animals. Despite the high VGCNs, no toxic or inflammatory effects were observed in the liver, likely due to the Schwann cell-specific expression of the transgene driven by the myelin-specific MPZ promoter. The same reason may also account for lack of DRG toxicity with this intrathecal approach, since DRG neurons do not express the transgene, in contrast to previous applications with reported DRG toxicity in primates with the neuronally overexpressed SMN transgene (61). This is also supported by our H\u0026amp;E staining that revealed no evidence of impaired tissue integrity in neural tissues or in relevant peripheral organs. Taken together, our results indicate that this AAV9 vector is overall safe and expected to neither cause any significant capsid-related toxicity when delivered intrathecally, as has already been demonstrated in the GAN clinical trial (62) nor any payload-related toxicity due to the cell-targeted expression. No or mild toxicity was also observed in a toxicology study after intra-cisterna magna injection in non-human primates (60) and high doses of AAV9 remained non-toxic (63). However, further studies and appropriate preventive immunosuppression protocols that have been established in other similar clinical stage gene therapy programs (62), are needed to ensure patient safety in future clinical applications for CMT4C. In particular, due to high percentage of CMT4C patients with biallelic premature stop codon variants likely resulting in lack of any SH3TC2 protein expression, stronger immunosuppressive protocols may be needed to avoid immune reactions to the virally expressed SH3TC3 protein itself.\u003c/p\u003e \u003cp\u003eTo address the beneficial effect of gene therapeutically based intervention in our CMT4C mouse model, we performed unbiased proteomic profiling on WT and \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice whereby for the latter group, untreated, mock treated and vector (different dosages) treated animals were included. Grouped protein analysis and heatmap-based visualization of proteomic findings enabled the determination of 58 relevant proteins covering different biological functions and thus cellular processes. Hereby, transcription and translation as well as mitochondrial function and associated oxidative stress as well as protein folding seem to represent the most vulnerable processes underlying denervation. Of note, these processes have already been linked to muscle denervation in studies of human musculature (64). Interestingly, our therapeutic intervention in particular corrected dysregulations of proteins involved in transcriptional and translational processes whereby the low dosage seems to have the most pronounced effect. One might speculate that this reflects the correction of the production of proteins associated with denervation such as \u0026ldquo;atrogenes\u0026rdquo; (65) or other proteins involved in atrophic remodelling of skeletal musculature. Along this line, DUSP29 showed a correction of increased abundance upon low-dosage treatment. DUSP29 is known to affect MAP kinase signalling though modulation of the MAPK1/2 cascade in skeletal muscle promoting muscle cell atrophy (66). Hence, our proteomic findings support the concept that re-innervation promoted by gene therapy corrected the pathobiochemical processes associated with muscle atrophy. These data are also in keeping with our findings showing reversal of muscular atrophy and improved muscle function in treated \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eAll data, analytical methods, and study materials are available from the corresponding author on request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eETHICAL APROVAL\u003c/h2\u003e \u003cp\u003e All experimental procedures were conducted in accordance with animal care protocols approved by the Cyprus Government\u0026rsquo;s Chief Veterinary Officer (project license CY/EXP/PR. L11/2022) according to national law and European guidelines (EC Directive 86/609/EEC).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe study is part of a PCT/EP2020/065312 application in which AK, IS and KAK are co-inventors.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis research was funded by Neurogene Inc and by Charcot-Marie-Tooth Association (CMTA Grant 2019-21 to KAK).\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eE.G. performed the experiments, acquired the data, analyzed the data, and wrote the manuscript. A.K. performed electrophysiology experiments. I.S. conducted cloning and mice PCR screening. R.P. performed paraffin embedding and sectioning. M.S. performed evaluations of control animal groups. C.T. and J.R. performed and analyzed VGCNs. A.H and A.R. performed and analyzed the proteomic analysis. K.A.K. conceptualization, supervision, project administration, writing-original draft, writing-review and editing. All authors critically reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank Dr.Kyriaki Michaelidou and Dr. Maria Zanti for their invaluable assistance with the statistical analysis of the data. Andreas Roos acknowledges the financial support of the German Society of Muscular Diseases (DGM). Andreas Hentschel gratefully acknowledges the financial support by the \u0026ldquo;Ministerium f\u0026uuml;r Kultur und \u0026ldquo;Wissenschaft des Landes Nordrhein-Westfalen\u0026rdquo;, the \u0026ldquo;Regierenden B\u0026uuml;rgermeister von Berlin- Senatskanzlei Wissenschaft und Forschung\u0026rdquo; and the \u0026ldquo;Bundesministerium f\u0026uuml;r Bildung und Forschung\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSenderek J, Bergmann C, Stendel C, Kirfel J, Verpoorten N, De Jonghe P, et al. Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy. Am J Hum Genet. 2003;73(5):1106-19.\u003c/li\u003e\n\u003cli\u003eSun B, He ZQ, Li YR, Bai JM, Wang HR, Wang HF, et al. Screening for SH3TC2 variants in Charcot-Marie-Tooth disease in a cohort of Chinese patients. 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Am J Physiol Cell Physiol. 2020;319(2):C441-C54.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CMT4C, gene therapy, SH3TC2, Schwann cells","lastPublishedDoi":"10.21203/rs.3.rs-8334328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8334328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCharcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the \u003cem\u003eSH3TC2\u003c/em\u003e gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage vector with a minimal human MPZ promoter driving expression of \u003cem\u003eSH3TC2\u003c/em\u003e. Groups of 1-month old \u003cem\u003eSh3tc2\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice were treated with 3 different doses of AAV9-\u003cem\u003ehMPZmini.SH3TC2.SV40pA\u003c/em\u003e or the formulation buffer by lumbar intrathecal injection. Outcomes were compared 8 weeks post injection by behavioral, electrophysiological, proteomics, morphological analysis and evaluation of tissue integrity and inflammatory responses. Vector biodistribution to the peripheral nerves and high rates of cell-specific therapeutic gene expression in Schwann cells resulted in significant therapeutic benefits in the CMT4C model. Treated mice showed improved motor performance in grip strength, rotarod testing and motor nerve conduction velocities. Morphological analysis revealed significant improvement in g-ratios, myelin thickness and ratios of demyelinated fibers in lumbar roots and femoral nerves of treated mice. Proteomic profiles showed correction of muscle denervation associated pathobiochemical processes in treated mice. Not observed tissue toxicity or immune reactions in neural tissues or peripheral organs. This study provides proof of principle for dose-dependent effectiveness and safety of intrathecal AAV9-mediated gene replacement paving the way for clinical translation.\u003c/p\u003e","manuscriptTitle":"A dose-escalation and safety study of gene therapy for CMT4C neuropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 18:34:48","doi":"10.21203/rs.3.rs-8334328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-02T15:07:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-25T10:39:46+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-22T11:44:31+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-20T16:35:45+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-13T19:52:38+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-09T21:56:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-08T12:46:37+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-08T12:26:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T14:13:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T14:13:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Gene Therapy","date":"2025-12-11T08:25:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3c26f118-d7f9-438c-b0c6-1203808ecbbc","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59560268,"name":"Biological sciences/Neuroscience/Peripheral nervous system"},{"id":59560269,"name":"Biological sciences/Biological techniques/Gene delivery/Genetic vectors"}],"tags":[],"updatedAt":"2026-05-12T07:14:59+00:00","versionOfRecord":{"articleIdentity":"rs-8334328","link":"https://doi.org/10.1038/s41434-026-00616-2","journal":{"identity":"gene-therapy","isVorOnly":false,"title":"Gene Therapy"},"publishedOn":"2026-05-12 04:00:00","publishedOnDateReadable":"May 12th, 2026"},"versionCreatedAt":"2026-01-13 18:34:48","video":"","vorDoi":"10.1038/s41434-026-00616-2","vorDoiUrl":"https://doi.org/10.1038/s41434-026-00616-2","workflowStages":[]},"version":"v1","identity":"rs-8334328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8334328","identity":"rs-8334328","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}