Methods and systems for organelle screening

Abstract

Method are providing such as a method for detecting axon length in a neuronal cell culture that can be used for high throughput drug screening by culturing neuronal cells derived from a subject or an induced pluripotent stem cell line; imaging the neuronal cells to determine axon length; and correlating the axon length to a presence or severity of a neurological or metabolic disorder.

Description

Atty Dkt.: 114198-0880METHODS AND SYSTEMS FOR ORGANELLE SCREENINGCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 734,006, filed December 13, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Organelle dynamics play a crucial role in cellular health as well as metabolic and neurodegenerative diseases. For example, Charcot-Marie-Tooth (CMT) disease is a debilitating inherited peripheral neuropathy that primarily affects myelination (Type I) and involves both motor and sensory neurons (Type II). It is characterized by progressive muscle weakness, atrophy, and sensory loss. In severe cases, individuals experience foot ulcerations, which can lead to amputation. Type II CMT can be caused by mutations in many proteins that impact organelle dynamics, most commonly MFN2 (i.e., “CMT2A”), which has been implicated in mitochondrial dynamics and inter-organelle contacts. A need exists in the art to understand these debilitating neurological and metabolic diseases and find safe and effective therapies. This disclosure satisfies this need and provides related advantages as well.SUMMARY OF THE DISCLOSURE

[0003] There remains an unmet need for screening platforms capable of identifying and ranking therapeutic candidates for neurological and metabolic disorders in disease-relevant neuronal models using quantitative, multiparametric phenotyping. In one aspect, the disclosed methods provide screening systems and associated methods in which patient-derived neuronal cultures, maintained in physiologically relevant formats such as neurospheres or microfluidic chips, are contacted with candidate therapeutic agents and assayed for both morphological parameters, including axon length and branching complexity, and functional parameters, including organelle mobility, morphology, and co-migration. Data from these multi-metric assays can be analyzed to generate quantitative efficacy scores that allow direct comparison and prioritization of diverse therapeutic modalities, including small molecules, protein variants, allele-specific antisense oligonucleotides, and targeted-1-4896-3340-4800.3Atty Dkt.: 114198-0880 intracellular modulators. This integrated, physiologically relevant, and quantitative screening approach enables the rapid identification of interventions capable of restoring key neuronal and organelle functions for subsequent in vivo validation.

[0004] In one aspect, Applicant studied fibroblasts from Charcot-Marie-Tooth 2 A (CMT2A) patients with mutations in the mitochondrial fusion protein MFN2 and age-matched controls and identified distinct patterns of mitochondrial dynamics. CMT2A patient cells exhibited increased mitochondrial fragmentation and clustering.

[0005] Applicant’s disclosure also provides methods and systems for the segmentation and tracking of mitochondria; however, the concepts and methods can be used to extend these capabilities to other organelles. These tools aim to enable high-throughput, unbiased quantification of complex organelle behaviors from label-free imaging data.

[0006] This platform combines cutting-edge optical engineering and machine learning to achieve an unprecedented view of organelle dynamics in the context of disease. The ability to detect cellular changes without fluorescent labels opens new avenues for studying dysfunction in living patient-derived cells with minimal perturbation. This approach has broad impact across cell biology and neuroscience, enabling new discoveries about how organelle dysfunction contributes to pathological states. Moreover, Applicant’s system can serve as a foundation for future high-throughput screening assays to identify therapeutics that modulate organelle dynamics, potentially accelerating drug discovery efforts for CMT and other neurodegenerative disorders.

[0007] Thus, in one aspect, a method of detecting at least one neurodegenerative phenotype in a neuronal cell culture is provided, the method comprising, or consisting essentially of, or yet further consisting of (a) culturing neuronal cells derived from a subject or an induced pluripotent stem cell line; (b) imaging the neuronal cells to determine a primary metric selected from: (i) axon length; (ii) organelle mobility of mitochondria or lysosomes within axons; or (iii) organelle morphology comprising mitochondrial fragmentation index, lysosomal dispersion index, or co-migration frequency; and (c) correlating the primary metric to presence or severity of a neurological or metabolic disorder. In one aspect, the neuronal cells are motor neurons or sensory neurons differentiated from patient-derived iPSCs. In another aspect, organelle mobility is measured by generating kymographs and quantifying-2-4896-3340-4800.3Atty Dkt.: 114198-0880 velocity, directionality, and pause frequency. In a yet further aspect, the disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis, or type II diabetes. In a further embodiment, organelle morphology is quantified by aspect ratio, form factor, branch length, and branch junction count.

[0008] Also provided are methods of screening candidate therapeutics for a neurological or metabolic disorder, comprising, or consisting essentially of, or yet further consisting of: (a) generating neuronal cell cultures from subjects diagnosed with, or at risk for, the disorder; (b) contacting the cultures with one or more candidate therapeutic agents; (c) imaging the cultures using quasi-simultaneous quantitative phase imaging and fluorescence microscopy to acquire high-spatiotemporal resolution time-series datasets; (d) analyzing the datasets using a deep learning model trained to extract at least one metric selected from axon length, axonal branching, neurite complexity, organelle transport velocity, or co-migration frequency; and (e) comparing the metrics in treated cultures to untreated control cultures to identify candidate agents that increase axon length and / or improve organelle transport relative to controls.

[0009] In one aspect, the candidate therapeutic agents are selected from MASM7, piperine, NGFAR100W, allele-specific antisense oligonucleotides, Rab7 GTPase inhibitors, or DeActs-ER. In another aspect, the deep learning model comprises a transformer architecture specialized for large-context morphological segmentation. In a further aspect, quantitative phase imaging and fluorescence imaging are acquired within 200 ms of each other to enable near-perfect overlay. In a yet further aspect, the disorder analysis further comprises generating a therapeutic efficacy score integrating axon length change and organelle transport improvement weighted by statistical significance.

[0010] In another aspect, methods are provided that screen candidate agents for peripheral neuropathy, comprising, or consisting essentially of, or yet further consisting of: (a) forming (b) embedding the cultures in extracellular matrix; (c) imaging axonal outgrowth networks for axon density, axon length, and organelle transport; and (d) identifying candidate agents that restore axon density or length and concomitantly improve mitochondrial or lysosomal mobility. In one embodiment, the neurospheres are embedded in Matrigel at a 1 :40 dilution in DMEM / F12. In another embodiment, the organelle transport metrics comprise both-3-4896-3340-4800.3Atty Dkt.: 114198-0880 mitochondria and lysosome velocity within axonal networks. In a further aspect, the candidate agents improve both axon branching complexity and organelle transport in treated cultures.

[0011] Microfluidic neuronal culture systems are provide, that comprise, or consist essentially of, or yet further consist of: (a) a soma compartment for neuronal cell bodies; (b) at least one axon compartment fluidly connected via microgrooves having a length between 500 pm and 1000 pm configured to permit selective axonal growth; (c) an imaging module configured for high-resolution, multimodal imaging of axons and subcellular organelles; and (d) an analysis module comprising non-transitory instructions to segment axons, quantify axon length, analyze organelle transport, and correlate the metrics to neurological or metabolic disorders. In one aspect, the imaging module comprises a confocal microscope with Airyscan super-resolution capability. In one embodiment, the analysis module includes instructions to apply pixel-wise logical operations to dual-channel images to quantify co-migration frequency. In a further embodiment, the axon compartment is configured to allow compartmentalized drug delivery for screening candidate agents.

[0012] Applicant also provides computer-implemented methods, comprising, or consisting essentially of, or yet further consisting of: (a) receiving label-free quantitative phase imaging datasets of neuronal cell cultures; (b) applying a trained deep learning model to generate virtual fluorescence signals for organelles selected from mitochondria and lysosomes; (c) automatically tracking the virtual organelles across frames to calculate transport metrics; and (d) correlating transport metrics to disorder severity or therapeutic efficacy.

[0013] In one aspect, the tracking is performed with an algorithm that surpasses IMARJS tracking accuracy in detecting lysosome mobility changes in CMT patient cells. In one embodiment, transport metrics include instantaneous velocity, mean velocity, displacement, and co-migration frequency. In another aspect, the deep learning model is trained on paired QPI and fluorescence datasets of mitochondria or lysosomes in live-cell imaging.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1: Pictorial depiction of impact of CMT2A mutations on MFN2 protein dynamics.-4-4896-3340-4800.3Atty Dkt.: 114198-0880

[0015] FIGS. 2A - 2C: Multiple CMT2A mutations disrupt mitochondrial dynamics in patient-derived fibroblasts. (FIG. 2A) MitoTracker-labeled mitochondria show shape differences between multiple CMT2A patient cells with MFN2 mutations and age-matched controls. (FIG. 2B) Mitochondrial network movement is reduced in CMT2A patients with multiple different MFN2 mutations. (FIG. 2C) Mitochondrial distribution is restricted in CMT2A patient fibroblasts, consistent with less anterograde mobility. All experiments are from at least n=3 exp. replicates, with n^ 10 cells per condition. *: p<0.05, ***: p<0.0005, ****: p<o 00005. All p-values calculated using ordinary one-way ANOVA.

[0016] FIGS. 3A - 3B: Lysosomes in CMT2A patient-derived fibroblasts display perturbed dynamics. (FIG. 3A) Lysosomal mobility was tracked in fibroblasts derived from CMT2A patients (CMT2A #1 : R364W, CMT2A #2: S249T) with multiple different MFN2 mutations. Lysosomes were segmented then tracked using Applicant’s software (FIG. 12, FIG. 2). (FIG. 3B) Lysosome transport shifted significantly in CMT2A patients but note the high amount of noise and variability in these data within and between patients, highlighting the need for higher throughput imaging methods Applicant are developing in this disclosure. N=6 biological replicates, t-test, *:p<0.05, **: p<0.01, ***: p<0.005, ****: pO.OOOl.

[0017] FIG. 4: Phototoxicity markedly impacts organelle mobility in unpredictable ways. Representative cells expressing a mitochondrial outer membrane targeted GFP (Fisl - EGFP) were imaged with simultaneous epifluorescence and quantitative phase imaging (QPI). The non-transfected neighboring cells quickly displayed reduced organelle mobility and eventual cell death, while the cells expressing Fisl-GFP remained normal over the same time. In contrast, cells expressing cytosolic GFP (EGFP) displayed reduced organelle mobility and death faster than neighboring non-transfected cells.

[0018] FIG. 5: Overview of QPI-fluorescence imaging system. A custom QPI+fluorescence microscope based on the Nikon Ti2e with a raised dual-wheel deck and PhiOptics SLIM module, with optimized optics for high spatiotemporal resolution with minimal lag between QPI and fluorescence image acquisition to ensure sufficient overlay of QPI+fluo imaging of rapidly moving organelles.

[0019] FIG. 6: Virtual staining of mitochondria from raw QPI input data. Live-cell QPI- fluorescence videomicroscopy data was used to test a deep learning-based model that predicts-5-4896-3340-4800.3Atty Dkt.: 114198-0880 mitochondria signal from raw QPI data. Fluorescence imaging of mito-GFP was used to evaluate the results, which showed a Pearson’s of 0.93.

[0020] FIG. 7: Representative QPI in CMT2A patient-derived fibroblasts reveals perturbed dynamics of all subcellular organelle features. Healthy age-matched control cells alongside CMT2A patient-derived cells show qualitative differences in organelle clustering and mobility as indicated by vector maps derived from particle image velocimetry analyses.

[0021] FIGS. 8A - 8J: Validation of Applicant’s R364W rat as a model for CMT2A. (FIGS. 8A-8B) Grip strength in WT and CMT2A rats. (FIG. 8A) Fore limb (FL) and (FIG. 8B) hind limb (HL) grip strength in CMT2A rats is significantly lower compared to WT at 16, 22, and 30w. (FIGS. 8C-8E) Compound action potential measurement (CAP) in WT and CMT2A HET rats. Peak amplitude at 22w (FIG. 8C) and 30w (FIG. 8D) is significantly lower in CMT2A rats. Onset latency did not differ between groups at 22w (FIG. 8E) but is shown to significantly increase with age (FIG. 8G) at 30w in CMT2A animals. Nerve conduction velocity at 22w (FIG. 8E) did not differ between groups but is shown to significantly decrease with age in CMT2A animals at 30w (FIG. 8H). Electron microscopic analysis of tibial nerve at 30w. In WT (FIG. 81), large motor axons (Ax) of diameters >5 pm are common, and axoplasm contain intact mitochondria and cytoskeleton. In CMT2A rats (FIG. 8J), axons were smaller, and axoplasm contained accumulations of disrupted mitochondria and other organelles or were undergoing Wallerian degeneration (w) and contained only flocculent material. Tibial nerves from CMT2A rats had only 53% of axons > 5 pm. Scale Bars: 10 pm, Bar graphs show mean+ / - S.E.M (** p<0.01, t test). n=l 1 animals per group.

[0022] FIGS. 9A - 9C: Single-particle MFN2-HALO dynamics in U2OS cell shows altered dynamics at ER-mitochondria dynamics. Single molecules of human MFN2 show distinct behavioral states in living cells. (FIG. 9A) A representative image showing the ER (white), the mitochondria (red), and the fluorescent signal from a few molecules of MFN2- HaloTag (yellow). Note associations with ER alone (blue arrows) and ER-mitochondria associations (yellow). Not shown in this frame but present are mitochondria-only associations. (FIG. 9B) A single representative trajectory and associated position vs time graph of a MFN2 molecule undergoing free diffusion in the ER (organelle not shown). (FIG.-6-4896-3340-4800.3Atty Dkt.: 114198-08809C) The trajectory and position vs. time graph of a representative ER-localized MFN2- HALO molecule that encounters a mitochondrion and undergoes a change in motion (site indicated by yellow arrow). The putative bound state is shaded in grey. FIG. 9B and FIG. 9C are trajectories from the cell shown in a and are drawn to scale relative to one another.

[0023] FIG. 10: MFN2 mutations studied in this application.

[0024] FIG. 11: Representative WB showing SNAP-biotin based purification of mitochondria. Mitochondrial targeting signal from COX8A fused to SNAP. VCP serves as a negative control.

[0025] FIGS. 12A - 12B: Quantitative TMT-based proteomic analysis of S249T MFN2 CMT2A patient fibroblasts reveals an inflammatory response. (FIG. 12A)Representative volcano plot depicting relative protein fold change compared to age and sex match healthy WT control cells. (FIG. 12B) ShinyGO Molecular Function analysis reveals candidate pathways of interest. N = 4 biological replicates, p value is based on B.H. procedure, i.e., FDR.

[0026] FIG. 13: Deep learning-based imaging of organelle mobility defects. LEFT:Applicant’s model tracks instances across frames by extracting features with convolutional neural networks and learning associations which are used to generate trajectories for each instance. RIGHT: Workflow integrating label-free QPI imaging with deep learning-based tracking of organelle mobility. Applicant’s tracking algorithm surpasses the accuracy of commercial IMARIS software when tracking lysosomes, which otherwise masks CMT pheno-types in mobility. Raw label-free QPI images are acquired, and either virtual organelles are extracted from the raw data or simultaneously imaged fluorescent organelles are directly tracked in healthy vs. CMT patient-derived neurons. Kymographs or organelle tracks can be used to quantify mobility.

[0027] FIGS. 14A - 14C: Representative proteomic analysis of mitochondria using Miltenyi Anti-TOM22 MicroBeads. (FIG. 14A) Coverage of mitochondrial proteome across immuno-isolated mitochondria. Cumulative number of proteins identified shown, n = 2-4 mice. (FIG. 14B) Immunoisolated mitochondria are substantially enriched in mitochondrial proteins, n = 4 mice. (FIG. 14C) Submitochondrial distribution of identified-7-4896-3340-4800.3Atty Dkt.: 114198-0880 mitochondrial proteins from cortical, heart, and spleen tissue extracts. Identified proteins are shown in orange; MitoCarta3.0 proteins are shown in gray, n = 3 mice.

[0028] FIG. 15: Superoxide activity is significantly elevated in CMT2A fibroblasts.

[0029] FIG. 16: qPCR analysis shows potential impact of MFN2 mutations in fibroblast on mtDNA maintenance. N = 3 independent cultures with technical quadruplicate. Bar graphs show mean+ / - S.E.M (** p<0.01, t test).

[0030] FIG. 17: Representative mtDNA proof or principal turnover studies using15N metabolic labeling with14N chase.

[0031] FIG. 18: iNs from CMT patients display neuronal swellings. iNs directly converted from CMT patients or age-matched healthy controls stained for tubulin. Bottom panels show magnified neurite examples. Neuronal swellings were manually scored for each individual neurite (dots on the graph). Neuronal swellings were significantly more frequent in the CMT condition. N = 3 biological replicates. Conditions were compared via t-test.

[0032] FIG. 19: iNs from CMT patients display altered microtubule PTMs: iNs directly converted from CMT patients or age-matched healthy controls were fixed and stained with antibodies against alpha tubulin, tyrosinated tubulin, and acetylated tubulin. The pixel intensities of tyrosinated tubulin and acetylated tubulin were measured and normalized to the alpha tubulin pixel intensity. Dots on the graph represent the normalized values from at least 10 different fields of view from two different healthy lines and 2 different CMT lines. The levels of acetylated tubulin and tyrosinated tubulin were increased in the CMT conditions. N= 2 biological replicates. Conditions were compared via t-test.

[0033] FIG. 20: Schematic of 30x genomic sequencing data from patient carrying the S249T CMT2A mutation. Patient sequencing data offers more potential binding sites for ASOs due to allele-specific variations that Applicant can target with different ASOs. ASO that can be used are al-PS backbone gapmers with 2’MOE modified nucleosides.

[0034] FIGS. 21A - 21B: Transfection of iPSC-Derived Motor Neurons with ASO (FIG.21A) Immunofluorescence of iPSC-derived motor neurons transfected with Cy3-labeled ASO. (FIG. 21B) Quantification of knockdown efficiency following transfection of ASO at concentrations of 500 nM and 2.5 pM.-8-4896-3340-4800.3Atty Dkt.: 114198-0880

[0035] FIGS. 22A - 22B: ER-associated actin accumulates at endosomal, lysosomal, and mitochondrial fission sites prior to organelle division. U2OS cells expressing fluorescent protein-tagged organelle markers and ER-targeted actin chromobodies (“AC -ER”) were imaged using timelapse microscopy. (FIG. 22A) Example organelles are shown at different time points prior to and immediately after the fission event. Arrowheads indicate the fission sites. White dotted lines have been drawn around the “daughter organelles” resulting from the fission event in the bottom panel for clarity. To aid visualization, line scans were drawn over the fission sites and the surrounding regions. The resulting pixel intensities associated with the line scans are shown. Lines indicate the region where the line scan was drawn (shifted so as not to block visualization of the organelle). Scale bars are 1pm. (FIG. 22B) Graph comparing the frequency of observed presence / absence of AC -ER at organelle fission events (bars) versus the possibility of AC -ER being at fission events by chance (bars). Actual observed AC -ER at fission events was determined by manually scoring fission events in a blinded fashion, followed by looking for AC -ER signal at the identified fission events. By chance values were determined by calculating the percent of organelle signal overlapped by AC -ER signal. Observed versus by chance values were statistically compared via Fisher’s exact test. All experiments were performed with N=3 biological replicates. Modified from Schiavon et al., 2024, bioRxiv.

[0036] FIG. 23: Putative interaction between INF2 and RAB7a Protein-proximity labeling with APEX2 alone vs APEX2-INF2 revealed Rab7a as a top hit in 3x biological repeat experiments (p<0.0002).

[0037] FIGS. 24A - 24B: CMT patient fibroblasts display actin-dependent organelle immobilization and aberrant actin assembly. (FIG. 24A) Lysosomes from two CMT patient and healthy control fibroblasts were imaged for 5 min. Rainbow “dragontail” tracks show organelle tracks. The mean distance for each organelle was quantified. Error bars: S.E.M. n>200 tracks / organelle for n=10 cells, n=3 biological replicates. Similar results were observed for endosomes & mitochondria (not shown) (FIG. 24B) INF2 CMT patient fibroblasts had significantly increased actin aggregates. These structures invariably contained organelles. n=100 cells, n=3 biological replicates per condition. ****indicate pO.OOOl, Welch’s t-test. Scale bars: 10pm.-9-4896-3340-4800.3Atty Dkt.: 114198-0880

[0038] FIGS. 25A - 25B: INF2 and RAB7A CMT mutations reduce organelle mobility in an actin-dependent fashion. (FIG. 25A) Lysosomes in INF2 CMT patient cells were significantly less mobile compared to control. LatB treatment restored INF2 CMT lysosome mobility. (FIG. 25B) Hippocampal rat neurons expressing Rab7a-GFP constructs and mito- dsRed were imaged using PSSR microscopy. Mitochondria in cells expressing CMT mutant RAB7[V162M] were less mobile compared to control. After LatB treatment mitochondrial mobility was increased. Scale bars: 5pm

[0039] FIGS. 26A - 26C: DeActs-ER selectively perturbs ER-associated actin and restores organelle mobility in INF2 CMT iNs. (FIG. 26A) Left: DeActs-ER architecture. Center: Cells expressing DeActs-ER (bottom) have disrupted AC -ER signal compared to controls (top). Right: DeActs-ER significantly decreases AC -ER, indicating loss of ER- associated actin, paired t-test. (FIG. 26B) Neurons expressing DeActs display normal cortical actin as measured by phalloidin staining. LatB treated cells shown as a positive control. Welch’s ttest (FIG. 26C) Lysosomes in INF2 CMT iNs expressing control mCherry-ER display reduced mobility compared to age-matched controls, but lysosome mobility is rescued in INF2 CMT iNs expressing DeActs-ER, Welch’s t-test.

[0040] FIGS. 27A - 27B: INF2 CMT iNs display significantly increased neurite swellings reversible by DeActs-ER expression. (FIG. 27A) INF2 CMT iNs displayed significantly more swellings per neurite compared to controls. n=5 fields of view, 2 coverslips each for INF2 L147, INF2 R91G (not shown), and control iNs. These swellings contained increased levels of actin and organelles. (FIG. 27B) Neurite swellings were quantified in age-matched control iNs expressing control mCherry-ER (n=37 neurites), INF2 CMT iNs expressing control mCherry-ER (n=59), and INF2 CMT iNs expressing DeActs-ER (n=69). Five replicates per condition, p-values calculated using Welch’s t-test.

[0041] FIG. 28A - 28N: Mitochondrial fragmentation in CMT2B patient fibroblasts. Healthy control (FIGS. 28A, B) and CMT2B patient (FIGS. 28C, D) fibroblasts were labeled with mitotracker. Mitochondrial area, perimeter, aspect ratio, form factor, branch junctions, branches, total branch length, and mean branch (FIGS. 28E-L) length were quantitated. The level of pDrpl-S616 was analyzed by SDS-PAGE / immunoblotting (FIGS. 28M) and quantitated (FIGS. 28N).-10-4896-3340-4800.3Atty Dkt.: 114198-0880

[0042] FIGS. 29A - 29D: Mitochondrial size and effect of Drpl inhibition in CMT2B E18 DRG neurons. (FIG. 29A) Representative images of axonal mitochondria from + / +, fl / +, fl / fl; (FIG. 29B) Aspect ratio for each group was measured (+ / +, n = 73; fl / +, n = 87; fl / fln, n = 91). (FIG. 29C), representative images of mitochondria in axons of DRG neurons were treated with vehicle or Mdivi-1 and mitochondria were analyzed (FIG. 29D) n = 257, n = 249, n = 262, n = 152, n = 235, n = 174, n = 220 in order. Results are shown as mean ± SEM. Statistical significances were calculated by One-Way ANOVA or unpaired t-test. All p values are shown.

[0043] FIG. 30: Deep learning-based imaging of organelle mobility defects. LEFT: Applicant’s model tracks objects across frames by associations that are used to generate trajectories for each objects. RIGHT: Workflow integrating label-free QPI imaging with deep learning-based tracking of organelle mobility. Applicant’s tracking algorithm surpasses the accuracy of commercial IMARIS software. Raw label-free QPI images are acquired, and simultaneously imaged fluorescent organelles are directly tracked in healthy vs. CMT patients. Kymographs or organelle tracks are used to quantify mobility. These results are representative of hundreds of organelles (small circles) from tens of cells imaged from n=4 biological replicates (large circles). p<0.05, two-way t-test.

[0044] FIG. 31: Primary fibroblasts from two CMT patients harboring mutations in INF2 and an age-matched healthy control were incubated with DQ-Red BSA and the intensity of the DQ-Red signal was measured over time. DQ-Red signal was normalized to Hoechst signal to correct for any differences in cell density. Both CMT lines displayed a marked delay in DQ-Red signal intensity signal increase as well as less overall DQ-Red signal intensity at later timepoints, indicating a defect in endocytic trafficking, n = at least 50 cells per condition.

[0045] FIG. 32: iNs were directly converted from INF2 CMTpatient or a healthy age- matched control fibroblast line. Culture media was collected from each condition and analyzed using the Quanterix SIMOA assay. Results showed a dramatic increase in the concentration of tau in CMT iN media, indicating poor neuronal health. Results were normalized to cell density, n = 3 biological replicates per condition.-11-4896-3340-4800.3Atty Dkt.: 114198-0880

[0046] FIG. 33: DRG neurons co-transfected with EGFPRab7N161T / TrkAmCherry followed by dual color-live cell imaging of axonal transport. Image series were analyzed for either EGFP (Top Left) or mCherry (Top Middle) or dual color (Top Right). Retrograde (R) or anterograde (A) transport speeds quantitated and shown. (Bottom panel).

[0047] FIG. 34: Microfluidic neuronal culture. Mouse neurons or human iPSCderived neurons can be cultured in the device. Neurites (dendrites, axons) can cross the microgrooves inro the distal chamber.

[0048] FIGS. 35A - 35B: Phenotypes with Treatment. (FIG. 35A) Length dependentCMTA phenotype. Mito / lyso anterograde / retrograde movement rations are inversely impacted and restored by MASM7. (FIG. 35B) WT vs. CMT2A vs. CMT-DIE patient derived motor neurons display rescued axons after MASM7 (and piperine) treatment.

[0049] FIG. 36A illustrates a flowchart of a method for detecting neurological or metabolic disorders based on image processing, in accordance with some embodiments.

[0050] FIG. 36B illustrates a flowchart of a method for screening candidate therapeutics for neuronal disorders or metabolic disorders based on high-resolution microscopy image data, in accordance with some embodiments.

[0051] FIG. 37A illustrates a block diagram of a computing device, in accordance with some embodiments.

[0052] FIG. 37B illustrates a block diagram of a computing device, in accordance with some embodiments.DETAILED DESCRIPTION

[0053] Definitions

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an-12-4896-3340-4800.3Atty Dkt.: 114198-0880 admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0055] Throughout and within this application technical and patent literature are referenced by a citation. For certain of these references, the identifying citation is found at the end of this application immediately preceding the claims. All publications are incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[0056] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0057] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood,-13-4896-3340-4800.3Atty Dkt.: 114198-0880 although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0058] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0059] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0060] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.-14-4896-3340-4800.3Atty Dkt.: 114198-0880

[0061] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0062] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0063] An equivalent or biological equivalent nucleic acid, polynucleotide or oligonucleotide or peptide is one having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference nucleic acid, polynucleotide, oligonucleotide or peptide.

[0064] “Detectable label”, “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein.-15-4896-3340-4800.3Atty Dkt.: 114198-0880

[0065] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0066] In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0067] As used herein, the term “axon” refers to the elongated projection of a neuron that conducts electrical impulses away from the neuronal cell body (soma) toward other neurons, muscles, or glands. In cultured neuronal models, the term includes both primary axons and axon-like neurites identifiable by morphological or molecular markers, such as acetylated tubulin or neurofilament proteins.

[0068] The term “axon length” means the measured distance from the proximal axon start point at the soma or at the microgroove entry in a microfluidic system to the distal axon tip. Axon length measurements are determined in vitro from microscopy images by automated or manual segmentation, skeletonization, or other image analysis methods. Axon length can be expressed in micrometers or millimeters, and may be normalized to cell density, culture duration, or other scaling parameters.

[0069] The term “branching metrics” refers to numerical measures of the complexity of axonal or neuritic branching patterns, including but not limited to branch number, branch length, branch point count, and results of Sholl analysis. Such metrics are obtained from image data using digital quantification methods.

[0070] The term “neurite complexity” refers to the extent of arborization formed by axons and dendrites in neuronal cultures, quantified by methods such as Sholl analysis or fractal dimension calculation.-16-4896-3340-4800.3Atty Dkt.: 114198-0880

[0071] The term “neuronal cell culture” means any in vitro preparation of neuronal cells, including primary neurons, neurons differentiated from induced pluripotent stem cells (iPSCs), motor neurons, sensory neurons, or directly converted induced neurons (“iNs”), maintained under conditions permitting neuronal morphology and function.

[0072] The term “neurosphere” or “spheroid culture” means a three-dimensional aggregate of neuronal cells formed under low-attachment conditions, used to model network formation and axonal outgrowth in vitro.

[0073] The term “microfluidic chip” means a culture device fabricated with microchannels and compartments allowing compartmentalized growth of neuronal cell bodies and axons. Such chips typically include microgrooves between compartments sized to permit axonal penetration while restricting cell bodies.

[0074] The term “imaging module” refers to any optical system capable of capturing high- resolution images of cultured neuronal cells, including confocal microscopy, super-resolution microscopy such as Airyscan, widefield tile microscopy, transmitted light imaging, or combinations thereof. Imaging modules may further comprise environmental control elements such as temperature or CO2 regulation to enable live-cell imaging.

[0075] The term “organelle” means any membrane-bound or membrane-less subcellular structure, including mitochondria, lysosomes, endosomes, and other intracellular bodies detectable by imaging methods.

[0076] The term “organelle transport” or “organelle mobility” refers to the movement of organelles along neuronal processes, including axons and dendrites, quantified by parameters such as velocity, displacement, directionality, and frequency of pausing.

[0077] The term “co-migration” refers to the simultaneous movement of two distinct organelle populations, such as mitochondria and lysosomes, along overlapping trajectories in neuronal processes, measurable via dual-channel microscopy and logical pixel-wise analysis.

[0078] The term “therapeutic agent” refers to any molecule, biologic, nucleic acid, genetic construct, molecular probe, or combination thereof administered, expressed, or otherwise applied with the intent to treat, prevent, delay progression, or ameliorate a disease, disorder, or pathological condition. Therapeutic agents may include small molecules, peptides,-17-4896-3340-4800.3Atty Dkt.: 114198-0880 proteins, nucleic acids (including antisense oligonucleotides and siRNAs), viral or non-viral gene therapy vectors, antibodies, or engineered biologic tools. For purposes of this disclosure, non-limiting examples of therapeutic agents tested or proposed herein include the mitofusin activator MASM7, the plant alkaloid piperine, wild-type nerve growth factor (NGF), NGFAR100W mutant, allele-specific antisense oligonucleotides targeting MFN2 mutations, Rab7 GTPase inhibitors such as CID 1067700, and the genetically encoded ER-actin perturbator DeActs-ER.

[0079] The term “therapeutic candidate” or “candidate agent” means any therapeutic agent under investigation for potential medical benefit, including the therapeutic agents exemplified above.

[0080] The term “therapeutic efficacy score” means a composite numerical metric generated by an analysis module, based on changes in one or more phenotypic measures including axon length change, branching complexity change, and organelle transport improvement, optionally weighted by statistical significance. The score can be used to rank candidate agents or correlate agent activity with disease amelioration.

[0081] MASM7 refers to a synthetic small molecule mitofusin activator that promotes mitochondrial fusion through direct or indirect activation of mitofusin proteins MFN1 and MFN2, thereby enhancing mitochondrial network connectivity and axonal transport. In this disclosure, MASM7 restored balanced anterograde and retrograde organelle movement and rescued axonal length in CMT2A and CMT-DIE patient-derived motor neurons.

[0082] Piperine refers to a naturally occurring alkaloid obtained from plants in the genus Piper, having reported bioactivities that include modulation of mitochondrial function, antioxidant effects, and enhancement of cellular transport processes. In this disclosure, piperine improved axonal morphology and organelle movement in CMT patient-derived motor neurons.

[0083] Wild-type NGF refers to the naturally occurring nerve growth factor polypeptide that binds and activates TrkA and p75NTR receptors to promote neuronal growth and survival. As described herein, wild-type NGF restored intraepidermal sensory fiber density and thermal sensory function in RAB7AV162M CMT2B mouse models, though with nociceptive side effects.-18-4896-3340-4800.3Atty Dkt.: 114198-0880

[0084] NGFAR100W refers to a mutant NGF having an arginine-to-tryptophan substitution at position 100 that preserves neurotrophic signaling capacity while reducing or eliminating nociceptive effects. In this disclosure, NGFAR100W restored sensory fiber density and function in RAB7AV162M CMT2B mice without inducing pain-associated behaviors.

[0085] The term “allele-specific antisense oligonucleotide” refers to a synthetic DNA or RNA oligomer complementary to a target RNA sequence containing a mutation, polymorphism, or unique haplotype, designed to selectively hybridize and induce RNase Fl- mediated degradation of mutant transcripts while sparing wild-type alleles. ASOs in this disclosure selectively knocked down mutant MFN2 transcripts in patient neurons and rat models.

[0086] The term “Rab7 GTPase inhibitor” means a small molecule that interferes with the GTP-binding or GTP-hydrolysis activity of Rab7A, thereby altering its regulatory effects on endosomal trafficking, lysosome positioning, and organelle contact dynamics. CID 1067700 is described herein as an exemplary competitive nucleotide binding inhibitor tested for CMT2B cellular phenotypes.

[0087] DeActs-ER refers to a recombinant protein or fusion construct that targets actindepolymerizing activity specifically to the endoplasmic reticulum, thereby selectively disrupting ER-associated actin filaments while leaving other actin pools intact. In INF2 CMT patient-derived neurons, DeActs-ER restored lysosome mobility and reduced neurite swellings.

[0088] The term “analysis module” refers to one or more processors or computing systems, optionally networked, executing software instructions to process microscopy image data, extract quantitative measurements such as axon length, perform statistical analyses, and generate outputs such as visualizations and ranked lists.

[0089] The term “non-transitory computer-readable medium” means a tangible storage device such as a hard disk, solid-state drive, flash memory device, or optical disc, storing executable instructions accessible by a computing system, and explicitly excludes transitory signals.

[0090] The phrase “correlating axon length to a disorder” means comparing measured axon length values obtained from neuronal cultures to reference values or statistical distributions-19-4896-3340-4800.3Atty Dkt.: 114198-0880 associated with known disease states or progression levels, thereby inferring the presence, severity, or risk of such disorder.

[0091] The term “time-series image dataset” means sequential microscopy images of a given field of view acquired at two or more time points, allowing observation of dynamic changes such as axonal growth or organelle movement over time.

[0092] The term “patient-derived” refers to cells obtained directly from a subject diagnosed with or at risk for a specific disorder or differentiated from such subject’s tissue-derived stem cells, for example fibroblast-derived induced pluripotent stem cells.

[0093] The term “subject” refers to any living organism in which the disclosed methods or systems can be applied, including mammals such as humans, non-human primates, domestic animals, livestock, laboratory animals (e.g., mice, rats), and other vertebrates. In certain embodiments, the subject is a human patient diagnosed with or at risk for a neurodegenerative disorder or a metabolic disorder such as Charcot-Marie-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes. The term “subject” may also encompass cells, tissues, or organoids derived from such organism, whether maintained in vivo, ex vivo, or in vitro, on which the methods and systems described herein are performed.

[0094] The term “patient” refers to a human subject who is diagnosed with, suspected of having, at risk for developing, or being treated for a condition, disease, or disorder described in this disclosure.

[0095] The term “sample” refers to any material obtained from a subject or patient that contains, or is suspected to contain, biological components of interest. Samples may include cells, tissues, organoids, organelles, bodily fluids, or explanted organs, and may be used in vivo, ex vivo, or in vitro in the context of the disclosed imaging, analysis, and screening methods. The sample can be derived from or isolated from animals, mammals such as humans, non-human primates, domestic animals, livestock, laboratory animals (e.g., mice, rats), and other vertebrates.

[0096] The term “culture module” refers to any physical component configured to hold, sustain, and maintain living cells or tissues under controlled environmental conditions-20-4896-3340-4800.3Atty Dkt.: 114198-0880 suitable for growth, differentiation, or functional activity, including but not limited to conventional culture ware and microfluidic devices.

[0097] The term “neurological disorder” refers to any disease, condition, or syndrome affecting the structure or function of the central or peripheral nervous system. Examples include Charcot-Mari e-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis, diabetic neuropathy, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple sclerosis, hereditary sensory autonomic neuropathy, spinal muscular atrophy, and optic neuropathies.

[0098] The term “metabolic disorder” refers to any disease, condition, or syndrome characterized by disruption of metabolic processes responsible for homeostasis and energy production. Examples include type II diabetes, type I diabetes, metabolic syndrome, mitochondrial disorders such as MELAS and Leigh syndrome, obesity-related metabolic dysfunction, lysosomal storage disorders, and phenylketonuria.

[0099] The term “organism” refers to any living entity capable of growth and reproduction. In this disclosure, organisms are preferably mammals such as humans, non-human primates, rats, and mice.

[0100] The term “ranking” refers to the ordering of candidate agents based on performance metrics including therapeutic efficacy score.

[0101] The term “correlation” refers to the statistical or logical relationship between two or more measured variables. In this disclosure, correlation includes linking quantitative metrics such as axon length or organelle transport to disease severity or treatment response.

[0102] The term “motor neuron” refers to a neuron whose axon innervates muscle tissue to produce movement.

[0103] The term “sensory neuron” refers to a neuron specialized to detect and transmit sensory signals to the central nervous system.

[0104] The term “time point” refers to a discrete moment for data acquisition allowing temporal comparison.-21-4896-3340-4800.3Atty Dkt.: 114198-0880

[0105] The term “compartmentalized growth” refers to the physical separation of cell bodies and extending neurites or axons into distinct culture regions, enabling independent manipulation or observation.

[0106] The term “biological replicate” refers to an independent experimental iteration using a distinct biological sample of the same type.

[0107] The term “therapeutic screening” refers to evaluating one or more candidate agents for measurable effects relevant to treating a disease, performed in vitro, ex vivo, or in vivo.

[0108] The term “high-resolution image” refers to an image with sufficient spatial detail to resolve fine structural features of cells, obtained by modalities such as confocal, superresolution, Airyscan, or tiled widefield microscopy.

[0109] Charcot-Marie-Tooth disease type 2A (CMT2A) is an axonal peripheral neuropathy caused by autosomal dominant MFN2 mutations, leading to mitochondrial fusion and transport defects and progressive length-dependent neuropathy.

[0110] Charcot-Marie-Tooth disease type 2B (CMT2B) is an axonal neuropathy caused by autosomal dominant RAB7A mutations, producing severe sensory loss, impaired neurotrophic signal trafficking, and organelle transport defects.[OHl] RAB7A is a small GTPase involved in late endosome trafficking, lysosome positioning, autophagy, and mitochondria-lysosome contacts, critical for neuronal function.

[0112] INF2 is an ER-anchored actin regulatory protein that facilitates mitochondrial fission by nucleating actin filaments at organelle contact sites; mutations lead to excess ER-actin, organelle immobilization, and CMT phenotypes.

[0113] CMT-DIE refers to Charcot-Marie-Tooth disease with intermediate electrophysiological features between demyelinating and axonal forms, sometimes involving INF2 mutations and combined axonal / myelin defects.

[0114] Modes For Carrying Out The Disclosure

[0115] Charcot-Marie-Tooth (CMT) disease is a debilitating inherited peripheral neuropathy that primarily affects myelination (Type I) and involves both motor and sensory neurons (Type II). It is characterized by progressive muscle weakness, atrophy, and sensory loss. In severe cases, individuals experience foot ulcerations, which can lead to amputation. Type II-22-4896-3340-4800.3Atty Dkt.: 114198-0880CMT can be caused by mutations in many proteins that impact organelle dynamics, most commonly MFN2 (i.e., “CMT2A”), which has been implicated in mitochondrial dynamics and inter-organelle contacts. Applicant describes herein the mechanisms and therapeutic potential of targeting dominant MFN2 mutations that cause CMT2A. CMT2A mutations in MFN2 are usually dominant-negative, yet there is little understanding of how different mutations in different MFN2 protein domains result in the same clinical phenotype. Applicant report that mutations in MFN2 result in not just mitochondrial but also lysosomal defects (data not shown). Without being bound by theory and in this disclosure, Applicant establishes that autosomal dominant CMT2A-associated point mutations in MFN2 disrupt organelle dynamics and function, ultimately resulting in progressive, length-dependent neuronal degeneration. See FIG. 1.

[0116] In one aspect, this disclosure provides methods of detecting a neurological or metabolic disorder phenotype in a neuronal cell culture, comprising, or consisting essentially of or yet further consisting of culturing neuronal cells derived from a subject, patient, or induced pluripotent stem cell (iPSC) line, imaging the neuronal cells to determine at least one primary metric selected from axon length, organelle mobility of mitochondria, lysosomes, or endosomes within axons, or organelle morphology including mitochondrial fragmentation index, lysosomal dispersion index, or co-migration frequency, and correlating the primary metric to presence or severity of the disorder. In one aspect, the neuronal cells are differentiated from iPSCs into motor neurons or sensory neurons. In other aspect, organelle mobility is measured by generating kymographs and quantifying instantaneous velocity, mean velocity, displacement, directionality, and pause frequency. In a further aspect, wherein organelle morphology is quantified using aspect ratio, form factor, branch length, and branch junction count. In a yet further embodiment, the disorder is selected from Charcot-Marie- Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis, and diabetic neuropathy.

[0117] In another aspect, provided are methods of screening candidate therapeutic agents for a neurological or metabolic disorder, comprising, comprising, or consisting essentially of or yet further consisting of, generating neuronal cell cultures from subjects diagnosed with, or at risk for, the disorder, contacting the cultures with one or more candidate therapeutic agents, imaging the cultures using quasi-simultaneous quantitative phase imaging (QPI) and-23-4896-3340-4800.3Atty Dkt.: 114198-0880 fluorescence microscopy to acquire multimodal time-series datasets, processing the datasets with a deep learning model trained to extract metrics selected from axon length, axonal branching complexity, neurite complexity, organelle transport velocity, organelle co-migration frequency, and organelle fragmentation index, and identifying candidate agents that increase axon length and / or improve organelle transport relative to untreated control cultures. In one aspect, of the method, the deep learning model comprises a convolutional neural network, a transformer-based architecture, or a hybrid thereof trained for neuronal morphometries. In a further aspect, the quasi-simultaneous QPI and fluorescence imaging are acquired within 200 milliseconds of each other to enable direct overlay. In a further aspect, the therapeutic agent is administered at two or more concentrations to derive a dose-response curve for axon length and organelle transport improvement. In one embodiment, the analysis further comprises calculating a therapeutic efficacy score that integrates axon length change with improvements in organelle mobility weighted by statistical significance.

[0118] In another aspect, methods are provided for identifying agents is provided that restore peripheral neuropathy phenotypes, comprising forming neurosphere or spheroid cultures from patient-derived motor or sensory neurons, embedding the cultures in extracellular matrix, imaging axon density, axon length, and organelle transport properties, and selecting agents that restore axon density or length and concurrently improve mitochondrial or lysosomal mobility. In one aspect, The method of claim 3, wherein the neurospheres are embedded in a Matrigel growth factor-reduced matrix at a 1 :40 dilution in DMEM / F12. In a further aspect, organelle transport metrics comprise velocity distributions for mitochondria and lysosomes separately and co-migration frequency. In another embodiment, agents that improve axonal branching complexity as measured by Sholl analysis.

[0119] In a further aspect, a microfluidic neuronal culture system is provided, the system comprising, comprising, or consisting essentially of or yet further consisting of a soma compartment configured to contain neuronal cell bodies, at least one axon compartment fluidly connected via microgrooves measuring between 500 micrometers and 1 millimeter in length, an imaging module configured for multimodal high-resolution imaging of axons and subcellular organelles, and an analysis module comprising non-transitory instructions to segment axons, quantify axon length, analyze organelle transport metrics, and correlate said metrics to neurological or metabolic disorders. In one embodiment of the system, the imaging-24-4896-3340-4800.3Atty Dkt.: 114198-0880 module comprises confocal microscopy with Airyscan super-resolution capability. In a separate embodiment of the system, the analysis module includes instructions to apply pixel-wise logical operations to dual-channel images to quantify co-migration events. In one aspect, the axon compartment is configured to allow compartment-specific application of candidate agents.

[0120] In one embodiment, computer-implemented methods are provided, the methods comprising, comprising, or consisting essentially of or yet further consisting of receiving label-free quantitative phase imaging datasets of neuronal cultures, applying a trained neural network model to generate predicted fluorescence images of organelles selected from mitochondria or lysosomes, automatically tracking the predicted organelles across sequential frames to calculate transport metrics including velocity, displacement, directionality, and pausing frequency, and correlating transport metrics to disease severity or therapeutic intervention efficacy. In one aspect, the deep learning model is trained on paired QPI and fluorescence datasets of mitochondria and lysosomes in live neuronal axons. In another aspect, transport metrics include changes in pausing frequency and displacement in response to candidate agents. In a further aspect, the tracking algorithms achieve accuracy exceeding IMARIS software in detecting mobility changes.

[0121] In another embodiment, methods for treating or ameliorating phenotypes of Charcot- Marie-Tooth (CMT) disease are provided, the methods comprising, comprising, or consisting essentially of or yet further consisting of contacting patient-derived neuronal cultures or animal model neurons with a therapeutic agent selected from MASM7, piperine, NGFAR100W, an allele-specific antisense oligonucleotide targeting MFN2, INF2, or RAB7A, a Rab7 GTPase inhibitor, or a DeActs-ER construct, imaging the cultures before and after contact to determine changes in axon length and organelle mobility, and identifying effective agents as those that restore axon length and organelle transport relative to untreated controls. In one embodiment, the MASM7 restores balanced anterograde and retrograde movement ratios of mitochondria and lysosomes in patient-derived motor neurons. In one embodiment, NGFAR100W restores intraepidermal sensory fiber density and thermal sensory response without nociceptive side effects in a RAB7A CMT2B model. In a further aspect, the allele-specific antisense oligonucleotides achieve selective knockdown of mutant mRNA by at least 90% while reducing wild-type mRNA by less than 10%. In another embodiment, a-25-4896-3340-4800.3Atty Dkt.: 114198-0880Rab7 GTPase inhibitor restores endosomal and lysosomal mobility in CMT2B neurons to levels observed in age-matched healthy controls. In another embodiment, DeActs-ER treatment depolymerizes ER-associated actin and rescues lysosome mobility and reduces neurite swellings in INF2 CMT patient-derived neurons.

[0122] In certain embodiments, the neuronal cultures are maintained in a microfluidic chip having microgrooves dimensioned to selectively pass axons while excluding neuronal cell bodies.

[0123] In one aspect of the above methods, the metrics are normalized to cell density and statistical comparisons are performed across at least three biological replicates.

[0124] In another aspect of these methods, the metrics are stored and aggregated to stratify patient populations for clinical trials.

[0125] This disclosure provides a method of detecting axon length in a neuronal cell culture, including: (a) culturing neuronal cells derived from a subject or an induced pluripotent stem cell line; (b) imaging the neuronal cells to determine axon length; and (c) correlating the axon length to a presence or severity of a neurological or metabolic disorder.

[0126] Also provided herein are methods of monitoring axon length over time in a neuronal cell culture following application of a therapeutic candidate, including: (a) culturing neuronal cells derived from a subject or patient population having or at risk for a neuronal disorder or a metabolic disorder; (b) applying the therapeutic candidate to the neuronal cells; (c) imaging the neuronal cells at two or more time points after applying the therapeutic candidate; and (d) determining changes in axon length between the time points, wherein a detectable increase in axon length relative to untreated control cultures is indicative of therapeutic efficacy.

[0127] Yet further provided are methods of screening candidate therapeutics for a neurological or a metabolic disorder using axon length as a phenotypic readout, including: (a) generating neuronal cell cultures from subjects diagnosed with the neurological or the metabolic disorder; (b) contacting the neuronal cell cultures with one or more candidate therapeutic agents; (c) imaging the neuronal cell cultures to determine axon length; and (d) comparing axon length in candidate agent-treated cultures to control cultures, wherein an increase in axon length relative to control identifies the candidate agent as having potential therapeutic efficacy.-26-4896-3340-4800.3Atty Dkt.: 114198-0880

[0128] In one aspect of these methods, the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Marie-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

[0129] In other embodiments of these methods, the neuronal cells are motor neurons or sensory neurons differentiated from patient-derived induced pluripotent stem cells.

[0130] In some aspects, imaging includes live-cell fluorescence microscopy, transmitted light microscopy, confocal microscopy, super-resolution microscopy, or widefield tile imaging.

[0131] In another aspect, the methods can further include quantifying axonal branching or neurite complexity in addition to axon length.

[0132] In a further embodiment of these methods, wherein monitoring axon length further includes measuring axonal transport of organelles selected from mitochondria and lysosomes.

[0133] In another embodiment of these methods, the therapeutic candidate is selected from a small molecule, antisense oligonucleotide, biologic, gene therapy construct, or neurotrophic factor. In some respects of these methods, the comparing step includes statistical analysis of axon length distributions across at least three biological replicates.

[0134] In other aspects, the screening further includes measuring co-migration of mitochondria and lysosomes as a secondary readout of neuronal health.

[0135] In further aspects, the neuronal cell cultures are maintained in microfluidic chip devices permitting compartmentalized growth of axons.

[0136] In one embodiment, the correlation to disorder presence or severity is used to stratify patient populations for clinical trials of the therapeutic candidates.

[0137] In a further embodiment, the disclosure provides a system for detecting axon length and correlating axon length to a neurological or metabolic disorder, including: (a) a neuronal cell culture chamber configured to accommodate cells derived from a subject or induced pluripotent stem cell line; (b) an imaging module configured to acquire images of axons in the neuronal cell culture at high spatial resolution; and (c) an analysis module including at least one processor and non-transitory computer-readable instructions configured to: (i) determine axon length from the images; and (ii) output a correlation of the axon length to a neurological or a metabolic disorder.-27-4896-3340-4800.3Atty Dkt.: 114198-0880

[0138] In one embodiment of the method, the neuronal cell culture chamber includes a microfluidic chip having: (a) a soma compartment for neuronal cell bodies; and (b) one or more axon compartments fluidly connected to the soma compartment via microgrooves permitting axonal growth.

[0139] Also provided are systems, wherein the imaging module includes at least one of: (a) a confocal microscope; (b) a super-resolution Airyscan microscope; (c) a widefield tile imaging microscope; or (d) a transmitted light imaging system.

[0140] In some aspects of the systems, the analysis module is further configured to monitor axon length at multiple time points after application of a therapeutic candidate and to calculate the change in axon length over time.

[0141] In some aspects of the systems, the analysis module is further configured to compare axon lengths between therapeutic candidate-treated cultures and untreated control cultures to identify candidate agents that increase axon length.

[0142] Further provided are systems, wherein the analysis module is further configured to: (a) detect and quantify axonal branching; (b) analyze neurite complexity; and (c) assess comigration or transport of axonal organelles selected from mitochondria and lysosomes. In one aspect, the neuronal cell cultures are motor neurons or sensory neurons differentiated from patient-derived induced pluripotent stem cells. In one embodiment, the systems further include a therapeutic application module configured to deliver candidate agents to cultured cells and record delivery parameters within the analysis module. In another aspect of the systems, the analysis module outputs both numerical and visualized metrics including kymographs, Sholl analysis plots, and axon length distributions for disease correlation or therapeutic screening. In another embodiment of the systems, the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Marie- Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

[0143] Also provided are methods to screen for candidate therapeutics, including: (a) culturing neuronal cells from a subject diagnosed the neurological or the metabolic disorder in the neuronal cell culture chamber; (b) applying one or more candidate therapeutic agents to the cultures; (c) acquiring high-resolution images of axons over a defined period; and (d) analyzing the images with the analysis module to determine changes in axon length relative-28-4896-3340-4800.3Atty Dkt.: 114198-0880 to untreated control cultures, wherein an increase in axon length identifies the candidate therapeutic agent as having potential efficacy. In one aspect, the method further includes correlating axon length changes to improvement in organelle transport metrics obtained by the system.

[0144] Also provided are non-transitory computer-readable medium storing instructions that, when executed by at least one processor, cause the processor to perform a method including: (a) receiving image data of neuronal cell cultures; (b) processing the image data to segment axons; (c) determining axon length for each segmented axon; and (d) correlating the axon length to one or more neurological or metabolic disorder.

[0145] In one aspect, the processing steps include: (a) skeletonizing segmented axons; (b) computing branching metrics; and (c) quantifying neurite complexity by Sholl analysis.

[0146] In another embodiment, the non-transitory computer-readable medium, further includes instructions that cause the processor to: (a) receive image data from two or more time points before and after application of a therapeutic candidate; (b) compute a change in axon length between the time points; and (c) output the change in axon length as a measure of therapeutic efficacy. In another embodiment, the instructions further cause the processor to: (a) measure co-migration of mitochondria and lysosomes in the image data; (b) quantify organelle transport metrics; and (c) correlate changes in axon length with changes in organelle transport metrics.

[0147] Further provided are non-transitory computer-readable mediums, wherein the instructions further cause the processor to: (a) normalize axon length measurements to cell density; (b) apply statistical analyses to compare treated vs. untreated cultures across at least three biological replicates; and (c) generate visualizations selected from kymographs, histograms, and scatter plots of axon length data.

[0148] In some aspects of the mediums, the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Marie-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

[0149] In some aspects, the disclosure provides a computer-implemented method for screening candidate therapeutics for neuronal disorders or metabolic disorders, including: (a) receiving high-resolution microscopy image data of neuronal cell cultures contacted with one-29-4896-3340-4800.3Atty Dkt.: 114198-0880 or more candidate agents; (b) segmenting axons in the image data; (c) computing axon length metrics; (d) comparing the axon length metrics to control cultures; and (e) identifying candidate agents that increase axon length relative to controls. In one aspect, the methods further include outputting a ranked list of candidate agents based on magnitude of axon length increase and statistical significance. In another aspect, the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Marie- Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes. In one embodiment, the method includes aggregating patient-derived culture data to stratify patient populations for clinical trials.

[0150] Also provided are methods to identify mutations in IFN2 and RAB7A in cells harboring these mutations including analyzing the cells for one or more of organelle mobility, morphology, and axonal function. In one aspect, organelle mobility is analyzed by a method including high spatiotemporal resolution. In another aspect, the methods further include analyzing the organelle mobility with deep learning-based point-scanning super-resolution microscopy (PSSR) image processing model trained for imaging organelle transport in the cells.

[0151] In some aspects of the above methods, the disclosure provides a method, wherein the cells are neurons or induced pluripotent stem cells (iPSCs).

[0152] In some aspects, the cells are mammalian cells, optionally human cells.

[0153] Experimental

[0154] Experiment No. 1: Impact of CMT2A mutations on MFN2 protein dynamics.

[0155] To study whether disease-associated point mutations in MFN2 disrupt its localization, dynamics, and stability, a comprehensive imaging and mass spectrometry (MS)-based proteomic analysis workflow can be used to compare healthy MFN2 variants with those associated with CMT2A. Applicant’s approach involves creating patient-derived cells and rat models harboring specific CMT2A MFN2 mutations. By utilizing endogenous knock-in (KI) HALO and SNAP tags for each allele, Applicant investigates their dynamics and interactions in live cells. Through quantitative proteomics, Applicant assesses the relative levels, turnover rates, and interactomes of the mutant MFN2 proteins. This integrated approach can provide-30-4896-3340-4800.3Atty Dkt.: 114198-0880 novel insights into the fundamental molecular and cellular mechanisms of MFN2 in health and disease.

[0156] The effects of CMT2A mutations on organelle composition, dynamics, and function.

[0157] Applicant hypothesized that MFN2 mutations disrupt organelle dynamics, including fission / fusion, mobility, and turnover, all of which significantly contribute to the progressive, length-dependent neuropathy characteristic of the disease. Label-free imaging and deep learning-based approaches that minimize phototoxicity and maximize spatiotemporal resolution can be used for tracking inter-organelle dynamics. It can assess turnover rates of mitochondria and lysosomes in normal vs. CMT2A cells and tissues. By identifying organellar dysfunctions associated with neurodegeneration in vitro and in vivo, a comprehensive view of cellular integrity and function can be obtained. Additionally, investigation of the effects of CMT2A mutations on post-translational modifications of microtubules, a critical component of neuronal architecture and transport, can lend new insight towards molecular mechanisms of CMT2A physiopathology. This comprehensive approach enhances the understanding of CMT2A mutations in MFN2 and disease.

[0158] Evaluate the efficacy of allele-specific genetic therapies in rescuing CMT2A phenotypes.

[0159] Applicant hypothesized that selectively knocking down mutant MFN2 can effectively rescue disease-relevant phenotypes associated with CMT2A. Evaluation of the efficacy and off-target effects of allele-specific antisense oligonucleotides (ASOs) as a potential therapeutic intervention for CMT2A caused by dominant-negative mutations in MFN2 can be used for these methods. This evaluation can be conducted using patient-derived iPSC neurons and relevant rodent models. ASOs provide a powerful approach for sequence-specific targeting of RNA transcript isoforms, enabling precise and patient-specific knockdown of the mutant gene expression. In addition to their therapeutic potential, ASOs can also serve as critical tools for validating the mechanisms driving CMT2A phenotypes, as outlined above. This will enable a new understanding of the role of organelle dynamics in CMT2A pathogenesis. The innovative imaging and therapeutic approaches have the potential to significantly impact the field of rare disease research and aid the development of novel treatments for CMT2A.-31-4896-3340-4800.3Atty Dkt.: 114198-0880

[0160] Charcot-Marie-Tooth Disease and the Critical Role of MFN2. Charcot-Marie-Tooth disease (CMT) is a progressive neuropathy that most profoundly impacts the longest neurons in the body, with the age of onset being directly proportional to the severity of the disease. In most patients, this manifests first as the loss of sensorimotor function in their feet, then their hands, and in more acute cases their vision and hearing can also suffer. Many genes associated with type II axonal CMT encode proteins involved in organelle dynamics. Without being bound by theory, it is believed that that disrupted organelle dynamics is particularly deleterious for the longest neurons in the body where organelle transport and turnover is exceptionally costly1. CMT2A, the most common subtype of axonal CMT, is primarily caused by mutations in MFN22, encoding Mitofusin 2, a key mitochondrial protein that mediates mitochondrial fusion / fission3, ER-mitochondria contacts4'6, mitochondria-lysosome contacts7, mtDNA maintenance8, and mitophagy4’7’9. This is important since, mitochondrial fusion, fission, and turnover is essential for maintaining cellular function. Applicant’s data show multiple mutations nMFN2 disrupt both mitochondrial (FIG. 2) and lysosomal (FIG. 3) mobility and dynamics, which Applicant hypothesized underlies the impaired axonal transport, energy deficits, and ultimately, neuropathy observed in patients. The devastating impact of CMT on patients’ lives underscores the urgent need for research into its underlying mechanisms and the development of effective therapies (see CMT Research Foundation LOS). That MFN2 is implicated in diabetes10, n, Parkinson’s7, 9, and Alzheimer’s disease12further underscores the importance of understanding MFN2’s cellular and molecular roles and mechanisms.

[0161] Unraveling the Complexities of MFN2 Mutations. Despite the identification of numerous CMT2A-causing MFN2 mutations, the precise mechanisms by which these mutations lead to disease remain poorly understood. This disclosure aims to address this knowledge gap by focusing on four MFN2 mutations for which Applicant have obtained either or both human patient and rat models: These mutations were selected based on the availability of valuable patient-derived cells and isogenic controls (R364W, T206I, R94W, S249T, see LOS from Bruce Conklin), and rat models (R364W and T206I), which facilitated Applicant’s data showing a significant impact on organelle dynamics and function.

[0162] Beyond Mitochondria: The Multi-Organelle Impact of MFN2 Mutations. The significance of this research extends beyond mitochondrial dysfunction. Emerging evidence-32-4896-3340-4800.3Atty Dkt.: 114198-0880 suggests interorganelle contacts between mitochondria, the ER, and lysosomes mediates different subtypes of mitochondrial fission and mitophagy. Applicant’s data unexpectedly reveals that some CMT2A-associated MFN2 mutations disrupt not only mitochondrial but also lysosomal dynamics (FIG. 3), highlighting a broader impact on organelle function. These observations can be extended to CMT-relevant neuronal cell models and in vivo rat models.

[0163] Detailed mechanistic investigations of organelle movements are hindered by challenges in observing and analyzing these dynamic processes. The highly dynamic nature of organelles within live cells necessitates both fast and precise monitoring and quantification strategies. While fluorescence microscopy offers exceptional resolution through fluorescent labeling, its inherent drawbacks, including perturbation of cellular processes and limitations in live-cell imaging, hinder comprehensive understanding. Moreover, the constrained number of observable channels restricts the breadth of investigation to usually just 2 or 3 organelles at a time, greatly reducing the throughput and context of each experiment. Further, labeling organelles with fluorescent proteins or dyes involves sample manipulations that are laborious, expensive, and often perturb cellular physiology in unpredictable ways. For example, Applicant discovered that organelle mobility decreases as an acute response to phototoxicity, introducing a potentially underappreciated confounding factor for all organelle studies to date. Just as importantly, Applicant discovered the phototoxic response is highly variable depending on the location of the GFP molecule; localizing GFP to the outer mitochondrial membrane paradoxically protected cells against phototoxicity, whereas cytosolic GFP sensitized cells to phototoxicity (FIG. 4).

[0164] To address these challenges, quantitative phase imaging (QPI) label -free imaging assays capable of visualizing organelles in their native state without the need for invasive techniques can be used13. QPI minimizes sample manipulation, enabling simultaneous observation of multiple biological structures with reduced phototoxicity and no photobleaching, rendering it ideal for continuous live-cell imaging. QPI enables the monitoring of all organelles within a cell simultaneously, yet they lack the specificity required to focus on individual organelles. To address this limitation, Applicant built a custom imaging system for quasi-simultaneous QPI+fluorescence microscopy (FIG. 5). Quasi-simultaneity is essential for capturing live moving organelles with near-perfect-33-4896-3340-4800.3Atty Dkt.: 114198-0880 overlays of QPI and fluorescence images that are suitable for deep learning models that virtually stain organelles in QPI datasets. Applicant’s data on mitochondria show feasibility of this approach (FIG. 6).

[0165] Deep learning strategies have been effectively applied in various label-free image systems, successfully resolving complex inverse problems that conventional methods cannot address14, 15. Applicant’s data indicate that the “fingerprint” of organelle mobility and clustering is clearly altered in CMT2A patient cells (FIG. 7). By leveraging deep learning for image / video classification, Applicant can significantly improve Applicant’s ability to monitor and analyze organelle dynamics in patient-derived cells.

[0166] Matching Rat Models for in vivo Studies: Applicant’s data shows that the R364W mutation leads to measurable defects in grip strength, compound muscle action potential (CMAP) measurements, and quantifiable differences in electron microscopy histology of the rat tibial nerve (FIG. 8, modified with permission from Mark Scheideler). These findings confirm the validity of this model for the in vivo characterization of CMT2A, enabling the study the progression of the disease and evaluate the efficacy of potential therapeutic interventions in a physiologically relevant context. The ability to study the disease in a model that closely mimics human CMT2A is a significant advantage, increasing the translational potential of Applicant’s research and strengthening the possibility of developing treatments for CMT2A.

[0167] Precision Medicine for CMT2A: Allele-specific ASO Therapies: The development of allele-specific genetic therapies, such as antisense oligonucleotides (ASOs), represents a timely and promising avenue for treating CMT2A and other autosomal dominant diseases. ASOs have already demonstrated clinical efficacy in other neurological disorders16, making them the most immediate option for patients. This disclosure can leverage these cutting-edge technologies to both elucidate the mechanisms underlying CMT2A and explore potential therapeutic strategies. By targeting specific MFN2 mutations in patient-derived cells and preclinical rodent models, precision medicine approaches can be designed. The successful development of such therapies would revolutionize the treatment of CMT2A, offering hope to patients and their families who currently lack hope.-34-4896-3340-4800.3Atty Dkt.: 114198-0880

[0168] Impact on rare disease research and beyond: This research has the potential to significantly advance Applicant’s understanding of CMT2A pathogenesis and pave the way for the development of targeted therapies. The innovative approaches and tools developed in this study, particularly the integration of advanced imaging and quantitative MS-based proteomics, can have broad applicability beyond CMT, enabling the study of organelle dynamics and their role in other neurodegenerative diseases. By addressing the unmet needs of CMT patients and their families, this research can make a lasting impact on the field of rare disease research and contribute to the development of novel therapeutic strategies for a range of neurological disorders.

[0169] Experiment No. 2: Deep Learning Microscopy for Enhanced Organelle Dynamics Analysis. This approach can leverage the emerging power of deep learning microscopy to achieve unprecedented spatiotemporal resolution in tracking organelle dynamics. This cutting-edge imaging technique can enable the ability to capture subtle changes in organelle mobility, morphology, and interactions that may be missed by conventional microscopy methods. By applying deep learning algorithms to analyze vast amounts of image data, Applicant can extract quantitative information about organelle behavior, providing a deeper understanding of how MFN2 mutations disrupt these processes.

[0170] Patient-Derived Cell Lines and Parallel Rat Models. The use of patient-derived cells and rat models carrying corresponding CMT2A-associated MFN2 mutations represent a significant conceptual innovation. The models allow the investigation and confirmation of the impact of these mutations in a human-relevant context and bridge the gap between basic research and clinical application. The ability to compare findings from both cellular and animal models can provide a more comprehensive understanding of the disease mechanisms and facilitate the translation of Applicant’s discoveries into potential therapies.

[0171] MS-based protein and nucleic acid analysis. The integration of stable isotope-based MS techniques can enable us to perform in-depth proteomic profiling and protein / nucleic acid turnover studies of normal and diseased states. This discovery-based proteomics can provide a holistic view of the cellular and molecular changes caused by MFN2 mutations, revealing novel protein-protein interactions, altered protein turnover rates, and dysregulated pathways. By identifying key biomarkers and therapeutic targets, MS can play a crucial role in guiding the development of precision medicine approaches for CMT2A.-35-4896-3340-4800.3Atty Dkt.: 114198-0880

[0172] Cutting-Edge Genetic Therapies: Allele-Specific ASOs. The exploration of allelespecific genetic therapies, including antisense oligonucleotides (ASOs), represents a major innovation in the field of CMT research. ASOs have already shown promise in clinical trials for other neurological disorders. By targeting specific MFN2 mutations, these approaches offer the potential for personalized treatment of CMT2A (ASOs). The use of preclinical rodent models can be crucial for evaluating the efficacy and safety of these therapies before their translation to the clinic, and for understanding the progressive, length-dependent nature of CMT2A.

[0173] Scientific rigor, statistics, and bioinformatics. Data analyses can be automated to minimize observer bias, mice can be assigned a random ID coded to the experimenter (blind to the different conditions). The size of each group can be estimated based on similar data sets with power analysis using G*Power. Applicant can consider sex as a biological variable in Applicant’s experimental design, collect sex-specific data, and perform Applicant’s analysis within and between equal sized groups of male and female mice and humans. For most analyses, Applicant can assess statistical significance by the Wilcoxon signed rank test or the Kolmogorov- Smirnov test for nonparametric comparison. For parametric data sets, the student’s / -test can be used for comparing two groups, or one-way ANOVA with Tukey’s post-hoc when comparing across multiple data sets. For multiple trial experiments, two-way re-peated measures ANOVA can be applied, and multiple testing adjusted with Benjamini- Hochberg. -values can be considered significant if <0.05. When possible, Applicant can use multiple independent assays.

[0174] The impact of CMT2A mutations on MFN2 protein dynamics. The overarching is that disease-associated point mutations in MFN2 disrupt the protein’s normal localization, dynamics, stability, and protein-protein interactions, leading to defects in organelle dynamics and function. A multi-faceted approach that combines cutting-edge imaging, MS, and the utilization of patient-derived cells and animal models can be applied._A workflows has been developed for analyzing protein dynamics with single-molecule tracking methods, ensemble super-resolution imaging approaches, and MS-based proteomics. Applicant’s data show that Applicant can capture trajectories of exogenously expressed single human MFN2-HALO molecules at inter-organelle contact sites (FIG. 9). Using CRISPR-mediated dual-allele endogenous MFN2 HALO vs. SNAP tags in Applicant’s patient cells generated by the-36-4896-3340-4800.3Atty Dkt.: 114198-0880Conklin lab (see LOS), Applicant can apply this methodology to distinguish single particle trajectories of normal vs. CMT2A MFN2 mutations. Applicant optimized super-resolution imaging of MFN2 and mitochondria dynamics, revealing distinct actin-dependent versus actin-independent mitochondrial fusion dynamics. Applicant also observed MFN2 at ER- mitochondria contacts during mitochondrial fission (data not shown). Similar quantitative imaging of MFN2 in the context of CMT2A mutations can be applied.

[0175] Experiment No. 3: CMT Patient-Derived Cells and Animal Models

[0176] Patient-derived neurons. Applicant obtained fibroblasts and produced induced pluripotent stem cells (iPSCs) from CMT2A patients harboring the MFN2 mutations of interest (R364W, T206I, R94W, and S249T, FIG. 10)19and healthy age-matched controls. Applicant also generated two rat models matching two of Applicant’s MFN2 mutations (R364W and T206I), from which also generated fibroblast cell lines for ASO design and validation. These iPSCs can be differentiated into sensory and motor neurons using standard protocols, the primary cell types affected in CMT2A, to create disease-relevant cellular models. Applicant can also identify age-dependent phenotypes masked by cellular reprogramming by investigating primary fibroblasts and neurons directly transdifferentiated from fibroblasts (“iNs”). Importantly, parallel experiments with age-matched and isogenic controls can be performed for each mutation to clarify background-dependent differences.

[0177] Endogenous Tagging and Imaging - CRISPR / Cas9-mediated Tagging. CRISPR / Cas9- mediated homology-directed repair can be utilized to introduce small molecule ligand binding domains (e.g., HALO, SNAP) at the endogenous gene loci of MFN2 in patient- derived iPSCs. This allows visualization of the localization and protein dynamics of both wild-type and mutant MFN2 proteins in their native cellular environment. These endogenous probes can also facilitate specific pulldown of mutant versus wild-type protein in the native heterozygote environment of patient cells for downstream biochemical and proteomic analyses (FIG. 11).

[0178] Live-Cell Imaging of Endogenous Tags. Applicant can employ advanced imaging techniques, including dual-color live-cell single-particle tracking microscopy and live-to- fixed correlative light and electron microscopy to track the movement and interactions of MFN2 and its associated organelles (i.e., mitochondria, lysosomes) in real-time. This enables-37-4896-3340-4800.3Atty Dkt.: 114198-0880 us to determine how CMT2A-associated mutations in MFN2 impact organelle dynamics and interorganelle contacts. Applicant can also utilize super-resolution microscopy (e.g., lattice- SIM) to visualize the nanoscale organization of MFN2 and its interaction with actin filaments and other proteins at ER-mitochondria contacts. This can provide insights into how MFN2 mutations disrupt these critical inter-organelle communication hubs.

[0179] MS-Based Proteomic Analysis - Bulk quantitative proteomics of cell and tissue extracts. This disclosure also provides methods and compositions to achieve precise measurements of relative protein abundance by utilizing tandem mass tags (TMT) in combination with Real-Time Search and synchronous precursor selection MS3 (SPS-MS3), as Applicant have successfully done in previous studies20, 21. This facilitates the study and comparison of the global cellular state of normal versus CMT2A patient-derived cells and rat tissues. By subjecting whole cell and bulk tissue extracts to Applicant’s established quantitative proteomic analysis pipeline, new information can be obtained regarding the effect of these mutations on thousands of individual proteins and potentially reveal unknown mechanisms contributing to disease pathogenesis (FIG. 12).

[0180] Protein Turnover, Orchestration of protein production and degradation (i.e., protein turnover) and the regulation of protein lifetimes play a central role in many basic biological processes. This is particularly true in com-plex organelles. Protein turnover is well known to be compromised during aging and numerous neurodegenerative diseases. Nearly all mammalian proteins are replenished by protein turnover in waves of synthesis and degradation, a process that ensures a highly functional proteome. To explore the possibility that MFN2 mutations destabilize the protein Applicant can use pulse-chase labeling with stable isotopes (13Ce-Lys and15N Spirulina) in conjunction with affinity purification-mass spectrometry (AP-MS) and MS to measure the turnover rates of wild-type and mutant MFN2 proteins. Mutated MFN2 can also be evaluated for its impact on the turnover of additional proteins in bulk extracts. This can provide insights into how CMT2A mutations impact protein synthesis and degradation.

[0181] Interactome Analysis. AP-MS can be used to identify the protein-protein interactions of wild-type and mu-tant MFN2. SNAP and Halo ligands can be used to selectively isolate the WT and mutated MFN2 proteins and use cells lacking the ligand binding domains as negative controls (FIG. 11). This can be used to control for non-specific proteins that stick to -38-4896-3340-4800.3Atty Dkt.: 114198-0880 the beads. By comparing first removing the non-specific back-ground and then comparing their interactomes, Applicant can uncover how these mutations alter MFN2’s binding partners, engage specific signaling pathways, and provide mechanistic insights into CMT2A pathogenesis.

[0182] Experiment No: 4 - The effects of CMT2A-associated MFN2 mutations on organelle composition, dynamics, and function.

[0183] Mitochondria and lysosomes are required for homeostasis and are key targets for many neuro-degenerative disorders where long-lived cells have more stringent requirements for properly regulated organelle turnover and maintenance. While much is known about MFN2 function in mitochondrial dynamics, little is known about how these specific MFN2 mutations impact other organelle’s dynamics. Surprisingly, Applicant’s data show these MFN2 mutations also impact lysosome mobility. Without being bound by theory, Applicant hypothesized that CMT2A-associated MFN2 mutations lead to defects in both mitochondria and lysosomal mobility, which then leads to defects in their turnover and function. A combination imaging, biochemical assays, and physiological analyses can be used to assess the impact of these mutations in vitro and in vivo to the end of filling the gap in understanding in how these dominant mutations in MFN2 lead to accelerated degeneration in extremely long peripheral neurons.

[0184] Using deep learning-based trans-former architectures that specialize in larger context windows, Applicant was successful in automated organelle tracking with accuracy far exceeding that of commercial software, which was able to detect alterations to organelle movement in CMT patient cells (FIG. 13).

[0185] Rat Models. There is some skepticism in the CMT field regarding the available CMT mouse models, which usually do display neurodegeneration, but do not recapitulate the progressive, length-dependent neuropathy typically observed in humans. In contrast, rats are thought to be a better model. Without being bound by theory, Applicant speculates this is due to their larger size, and therefore longer neurons. For this disclosure, Applicant acquired a CMT2A rat model with an R364W mutation matching one of Applicant’s patient fibroblasts. Applicant’s in vivo and histological data show this rat displays all the hallmarks of CMT2A. Applicant also generated a T206I CMT2A rat model to match another patient, a 6-year-old-39-4896-3340-4800.3Atty Dkt.: 114198-0880 girl with very severe, early onset CMT2A symptoms. To date, at least 63 FO pups were bom. These rats are models for in vivo studies, histology, primary DRG neuron live cell imaging, and rat embryonic fibroblasts can be used for in vitro ASO screening. These models can enable the investigation of the in vivo consequences of these mutations on neuronal function, morphology, and overall CMT2A progression, while also providing a platform for testing the genetic tools described in 3, below.

[0186] Organelle and Protein Turnover and Function - Organelle Dynamics. Live-cell imaging and deep learning-based analysis can be used to track and quantify the dynamics of mitochondria and lysosomes in patient-derived iPSCs and rat models expressing CMT2A-as- sociated MFN2 mutations. This allows assessment of changes in organelle velocity, directionality, pausing, and interactions with other cellular components.

[0187] Organelle protein composition. Previously established mitochondria and lysosome purification techniques (i.e., Tom-22 affinity capture and Lyso IP) can be utilized to obtain highly enriched material for deep proteomic analysis27(FIG. 14). In this way, Applicant can obtain a much comprehensive assessment of protein composition. Furthermore, by performing these experiments in metabolic labeled Mfn2a model cells, rats, and the relevant controls a new understanding of how MFN2 mutations impact organellar protein homeostasis can be discovered.

[0188] Organelle Function. Mitochondrial function can be assessed using assays such as Seahorse XF analysis (to measure oxygen consumption rate and extracellular acidification rate), mitochondrial membrane potential measurements, and ATP production assays. Lysosomal function can be evaluated using assays for lysosomal pH, enzyme activity, and autophagic flux. It was found that CMT2A fibroblasts have elevated superoxide activity at baseline and after inhibition of mitochondrial oxidative phosphorylation with CCCP (FIG. 15).

[0189] mtDNA Quantification and turnover. MFN2 defects can have downstream consequences on mitochondrial function. For example, fusion is important for cristae maintenance, and thus defects could impact oxidative phosphorylation28, 29. Meanwhile, mitochondria-organelle contact alterations can influence lead to Ca2+mediated defects30. Additionally, properly regulated mitochondrial fission and fusion can impact the stability of-40-4896-3340-4800.3Atty Dkt.: 114198-0880 mtDNA, which is required for proper oxidative phosphorylation. Damaged and depleted mtDNA is associated with MFN2 dysfunction31, 32, likely via its fusion role, but perhaps also via overall impact on mitochondrial dynamics33. Together, it is perhaps not surprising there is reduced mitochondrial respiration in cells lacking MFN234. Applicant can measure mtDNA copy number and integrity in patient-derived neurons and CMT2A rat models using qPCR and long-read sequencing (FIG. 16). Furthermore, by extracting the mtDNA from the mitochondria isolated from the15N pulse-chase tissues and cells the purified mitochondria digesting it into individual nucleoside bases and by using MS analysis Applicant can measure the level and turnover of the14N and15N containing bases as Applicant have done in the past (FIG. 17). Applicant can also assess the release of mtDNA into the cytoplasm and circulation, which can trigger innate immune responses. Evaluation of the activation of the cGAS-STING pathway can be assessed, a key mediator of innate immune responses to cytoplasmic DNA, in patient-derived neurons and CMT2A rat models.

[0190] Neurodegenerative Phenotype Assessment. Neuronal health and viability in patient- derived iPSC-derived neurons can be assessed using assays such as neurite outgrowth, mitochondrial morphology, axonal swellings and markers of apoptosis and oxidative stress (FIG. 18)._Peripheral neuropathy can be longitudinally evaluated n=14 WT vs. CMT2A rats at 16, 22, and 30 weeks of age. An additional n=3 animals of each sex can be evaluated for ages 16 and 22 for endpoint experiments. All assays can be conducted as follows:

[0191] Grip strength. The strength of all four limbs can be evaluated at 16, 22 and 30 weeks using an automated Grip Strength Meter (Columbus Instruments). Within 1 week after training (10 practice trials using a mesh bar), peak force exerted by each rat is measured 10 times consecutively with 10s resting periods and averaged.

[0192] Electrophysiology. Rats aged 16, 22, and 30 weeks can be anaesthetized in Manor lab and placed on a heating pad to maintain constant body temperature throughout the electrophysiological study. Sciatic nerve motor nerve conduction velocity and compound action potential (CAP) can be recorded.

[0193] Morphology. After in vivo experiments are completed, sciatic nerve samples can be processed in the Manor lab for imaging. For morphometric analysis, semithin sections can be-41-4896-3340-4800.3Atty Dkt.: 114198-0880 examined. Ultrathin sections can be examined by electron microscopy to measure changes to myelin / axons in SCs and to axonal organelle ultrastructure.

[0194] Microtubule Post-translational Modifications (PTMs) Immunofluorescence, Western Blotting and MS analysis. One can assess the levels and distribution of key microtubule protein (i.e., alpha- and beta-tubulin) PTMs, such as acetylation and detyrosination, in patient-derived neurons and CMT2A rat models using AP-MS analysis, Immunofluorescence and Western blotting with commercially available antibodies, and Western blotting. These PTMs are known to regulate microtubule stability and dynamics, which are crucial for axonal transport and neuronal function (FIG. 19).

[0195] Experiment No.: 5 - Evaluate the efficacy of allele-specific genetic therapies in rescuing CMT phenotypes

[0196] Targeted depletion of mutant MFN2 transcripts using allele-specific ASOs can normalize disease phenotypes in rat and human iPSC models are also evaluated. Applicant’s model filters out ASO sequences predicted to elicit innate immune responses, incorporates ENCODE protein-RNA interaction data to prioritize RNA regions accessible for ASO targeting, and also considers genetic variation. This can reduce the number of ASOs required for screening.

[0197] Applicant performed 30x phased long-read whole genome sequencing on a CMT2A patient carrying the S249T mutation. This analysis revealed multiple sequence variations, including insertions, SNVs, and deletions (FIG. 20), within the CMT2A-causing allele (haploblock). These multiple sites offer additional target sequences for ASO design beyond the disease-causing mutations. Additionally, these mutations provide distinct sequences for qPCR primers, enabling precise differentiation between alleles.

[0198] Evaluation of phenotypic rescue in rat models. Applicant’s ASO design tool can be used to generate 10-20 ASOs of varying lengths (18-25 nt) each, tiled across the pathogenic mutations c, 1090C>T / R364W, and c.617C>T / T206I) of Applicant’s rat CMT2A models and prioritized by predicted allele selectivity. Using fibroblasts isolated from embryonic day 13 rats, Applicant can test ASOs by transfecting the cells with 500 nM of fully phosphorothioate (PS) backbone modified, 2’-O-methoxyethyl (MOE) “gapmer” (RNA-DNA-RNA) ASOs that recruit endogenous RNase H for target RNA degradation. After 48 hours, RNA can be-42-4896-3340-4800.3Atty Dkt.: 114198-0880 extracted, and depletion evaluated using allele-specific rt-qPCR for the transcripts produced from the mutant and normal alleles. Specificity of allele-specific qPCR primers can be validated using wildtype and mutant in vitro RNA transcripts. One can prioritize 3-5 ASOs for in vivo studies that show selective reduction (>90%) of mutant MFN2 mRNA while minimally (<10%) affecting wild-type levels. These conservative metrics are informed by successful development of allele-specific ASOs that achieved as much as 100-fold selectivity for reduction of mutant huntingtin mRNA by ASO-targeting of SNPs linked to the CAG repeat expansion allele that is the cause of Huntington’s disease36.

[0199] Candidate ASOs \n Mfii2 mutant rats (n=4) can be delivered via intrathecal delivery. Intrathecal injections can be performed with 30 pl ASO solution or vehicle (PBS) into the subarachnoid intrathecal space via catheter, followed by 40 pL of artificial cerebrospinal fluid37. The procedure involves puncturing the at lantooccipital membrane, delivering the solution into the cisternal space, and closing with staples. After the initial injection, subsequent injections can be performed every other day under Isoflurane anesthesia. Guided by preclinical studies using ASOs in neurodegeneration (e.g. Huntington’s disease, SMA, ALS caused by SOD1 mutation), one can initially test four biweekly doses, 100 pg ASO or vehicle (PBS) each, starting at postnatal day 30. After four weeks, one can dissect sciatic nerve and CNS tissue and measure the levels of mutant and wild-type Mfn2 RNA using allele-specific rt-qPCR following tissue harvest and RNA ex-traction. The most highly efficacious ASO can be used to assess amelioration of disease-relevant molecular, cellular and functional phenotypes; this can involve assessing behavior, electrophysiology, and histological markers of neurodegeneration as described in 2, above. The minimum extent of depletion of the mutant transcript expected to achieve therapeutic efficacy in CMT2A patients is also evaluated.

[0200] Validation of therapeutic efficacy of allele-specific ASOs in CMT2A human cellular models as candidate ASO therapeutics. Applicant previously performed phased long-read whole genome sequencing (WGS) at 30x depth on a CMT2A patient carrying the S249T [T- >A] mutation. This analysis revealed multiple sequence variants - SNVs, insertions and deletions (FIG. 20) - in intronic and exonic regions of the CMT2A-causing allele (haploblock). These sites offer additional target sequences for ASO design beyond the disease-causing mutation. Similarly, One can perform WGS of the other CMT2A patients to-43-4896-3340-4800.3Atty Dkt.: 114198-0880 identify variants linked to each patient’s pathogenic mutation to expand the ASO targeting space.

[0201] Applicant can transfect patient iPSC-derived motor neurons with prioritized ASOs (30-50 per patient; n=3 replicates; 24-well plates). Applicant has developed a robust protocol for efficient transfection of ASOs into iPS-motor neurons and demonstrated successful depletion of target RNAs (FIG. 21). After transfection (48-72 hours), RNA can be extracted and allele-specific transcript depletion assessed by rt-qPCR. Depletion can be validated on the protein level by immunoprecipitation of MFN2 followed by quantitative mass spectrometry to measure levels of MFN2 peptides containing the mutant vs wildtype amino acid.

[0202] For ASOs that demonstrate at least 80% reduction in mutant mRNA without or minimally (<10%) affecting wild-type levels, one can proceed with dose-response assays to determine potency (IC50) and selectivity. Once the IC50 is established, one can measure off- target effects at both IC50 and IC90 concentrations using next RNA-seq, using the number of non-target transcripts with <5 nucleotide mismatches changed >2-fold at FDR-adjusted p- value <0.01 as the primary metric. After validating candidate ASOs and determining the most potent ASO candidates, one can assess the impact of mutant MFN2 knockdown on the cellular phenotypes observed in Aim 2, focusing on organelle dynamics, function, and neuronal health.4896-3340-4800.3Atty Dkt.: 114198-0880

[0203] Experiment No.: 6 - Organelle Dysfunction at the Nexus of Neurodegeneration

[0204] The precise regulation of organelle activity, positioning, and turnover is paramount for cellular health and survival. Organelle dynamics, including fission / fusion and mobility, are orchestrated by the actin cytoskeleton and its regulatory proteins. In peripheral neurons, with their extensive projections demanding meticulously positioned organelles and extraordinarily long travel distances, the importance of organelle mobility and turnover is amplified1-4. Consequently, mutations that disrupt organelle mobility / turnover are implicated in a multitude of peripheral neurodegenerative diseases, including Charcot-Marie-Tooth (CMT) disease5'7, amyotrophic lateral sclerosis (ALS), and others. However, significant knowledge gaps remain to be filled to fully understand the fundamental mechanisms5’6’8'10. For instance, conflicting conclusions have been drawn regarding the impact of RAB7A mutations in CMT2B: 1) the functional consequences of these mutations on RAB7A activity are haploid insufficiency11vs gain of toxicity12'14; 2) The RAB7A mutations promote mitochondrial elongation15, 16vs fragmentationl 7‘18. By using innovative techniques and physiological models, Applicant’s disclosure is aimed at resolving these important mechanistic issues and informing future therapies.

[0205] CMT: A Focus on Axonal Degeneration - CMT, the most prevalent inherited neurological disorder, manifests as a progressive deterioration of peripheral nerves, leading to motor and sensory impairments6, 7’9. Despite the ubiquitous presence of CMT-implicated proteins throughout the body, the disease’s predilection for peripheral nerves with long projections underscores the critical role of organelle mobility in its pathogenesis. Notably, genes like INF2l 9‘20and Rab7a ’13, 21, 22, known to influence organelle fission / mobility / turnover, are mutated in CMT.

[0206] INF2 & RAB7 at the Intersection of Actin, ER, and Organelles. Applicant’s data show organelle fission necessitates the transient accumulation of actin filaments on organelles like mitochondria, lysosomes, and endosomes. The endoplasmic reticulum (ER), through its extensive organellar contacts, designates fission sites where actin’s force facilitates the process. Applicant’s data shows ER-mediated, actin-dependent fission mechanism appears conserved across various organelles, suggesting a universal ER / actin- dependent process for regulating organelle dynamics (FIG. 22).23-45-4896-3340-4800.3Atty Dkt.: 114198-0880

[0207] However, the precise coordination between the ER, actin, and the organelle during fission remains enigmatic. Applicant postulates that actin regulatory proteins on both the ER and the organelle may hold the key to this coordination, and their dysfunction could trigger neurodegenerative disorders like CMT24 25. Recent work shows that lysosomes also mark mitochondrial fission sites16, indicating fission depends on a 4-way intersection between mitochondria, lysosomes, the ER, and the actin cytoskeleton.

[0208] That INF2 is an ER-anchored protein and RAB7A a lysosomal protein, and Applicant’s data showing INF2 and RAB7 interact with one another ( FIG. 23) and with actin strongly suggests these two proteins act within the same pathways controlling mitochondrial dynamics. Together, these observations motivate investigation of the intriguing fact that gain-of-function mutations in each protein leads to CMT.

[0209] Applicant’s data show INF2 CMT patient fibroblasts display actin-dependent organelle immobilization and aberrant actin assembly. Live cell imaging of lysosomes in CMT patient and healthy control fibroblasts shows decreased lysosome mobility in CMT patients. This mobility defect is rescued by treatment with Latrunculin B, an actin depolymerizing drug. Additionally, INF2 CMT patient fibroblasts display a significant increase in actin aggregates compared to healthy controls. These actin aggregates often contain organelles, suggesting a link between aberrant actin assembly and organelle immobilization in INF2 CMT (FIG. 24).

[0210] In further support of Applicant’s hypothesis that INF2 and RAB7A act in the same mechanisms impacting organelle defects in CMT, Applicant observed a strong actindependent reduction in organelle mobility in INF2 CMTDIE patient-derived neurons and in primary neurons expressing RAB7A CMT2B mutants ( FIG. 25).

[0211] Given the emerging evidence for an important role for ER-associated actin in both INF2 and RAB7A patients, Applicant sought to manipulate ER-associated actin while leaving the rest of the actin cytoskeleton intact. Applicant hypothesized that in the same way Applicant’s AC -ER probe specifically labels ER-associated actin, ER-anchored DeActs (“DeActs-ER”) might specifically destroy ER-associated actin. In support of this hypothesis, Applicant found that expressing DeActs-ER left cortical actin and stress fibers intact (as measured using phalloidin) in U2OS cells yet caused significant disruption to ER-associated-46-4896-3340-4800.3Atty Dkt.: 114198-0880 actin (as measured using Applicant’s AC -ER probe) ( FIG. 26A). Applicant’s DeActs-ER and DeActs-mito tools were further validated as not disrupting organelle dynamics with a large array of control experiments, which are reported in Applicant’s recent preprint publication investigating the role of actin in mitochondrial fusion. These experiments also showed that mitochondrial fusion depends on actin, ER-mitochondria contacts, and the dynamin-related protein MFN2, which intriguingly is yet another protein involved in both organelle dynamics and in Charcot-Marie-Tooth disease (CMT2A)24.

[0212] To test Applicant’s hypothesis that ER-associated actin mediates defects in organelle mobility in CMT patient neurons, Applicant expressed DeActs-ER in patient derived neurons. Similarly, cortical actin remained largely unchanged in DeActs-ER expressing neurons ( FIG. 26B). In support of Applicant’s hypothesis that ER-associated actin mediates organelle immobilization in INF2 CMT neurons, Applicant’s results show lysosome mobility was restored in INF2 CMT iNs expressing DeActs-ER ( FIG. 26C). Remarkably, Applicant found that expression of DeActs-ER in INF2 CMT iNs also reversed neuronal swellings to healthy control levels four days after lentiviral transduction ( FIG. 26B).

[0213] INF2 and RAB7A: Key Players in Axonal Health. INF2, an ER-tethered actin regulatory protein, promotes actin assembly in a regulated fashion through autoinhibitory mechanisms. Its depletion curtails mitochondrial fission, while a constitutively active mutant leads to excessive mitochondrial fission and restricted mobility. Numerous gain-offunction, autosomal dominant CMTassociated INF2 mutations exist, predicted to yield a constitutively active protein. As shown above, Applicant’s data show these mutations alter organelle dynamics and neuronal health due to excess actin assembly at the endoplasmic reticulum ( FIG. 27)

[0214] RAB7A, an endo-lysosomal protein 26-29, participates in regulating mitochondrial mobility ( FIG. 28). Applicant’s data and other recent studies reveal that CMT-associated RAB7A mutations disrupt organelle mobility in an actin-dependent fashion, and that RAB7A may interact with INF2. Both RAB7A and INF2 have also been implicated in mitochondrial dynamics and function15-20>24>25>30RAB7A endosomes carry and translate mRNAs encoding mitochondrial proteins15. Interestingly, both INF2 and RAB7A appear to recruit dynaminrelated protein 1 (Drpl) onto the organellemitochondria contact sites to promote mitochondria fission 16, resulting in excessive mitochondrial fragmentation in CMT2B-47-4896-3340-4800.3Atty Dkt.: 114198-0880RAB7AV162Mfibroblasts from patients as well as primary sensory neurons from a knock-in mouse model ( FIG. 29)17, 20, 25. The effect was partially rescued with small molecular inhibitor targeted Drpl17, further highlighting the interconnectedness of these proteins, organelles, and cellular processes.

[0215] Data has shown that RAB7A mutations reduce TrkA receptors and inhibit nerve growth factor (NGF)-induced neurite outgrowth, further underscoring the importance of RAB7A in axonal function and neurotrophic signaling14, 29, 31. Understanding the precise mechanisms by which INF2 and RAB7A regulate organelle dynamics and their interplay with the actin cytoskeleton may contribute to understanding their contribution to CMT pathogenesis.

[0216] A Focus on Axonal Mechanisms. One aspect of this disclosure provides the mechanisms by which INF2 and RAB7A, in conjunction with actin and ER-organelle interactions, regulate organelle dynamics and contribute to CMT pathogenesis and their application to the develop of new therapies. Leveraging cutting-edge imaging techniques and patient-derived neuronal models, one can dissect the impact of CMT-associated mutations on axonal transport, organelle morphology, and health in both mitochondria and endosomes. Applicant’s data reveals that INF2 CMT mutations not only disrupt mitochondrial mobility, but also impact endolysosomal transport, underscoring the interconnectedness of these cellular processes. Furthermore, it is demonstrated that depolymerizing ER-associated actin with Applicant’s novel DeActs-ER tool reverses neuronal swelling and restores organelle mobility in INF2 CMT patient-derived neurons, providing a potential therapeutic avenue for CMT.

[0217] RAB7A also regulates retrograde axonal transport of neurotrophic signals such as NGF / TrkA14or BDNF / TrkB27in motor neurons, and trafficking and signaling of the NGF receptor-TrkA in PC 12 cells29. Retrograde axonal transport of trophic signals is critical for neuronal function and maintenance32'43. Thus, RAB7A controls axonal trophic signaling by downregulating the signaling machinery in a spatial and temporal manner. By disrupting the tight regulation, CMT2B RAB7A mutations result in NGF / TrkA signaling deficits leading to axonal degeneration13,14,44. Although the exact mechanism is not well understood, it is possible that peripheral sensory neurons with long axons are most susceptible to neurotrophic deficits, thus leading to severe axonal degeneration or “axonopathy”, as seen in CMT213.-48-4896-3340-4800.3Atty Dkt.: 114198-0880

[0218] By elucidating the interplay of actin, the ER, and organelles in the context of CMT, with a particular focus on axonal transport and the roles of INF2 and RAB7A, Applicant’s disclosure can identify fundamental principles governing organelle dynamics in both health and disease. Novel therapeutic strategies targeting organelle dysfunction in CMT and other neurodegenerative disorders can be identified, ultimately improving the lives of countless patients.

[0219] Pioneering CMT Patient-Derived Cell Models and Therapeutic Exploration. This disclosure breaks new ground by utilizing CMT patient fibroblasts, direct deriving neurons and primary rodent neurons that endogenously express INF2 and RAB7A mutations. These novel cellular models provide a highly relevant platform to investigate the pathophysiology of CMT, offering insights into mechanisms and potential therapeutic targets.

[0220] Deep Learning-Powered Microscopy and Analysis. Applicant’s deep learning-based point-scanning super-resolution microscopy (PS SR) enables high spatiotemporal resolution imaging of neuronal organelles with minimal phototoxicity. Subtle changes in organelle dynamics can be discovered which is crucial for understanding the impact of CMT mutations.

[0221] Furthermore, Applicant has developed deep learning-based quantitative organelle tracking, automating the challenging task of tracking organelle movements in neurons. This innovation overcomes the limitations of manual tracking and conventional software, providing efficient quantification of organelle dynamics.

[0222] Novel Tools for Probing ER-Actin Dynamics. Applicant has engineered groundbreaking genetically-encoded tools to visualize ( FIG. 25) and manipulate ( FIG. 26) ER-associated actin filaments. Applicant’s organelle-targeted actin probes offer exceptional clarity in monitoring ER-actin dynamics, while Applicant’s DeActs-ER probes enable selective disruption of ER-associated actin without affecting the broader actin cytoskeleton. These unique and innovative tools are instrumental for deciphering the role of ER-associated actin in organelle mobility and CMT.

[0223] Unraveling the RAB7A-INF2 Axis in CMT, Applicant’s data reveals a novel and intriguing interaction between INF2 and RAB7A, an endo-lysosomal protein implicated in CMT. This discovery opens new avenues for investigating the interplay between these proteins in regulating organelle dynamics and contributing to CMT pathogenesis. Applicant’s-49-4896-3340-4800.3Atty Dkt.: 114198-0880 data demonstrates that Rab7 mutations inhibit NGF-induced neurite outgrowth, further emphasizing the importance of Rab7 in axonal function and neurotrophic signaling.Applicant can build on these findings to explore the functional consequences of this interaction and its implications for CMT. Applicant can also test whether a modified version of exogenous NGF that does not induce pain can rescue axonal defects in the Rab7 CMT2B mouse model, providing potential therapeutic avenues.

[0224] Translational Potential: ASO-Based Therapeutics, Small Molecular Inhibitor and Nerve Growth Factor. One can use these finding to explore innovative therapeutic strategies, such as antisense oligonucleotides (ASOs), small molecular inhibitor and novel NGF mutant, to target the RAB7A-INF2 axis in CMT. The efficacy of ASOs can be rigorously evaluated in both patient-derived cell lines and a CMT2B mouse model, providing crucial preclinical data for potential clinical translation. This approach holds promise for developing targeted therapies that restore organelle dynamics and mitigate neurodegeneration in CMT.

[0225] Animal Protocol. All animal experimental protocols have been reviewed and approved by UCSD IACUC. All animal experiments can be blind-coded and repeated for > 3 times to ensure reproducibility and all information regarding the test samples such as the age, sex, genotype of mice can be purposely withheld from the scientist s) who performs any specific tests. These data can be only revealed at the final tubulation and analysis.

[0226] Sex difference and power analysis. Male and female can be measured separately to test for potential sex differences in all studies. The minimal number of mice needed can be powered by using GPower3.1(gpowersoftware.informercom / 3.1) to meet statistical analysis standards. Based on data and past studies45, 10-15 mice / group can be needed for behavioral assays, and 3-5 mice for neuropathological analysis. A total number of 300 mice can be needed for completion of the proposed study.

[0227] Scientific Rigor, Transparency, Statistical Analysis. Applicant can use reagents (chemicals, antibodies) that have been authenticated extensively; all experiments can be performed blindly. All data can be collected from > 3 independent experiments.

[0228] Data can be analyzed using GraphPad Prism (GraphPad, La Jolla, CA). The significance of differences can be generated using the “Analyze” function in Prism with appropriate test methods (student / -test, One-Way or Two-Way ANOVA tests) including-50-4896-3340-4800.3Atty Dkt.: 114198-0880 post-hoc tests (Dunnett, Tukey or Bonferroni etc.). Results can be expressed as mean ± SEM and significance can be rejected at p>0.05.

[0229] Experiment No: 7 - The Impact of INF2 and RAB7A CMT Mutations on Neuronal Organelles.

[0230] Applicant’s data show CMT-associated mutations in INF2 an RAB7A disrupt organelle mobility8,46. The precise mechanisms by which these mutations disrupt organelle dynamics and contribute to CMT can be probed in this aim by measuring both organelle dynamics and function in patient-derived cells.

[0231] Multiple deep learning-based workflows that integrate classical fluorescence microscopy with label-free quantitative phase imaging can be used to enhance Imaging speed and content while minimizing phototoxicity. Using deep learning-based transformer architectures that specialize in larger context windows, Applicant automated organelle tracking with accuracy far exceeding that of commercial software, which was able to detect alterations to organelle movement in CMT patient cells ( FIG. 30).

[0232] To understand the impact of INF2 and RAB7A mutations on neuronal health and function, as implicated in CMT and other neurodegenerative diseases, Applicant can utilize patient-derived induced pluripotent stem cells (iPSCs) and rodent primary neurons to investigate how CMT2-associated mutations in INF2 and RAB7A affect organelle mobility, morphology, and axonal function. To capture fast neuronal organelle movements with high spatiotemporal resolution, Applicant can combine confocal microscopy with Applicant’s deep learning-based point-scanning super-resolution microscopy (PSSR) image processing model trained for imaging organelle transport in neurons. Applicant can image organelles in the axons and dendrites to measure their mobility. These neurons can also be assessed for changes in organelle health. To probe alterations in specific subpopulations of organelles, Applicant can use pre-synaptic markers to measure the extent to which organelle distribution relative to synapses is altered.

[0233] Assess the impact of INF2 and RAB7A CMT2 mutations on organelle mobility in neurons. Applicant has directly converted INF2 CMT patient fibroblasts into neurons, as well as age-matched healthy patient controls. To determine the changes in mobility that may be conserved across organelles, mitochondria and endosomes can also be labeled with organelle-51-4896-3340-4800.3Atty Dkt.: 114198-0880 markers. This approach can be applied to CMT2B patient cells. The mutant alleles of INF2 / RAB7A can be knocked down with siRNA, and control cells can be transfected with INF2 / RAB7A CMT mutant constructs. Applicant can image and analyze organelle mobility in the axons and dendrites of patient-derived and primary mouse motor neurons to measure their mobility.

[0234] Assess the impact of INF2 and RAB7A CMT2 mutations on organelle and neuronal health. To investigate how alterations to INF2 and RAB7A-mediated organelle mobility affect overall organelle function, Applicant can address CMT2-associated mutation effects on mitochondria’ and endosomal functions. In mitochondria, Applicant can assay mitophagic flux using the MitoQC probe, which changes from yellow (green+red) to red during mitophagy, ATP production (JC9 ratiomic dye for measurement of mitochondrial membrane potential), and oxidative phosphorylation (OxPhos) capacity by utilizing the Seahorse assay. To measure endosomal function and retrograde axonal transport of NGF, Applicant can culture neurons in the microfluidic chamber47,48and track transport of endocytic cargo using either quantum dot-labeled NGF(QD-NGF)47'49or the Dye Quenched-Bovine SerumAlbumin trafficking assay (“DQ-Red”) ( FIG. 31), which is endocytosed and fluoresces when endosomes fuse with lysosomes. Lysosomal pH can be measured by imaging cells labeled with a combination of pH-sensitive and pH-insensitive dyes or proteins.

[0235] Neuronal health can be assessed three days after transfection as well as neuron viability using propidium iodide / Annexin V staining and flow cytometry to measure apoptosis and the proportion of dead / dying cells. Applicant can measure neurite growth and axonal swelling by imaging fixed neurons labeled with DAPI, Tuj 1, and phalloidin. As an additional assay for neuronal health, Applicant can use the Quanterix SIMOA assay to measure levels of neurofilament light and tau leaked into the cell media ( FIG. 32). Each experiment can be repeated with DeActs-ER to determine if DeActs-ER can reverse neuronal health defects. As a positive control, Applicant can also use LatB. The neuron viability, neurite swelling, and SIMOA assays can be planned as endpoint experiments for the live imaging experiments in Aim 1 A. All experiments can have 6 biological replicates.

[0236] Data Analysis. All assays below are performed with at least three biological replicates. One can image at least 45 cells from 3 biological replicates per organelle / condition and use deep learning-based object tracking to determine both instantaneous and average-52-4896-3340-4800.3Atty Dkt.: 114198-0880 velocity, flux, moving directionality, and percentage of time moving. Applicant utilizes high- resolution Airyscan confocal microscopy images to measure organelle number, shape, and size. Comparisons are assessed using Student’s t-test.

[0237] Organelle and Neuron Health. Differences between INF2 CMT patients and controls can be compared via Student’s t-test with at least three biological replicates.

[0238] Fixed neuron imaging. At least 100 cells per condition can be quantified. Applicant can measure neurite length and neurite area by subtracting the DAPI-labeled area from the Tuj 1 -labeled area. Applicant can count the number of axonal swellings (determined by an axonal diameter at least five times larger than normal) per mm of acetylated-tubulin-positive axons. The intra-assay and inter-assay coefficient of variance for neurofilamin levels can be calculated for each condition along with positive controls. Images for each condition can be collected under identical settings, and results can be statistically compared via Student’s t- test.

[0239] Mitochondrial health. The ratio of greemred signal at mitochondria can be measured for the MitoQC assay. ATP production and OxPhos capacity differences between CMT mutant conditions can be compared to age-matched and isogenic controls with a student’s t- test.

[0240] Endosomal health. The number of QD-NG F47’48, DQ-Red-positive organelles and DQ-Red signal intensity can be measured. Applicant can compare the timing of fluorescent signal following the addition of DQ-Red BSA. Imaging conditions can be kept identical between all conditions for imaging experiments. Comparisons can be made based on a student’s t-test. As in Experiment 1 A.1, Applicant can use at least 45 cells from at least 3 biological replicates.

[0241] Lysosomal health. Lysosomal pH can be measured by imaging cells labeled with a combination of pH-sensitive and pH-insensitive dyes or proteins.

[0242] INF2 / RAB7A CMT mutations may cause reduced mobility in neurons.

[0243] INF2 / RAB7A CMT mutations may increase organelle fragmentation and reduce mitochondrial health. Thus, it is expected that reduced ATP production and reduced OxPhos capacity in CMT patient mitochondrial cells. Increased mitochondrial damage associated-53-4896-3340-4800.3Atty Dkt.: 114198-0880 with aberrant mitochondrial fission is predicted to cause an upregulation of mitophagy. However, decreased mitochondrial mobility could also impede mitochondrial turnover.

[0244] In endosomes, Applicant expects a decrease in endocytic cargo transport.

[0245] INF2 / RAB7A CMT mutations cause degeneration of peripheral neurons, so Applicant predict increased axonal swelling, reduced neurite growth, and reduced viability in INF2 CMT patient-derived cells and controls expressing INF2 / RAB7A CMT mutant constructs.

[0246] Applicant anticipates that INF2 / RAB7A siRNA may negatively impact axonal swelling, neurite growth, and viability due to alteration of normal organelle dynamics. However, these readouts may be unaffected since INF2 siRNA is not yet known to inhibit organelle mobility specifically.

[0247] Transfecting patient-derived neurons is challenging yet routine in the lab. If needed, Applicant can utilize electroporation methods in lieu of lipid-based transfections or lentiviral transduction. To avoid potential artifacts associated with transient expression, Applicant can also supplement the study with primary DRG neurons cultured from the KI RAB7V162Mmouse model. These results may mirror those from Applicant’s data in fibroblasts, organelle mobility in axons uses neuron-specific mechanisms that, if illuminated, can provide insight into the organelle physiology specific to neurons, as well as the pathology of CMT.

[0248] Dissecting Molecular Mechanisms Underlying INF2 and RAB7A-Mediated Regulation of Organelle Dynamics and Their Interplay with the Actin Cytoskeleton. The precise mechanisms by which INF2 and RAB7A regulate organelle dynamics and contribute to CMT pathogenesis remain elusive. Understanding these mechanisms is crucial for developing targeted therapeutic interventions for CMT and other neurodegenerative diseases associated with impaired organelle dynamics. Applicant’s data suggests a complex interplay between INF2, RAB7A, and the actin cytoskeleton in regulating organelle mobility and function.

[0249] CMT2B-RAB7 mutant retards axonal transport of TrkA. Following co-transfection, DRG neurons were assayed for axonal transport. RAB7AN161Tand TrkA endosomes were largely separated from each other with distinct transport speed with TrkA moving at a faster speed in both retrograde and anterograde directions. However, when the two populations of vesicles merged, axonal transport of TrKA was significantly reduced ( FIG. 33).-54-4896-3340-4800.3Atty Dkt.: 114198-0880

[0250] To dissect the molecular mechanisms underlying INF2 and RAB7A-mediated regulation of organelle dynamics, a critical step towards developing targeted therapies, Applicant can employ a multifaceted approach, combining genetic manipulations, biochemical assays, and advanced imaging techniques, to investigate how INF2 and RAB7A interact with actin, molecular motors, and other cytoskeletal components to regulate organelle fission, axonal transport, and positioning. Applicant can utilize patient-derived iPSCs, primary neurons from the KI mouse model, and genetically modified cell lines to elucidate the molecular pathways involved in INF2 and RAB7A-mediated organelle dynamics.

[0251] Determining the Role of the INF2-RAB7A Interaction in Regulating Organelle Dynamics. Applicant can investigate the functional consequences of the INF2-RAB7A interaction and its impact on organelle dynamics. Co-immunoprecipitation, proteomic analysis and proximity ligation assays can be employed to confirm and characterize the interaction between INF2 and RAB7A in neuronal cells. Applicant can assess the effects of disrupting this interaction, using either dominant-negative mutants or targeted protein depletion, on organelle mobility, fission, and morphology. Live-cell imaging and quantitative organelle tracking can be used to monitor changes in organelle dynamics in response to perturbations of the INF2-RAB7A interaction.

[0252] Investigate the Role of the Actin Cytoskeleton in INF2 and RAB7A-Mediated Organelle Dynamics. Applicant can examine how INF2 and RAB7A influence actin dynamics and how these changes impact organelle mobility and function. Applicant can utilize Applicant’s genetically encoded actin probes, such as AC-ER, to visualize and quantify actin filament assembly and dynamics in the presence of CMT2-associated mutations in INF2 and RAB7A. Applicant can also employ Applicant’s DeActs-ER tool to selectively perturb ER-associated actin and assess its effects on organelle mobility and function. Additionally, Applicant can investigate the role of myosin motors, which interact with actin filaments, in INF2 and RAB7A-mediated organelle transport.

[0253] Exploring the Impact of INF2 and RAB7A Mutations on Axonal Transport and Organelle Positioning. Applicant can assess how CMT2-associated mutations in INF2 and RAB7A affect axonal transport and organelle positioning. Neurons can be cultured in microfluidic chambers47,48’50( FIG. 34) and live-cell imaging and quantitative organelle tracking can be used to monitor the movement of mitochondria and NGF-endosomes4749 in -55-4896-3340-4800.3Atty Dkt.: 114198-0880 axons and dendrites of patient-derived neurons and rodent primary neurons expressing mutant INF2 or RAB7A. Applicant can also investigate the impact of these mutations on the distribution of organelles and movement.

[0254] Protein-protein interactions. Co-immunoprecipitation, proteomic analysis and proximity ligation assay results can be analyzed by densitometry and statistical comparisons using Student’s t-test or ANOVA.

[0255] Actin dynamics. Actin filament assembly and dynamics can be quantified using Applicant’s AC -ER probe and image analysis software. Statistical comparisons can be performed using Student’s t-test or ANOVA.

[0256] Organelle dynamics. Organelle mobility, fission, and morphology can be analyzed using live-cell imaging, quantitative organelle tracking, and high-resolution microscopy. Statistical comparisons can be performed using Student’s t-test or ANOVA.

[0257] Axonal transport and organelle positioning. The movement of organelles in axons and dendrites can be tracked and quantified using live-cell imaging and image analysis software. The distribution of organelles relative to synapses can be assessed using immunofluorescence and confocal microscopy. Statistical comparisons can be performed using Student’s t-test or ANOVA.

[0258] Without being bound by theory, it is expected that disrupting the INF2-RAB7A interaction can impair organelle mobility, fission, and morphology and that CMT2-associated mutations in INF2 and RAB7A can lead to aberrant actin dynamics, affecting organelle mobility and function. It is expected that perturbing ER-associated actin with DeActs-ER can rescue organelle mobility defects in CMT2 patient-derived neurons and that CMT2- associated mutations in INF2 and RAB7A can impair axonal transport and disrupt organelle positioning in neurons.

[0259] Complementary approaches, such as immunofluorescence staining or phalloidin labeling can be used to visualize and quantify actin dynamics.

[0260] One can control for variability in patient-derived iPSCs and neurons by using multiple clones and isogenic controls.-56-4896-3340-4800.3Atty Dkt.: 114198-0880

[0261] Overall, this approach can provide critical insights into the molecular mechanisms underlying INF2 and RAB7A-mediated regulation of organelle dynamics and their contribution to CMT pathogenesis. These findings can lay the foundation for developing targeted therapeutic interventions for CMT and other neurodegenerative diseases.

[0262] Target the INF2-Rab7A nexus for developing therapies. Gain-of-function autosomal dominant mutations in INF2 and Rab7 cause CMT2. Currently, there are no effective treatments for CMT2, highlighting the urgent need for novel therapeutic interventions. Applicant’s data and disclosure of 1 and 2 can provide a strong foundation for identifying and validating potential therapeutic targets within the INF2-Rab7A pathway. This aim (3) focuses on developing and evaluating innovative therapeutic strategies to restore organelle dynamics, mitigate neurodegeneration, and improve axonal function in CMT2.

[0263] Data: Applicant tested the hypothesis that restoring NGF signals rescued small sensory fiber degeneration induced by CMT2B RAB7A. Applicant explored NGFR100W, a missense mutation discovered in hereditary sensory autonomic neuropathy V (HSAN V)51that retains its trophic support function while no longer cause pain45,52’53. Applicant first carried out dose studies to confirm that NGFRR100Wdid not cause significant pain response in mice even at 5.0 pg compared to 0.5 pg wtNGF (data not shown). Applicant then carried out a 6 week treatment regimen of 9 months old CMT2B RAB7AV162Mmice that show significant loss of small intraepidermal sensory fibers (lENFs) in the hind paw skin sections when compared to that in same age of WT mice (C: b vs a). At completion, both wtNGF-(C: c, d) and NGFRR100W-treated CMT2BV162Mmice showed significant recovery of lENFs (C: e, f) (data not shown). More importantly, sensory function to thermal stimuli showed a full recovery by both treatments (data not shown).

[0264] Experiment No: 8 - Evaluating the Therapeutic Potential of ASOs Targeting INF2 or RAB7A

[0265] Gain-of-Function Autosomal Dominant Mutations in INF2 and Rab7 Causing CMT2, Reducing the expression of these mutant alleles using allele-specific ASOs may mitigate their dominant-negative effects and restore normal organelle dynamics and axonal function.

[0266] ASOs can be designed that specifically target the mutant alleles of INF2 or RAB7A. The efficacy of these ASOs can be evaluated in patient-derived iPSCs and neurons, assessing-57-4896-3340-4800.3Atty Dkt.: 114198-0880 their impact on organelle mobility, morphology, and function, as well as neuronal health and viability. In one approach, ASOs targeting mutant INF2 or RAB7A can reduce their expression, leading to improved organelle dynamics, enhanced axonal transport, and increased neuronal survival in CMT2 models.

[0267] Assessing the Therapeutic Efficacy of Rab7 GTPase Inhibitors. Rab7 GTPase activity is crucial for its function in regulating organelle dynamics and trafficking. Inhibiting Rab7 GTPase activity with CID 106770054or may restore normal organelle function and mitigate the effects of CMT2-associated Rab7A mutations.

[0268] Applicant can screen drugs to identify and characterize small molecule inhibitors of Rab7 GTPase activity54. The efficacy of these inhibitors can be evaluated in patient-derived iPSCs and neurons, as well as in rodent primary neurons expressing CMT2-associated Rab7A mutations. One can assess their impact on organelle dynamics, axonal transport, and neuronal health. In one aspect, Rab7 GTPase inhibitors can restore normal organelle dynamics and improve axonal function in CMT2 models by modulating Rab7A activity.

[0269] Assessing Therapeutic efficacy of exogenous NGFR100Wtreatment. NGF is a powerful trophic factor that spurs the regeneration of small sensory fibers56, that are lost in CMT2. However, previous clinal trials failed Phase III due to an unbearable pain experienced during treatment. Since then, studies have reported an NGFR100Wmutant that does not induce pain while retaining its trophic support function45’52, and discovered that exogenous treatment with NGFR100Wmay restore sensorineural function in the RAB7V162MCMT2B mouse model.

[0270] In one aspect, Applicant can establish the efficacy of NGFR100Wtreatment in Applicant’s mouse model and measure the effects of NGF treatment on the same functional and cellular readouts described for all other methods. Without being bound by theory, Applicant expects that NGFR100Wtreatment can restore sensorineural function and improve axonal health in the RAB7V162MCMT2B mouse model by promoting neurite outgrowth and enhancing neurotrophic signaling.

[0271] While NGFR100Wis designed to be painless, unexpected side effects or limited efficacy in vivo may occur. Applicant can carefully monitor the mice for any adverse effects and explore alternative neurotrophic factors or signaling pathways if needed.-58-4896-3340-4800.3Atty Dkt.: 114198-0880

[0272] Therapeutic Potential of ER-Associated Actin Perturbators. Applicant’s data demonstrate that depolymerizing ER-associated actin with Applicant’s novel DeActs-ER tool reverses neuronal swelling and restores organelle mobility in INF2 CMT patient-derived neurons. Thus, modulating ER-associated actin dynamics may be a promising therapeutic strategy for CMT2.

[0273] In one aspect, Applicant’s DeActs-ER tool is used to selectively depolymerize ER- associated actin filaments in CMT2 models. One can evaluate the efficacy of DeActs-ER in rescuing organelle mobility defects, improving axonal transport, and mitigating neurodegeneration in patient-derived iPSCs and neurons, as well as in rodent primary neurons expressing CMT2-associated INF2 mutations.

[0274] Without being bound by theory, it is expected that DeActs-ER treatment can restore normal organelle dynamics, improve axonal transport, and promote neuronal survival in CMT2 models by modulating ER-associated actin.

[0275] Validating the Most Promising Therapeutic Strategy in the RAB7 CMT2B Mouse Model. The most potent and promising therapeutic approach identified in the in vitro studies can be further evaluated in vivo using Applicant’s CMT2B mouse model. For ASO’s Applicant can use intrathecal injection. One can assess the impact of the therapeutic intervention on disease-relevant phenotypes, such as motor and sensory function, nerve conduction velocity, and muscle strength. Additionally, one can examine the effects of the therapy on axonal transport, organelle dynamics, and neurodegeneration in the peripheral nervous system.

[0276] It is anticipated that selected therapeutic intervention to demonstrate efficacy in ameliorating disease-relevant phenotypes in the CMT2B mouse model.

[0277] Experiment No: 9

[0278] Applicant have adapted a previously published protocol for “neurosphere” / ” spheroid” cultures of IPSC patient derived motor and sensory neurons (slightly different differentiation protocols) from patients with different forms of Charcot-Mari e-Tooth disease and ALS. The results show a striking phenotype of reduced axon density and length, which reflects the length-dependent neuropathy observed in the patients. Furthermore, Applicant observe a significant decrease in axonal organelle transport in direct proportion to axon lengths. (FIG.-59-4896-3340-4800.3Atty Dkt.: 114198-088035A) Finally, Applicant found that treating with small molecules that restore organelle movement also restores axonal outgrowth. (FIG. 35B). Hence, these results demonstrate an imaging-based screen for therapeutics for peripheral neuropathy in patient-derived cell lines.

[0279] Cell Culture. Primary human fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11965-092) supplemented with 15% fetal bovine serum (FBS, Gibco, 10437-028) and 1% GlutaMAX (Gibco, 35050-061) at 37°C in a humidified atmosphere of 5% CO2. Cells were routinely passaged at 80% confluence using trypsin- EDTA and maintained for no more than 20 passages to ensure genomic stability and optimal growth characteristics. Medium was replaced every two to three days, and cells were visually monitored for morphology and density prior to each passage.

[0280] Induced pluripotent stem cells (iPSCs) were maintained in mTeSR Plus medium (STEMCELL Technologies, 100-0276) on culture dishes coated with Matrigel Growth Factor Reduced (GFR, Coming, 354230) diluted 1 :40 in DMEMZF12 (Gibco, 11320-033). Plates were coated by adding the diluted Matrigel solution and incubating at room temperature for at least 1 hour. After aspirating the coating solution, iPSCs were plated and maintained under feeder-free conditions. Colonies were passaged as clusters every four to seven days using ReLeSR (STEMCELL Technologies, 100-0483) according to the manufacturer’s instructions, and only lines at passages no higher than 20 were used for experiments to minimize genetic drift and maintain pluripotency.

[0281] Human fibroblasts were reprogrammed into induced pluripotent stem cells using the ReproRNA™-OKSGM kit (STEMCELL Technologies, 05924) in combination with ReproTeSR™ medium (STEMCELL Technologies, 05920) according to the manufacturer’s protocol.

[0282] The motor neurons were differentiated as described by Kawada et al. Undifferentiated human iPSCs were maintained in culture in iPSC culture medium (mTeSR, STEMCELL) until -70% confluency in a 6-well plate, followed by PBS wash and dissociation with TrypLE Select (Thermo Fisher Scientific). Cells were resuspended in iPSC culture medium with 10 pM Y-27632 dihydrochloride (Tocris) and seeded at 40,000 cells / well in low- attachment 96-well U-bottom plates (Corning) with iPSC culture medium containing 10 pM Y-27632 dihydrochloride. Differentiation was initiated using DMEM / F12 (Gibco)-60-4896-3340-4800.3Atty Dkt.: 114198-0880 supplemented with 15% Knockout Serum Replacement (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (Thermo Fisher Scientific), 10 pM SB-431542 (Sigma-Aldrich), and 100 nM LDN-193189 (Sigma- Aldrich) for 2 days, followed by a gradual transition to N2 medium over 8 days with 1 pM retinoic acid (Sigma- Aldrich), 1 pM Smoothened Agonist (Sigma-Aldrich), 5 pM SU5402 (Sigma-Aldrich), and 5 pM DAPT (Sigma-Aldrich). On day 12, spheroids were transferred to a tissue culture plate in maturation medium, 20 ng / mL BDNF (Preprotech). Prior to transfer, spheroids were embedded in Matrigel (1 :40 in DMEM / F12) in an 8-well chambered glass-bottom slide using wide-bore pipette tips. Medium was changed every 2-3 days, and axon networks formed within 1 week.

[0283] Microfluidic Chip Fabrication. SU-8 master molds for microfluidic chip production were fabricated by Hicomp (Hicomp Microtechnology, China) using standard photolithography techniques for high precision patterning. Polydimethylsiloxane (PDMS) chips were cast in the laboratory by mixing Sylgard 184 prepolymer and curing agent at a 10: 1 ratio, followed by thorough vacuum degassing to remove air bubbles. The degassed mixture was poured onto the SU-8 mold and cured at 70°C for 3 hours. After curing, the PDMS chips were carefully peeled from the master, plasma cleaned (Harrick Plasma) for 45 seconds, and irreversibly bonded to glass coverslips. Finished devices were inspected for integrity and ready for cell culture or imaging applications.

[0284] Live-cell Imaging. Live-cell fluorescence imaging was performed using a Zeiss LSM 980 confocal microscope with Airyscan super-resolution (SR) mode. For all imaging experiments, the cell culture medium was replaced with phenol red-free imaging medium to minimize background fluorescence. Imaging was conducted with a 63X Plan-Apochromat 1.40 NA oil DIC M27 objective, maintaining cells at 37°C on a heated stage. MitoTracker Green and LysoTracker Deep Red were excited with 488 nm and 647 nm lasers and their emissions were detected using optimized bandpass filters for each probe. Acquisition was performed at one frame per second, with two-minute time-lapse sequences for each experiment. All microscope parameters, including laser power, detector gain, and pixel sampling for super-resolution, were meticulously standardized and kept constant for every experiment to ensure reproducibility and quantitative consistency.-61-4896-3340-4800.3Atty Dkt.: 114198-0880

[0285] For widefield tile imaging, experiments were performed using either a transmitted light (TL) halogen lamp set to 2 volts for brightfield acquisitions or a fluorescence LED light source for fluorescent imaging, as required by the specific experimental protocol. Imaging was performed with a 10X Plan-Apochromat 0.45 NA M27 air objective and 10% tile overlap, and acquired with a Zeiss AxioCam 820 widefield camera. Image tiles were automatically stitched for quantitative analysis of axonal networks.

[0286] Image Analysis. Kymographs were generated using the KymoClear plugin in Fiji / ImageJ to analyze dynamic organelle movement. For motility classification, organelles were considered static if their net displacement was less than 2.5 pm. Co-migration analysis of mitochondria and lysosomes was performed by applying a pixel-wise AND operation to dual-channel kymographs, yielding the fraction of overlapping pixels and quantifying organelle colocalization. This approach allowed the detection of co-localization events, which predominantly occurred when both organelles were static. Mitochondrial morphology was quantified with the mitoMorph plugin, and organelle segmentation and tracking were performed using Ilastik and TrackMate, respectively. Neurite complexity and axonal branching were evaluated using Sholl analysis in NeuriteJ.

[0287] Referring now to FIG. 36A, illustrated is a flow chart of an implementation of a method 3600 for detecting neurological or metabolic disorders based on image processing, according to some implementations. The method 3600 may be performed by a data processing system, such as the computing device 3700, shown and described with reference to FIGS. 37A and 37B. The method 3600 may include any number of steps and the steps may be performed in any order. The data processing system may perform the method 3600 using the methods described herein.

[0288] At operation 3602, the data processing system can receive image data of neuronal cell cultures. The data processing system can obtain the image data through advanced live-cell fluorescence imaging techniques. These images may be captured using a confocal microscope equipped with super-resolution capabilities, such as the Zeiss LSM 980 with Airyscan SR mode, to ensure high spatial and temporal resolution of cellular structures. Prior to imaging, neuronal cultures — often derived from patient-specific induced pluripotent stem cells (iPSCs) differentiated into motor and sensory neurons — are prepared under carefully controlled conditions, including the use of phenol red-free imaging medium to reduce background-62-4896-3340-4800.3Atty Dkt.: 114198-0880 fluorescence. Fluorescent probes like MitoTracker Green and LysoTracker Deep Red are applied to visualize specific organelles within axons, and images are acquired at defined intervals for time-lapse analysis. The resulting image data may also include widefield tile scans for comprehensive mapping of axonal networks, which are then stitched together for quantitative assessment. By standardizing imaging parameters across experiments, the system ensures reproducibility and accurate downstream analysis of axonal morphology and organelle dynamics.

[0289] At operation 3604, the data processing system can process said image data to segment axons. The data processing system can process the acquired image data to segment axons by applying advanced image analysis techniques. The system may utilize specialized software tools, such as Fiji / ImageJ with plugins like Ilastik and TrackMate, to identify and delineate individual axons within the neuronal cultures. These tools can leverage pixel-based algorithms and machine learning models to distinguish axonal structures from background and other cellular components, enabling accurate segmentation even in complex networks. The segmentation process may involve thresholding, edge detection, and region-growing methods to trace axon boundaries, and can be further refined through manual or automated validation steps to ensure consistency and reproducibility. By standardizing analysis parameters and employing robust computational workflows, the data processing system can generate reliable segmented axon datasets for subsequent quantitative assessment.

[0290] At operation 3606, the data processing system can determine axon length for each segmented axon. The data processing system can determine axon length for each segmented axon by applying quantitative image analysis algorithms to the segmented image data. The system may utilize specialized software tools, such as Fiji / ImageJ equipped with plugins like TrackMate, to trace the path of each identified axon and calculate its length with high spatial accuracy. The process can involve skeletonizing the segmented axon structures to reduce them to their central lines, followed by measuring the total pixel distance along these paths. The data processing system may standardize measurement parameters to enhance consistency across samples and experiments, and can automate the extraction of axon length metrics for large datasets. Additionally, results may be validated or refined through manual inspection or automated quality control steps, ensuring reliable quantification suitable for downstream analysis of neuronal morphology and function.-63-4896-3340-4800.3Atty Dkt.: 114198-0880

[0291] At operation 3608, the data processing system can correlate said axon length to one or more neurological or metabolic disorders. The data processing system can correlate axon length measurements to one or more neurological or metabolic disorders by applying statistical analysis and pattern recognition techniques to the quantified axon length data derived from segmented neuronal images. The system may compare axon length distributions across samples from healthy and disease-model cultures, identifying deviations that are characteristic of specific disorders. Additionally, the data processing system can integrate axon length metrics with other morphological and functional parameters, such as organelle motility, neurite complexity, and axonal branching, to strengthen associations with disease phenotypes. By referencing established clinical datasets or literature, the system may further validate correlations between observed axonal changes and known neurological or metabolic conditions. This approach can facilitate the identification of biomarkers and support the diagnosis or monitoring of disease progression in patient-derived cell models.

[0292] The method 3600 offers several technical advantages for detecting neurological or metabolic disorders through advanced image processing of neuronal cell cultures. By integrating high-resolution live-cell fluorescence imaging with standardized acquisition protocols, the approach ensures reproducibility and quantitative consistency across experiments. The use of specialized software tools and machine learning-based segmentation enables accurate identification and measurement of axon morphologies, even in complex cellular networks. Furthermore, the automated workflows for quantifying axon length and correlating these metrics with disease phenotypes facilitate robust, high-throughput analysis, supporting both early detection and comprehensive characterization of neurological or metabolic conditions.

[0293] Referring now to FIG. 36B, illustrated is a flow chart of an implementation of a method 3610 for screening candidate therapeutics for neuronal disorders or metabolic disorders based on high-resolution microscopy image data, according to some implementations. The method 3610 may be performed by a data processing system, such as the computing device 3700, shown and described with reference to FIGS. 37A and 37B. The method 3610 may include any number of steps and the steps may be performed in any order. The data processing system may perform the method 3610 using the methods described herein.-64-4896-3340-4800.3Atty Dkt.: 114198-0880

[0294] At operation 3612, Receive high-resolution microscopy image data of neuronal cell cultures contacted with one or more candidate agents. The data processing system can receive high-resolution microscopy image data of neuronal cell cultures that have been contacted with one or more candidate agents. The data processing system may obtain these images through advanced live-cell fluorescence imaging techniques, such as confocal microscopy equipped with super-resolution capabilities. Prior to imaging, neuronal cultures, often derived from patient-specific induced pluripotent stem cells differentiated into motor and sensory neurons, may be carefully prepared, including replacing the cell culture medium with a phenol red-free imaging medium to reduce background fluorescence. Fluorescent probes, such as MitoTracker Green and LysoTracker Deep Red, can be applied to label and visualize specific organelles within axons. The data processing system may acquire images at defined time intervals to enable time-lapse analysis, ensuring high spatial and temporal resolution of cellular structures. Additionally, the data processing system can collect widefield tile scans for comprehensive mapping of axonal networks, which may then be automatically stitched for quantitative assessment. By standardizing imaging parameters across experiments, the data processing system may ensure reproducibility and facilitate accurate downstream analysis of the effects of candidate agents on neuronal morphology and organelle dynamics.

[0295] At operation 3614, the data processing system can segment axons in said image data. The data processing system can segment axons in said image data by applying advanced image analysis techniques tailored for neuronal cultures. The data processing system may utilize specialized software tools, such as Fiji / ImageJ equipped with plugins like Ilastik and TrackMate, to identify and delineate individual axons within the acquired microscopy images. These tools can leverage pixel-based algorithms and machine learning models to distinguish axonal structures from background and other cellular components, enabling accurate segmentation even within complex neuronal networks. The segmentation process may involve thresholding, edge detection, and region-growing methods to trace axon boundaries, and the data processing system can refine results through manual or automated validation steps to ensure consistency and reproducibility. By standardizing analysis parameters and employing robust computational workflows, the data processing system can-65-4896-3340-4800.3Atty Dkt.: 114198-0880 generate reliable segmented axon datasets that support subsequent quantitative assessment of neuronal morphology and function.

[0296] At operation 3616, the data processing system can compute axon length metrics. The data processing system can compute axon length metrics by leveraging sophisticated image analysis algorithms applied to segmented axon data derived from high-resolution microscopy images. The data processing system may utilize specialized tools, such as Fiji / ImageJ with plugins like TrackMate, to trace the paths of individual axons and measure their lengths with precise spatial accuracy. This process can involve skeletonizing the segmented axonal structures to reduce them to their central lines, followed by calculating the total pixel distance along these paths. The data processing system may standardize measurement parameters to ensure consistency across different experimental samples and conditions, and can automate the extraction of length metrics for large datasets. Additionally, the data processing system can validate or refine the computed measurements through manual inspection or automated quality control steps, thereby supporting reliable and reproducible quantitative analysis of neuronal morphology and the effects of candidate agents on axon growth.

[0297] At operation 3618, the data processing system can compare said axon length metrics to control cultures. The data processing system can compare the computed axon length metrics from neuronal cell cultures contacted with candidate agents to those from control cultures. The data processing system may leverage standardized quantitative analysis workflows to ensure consistency in measurement parameters across both experimental and control samples. It can use advanced image analysis tools, such as Fiji / ImageJ with TrackMate plugins, to extract and organize axon length data for statistical comparison. The data processing system may apply pattern recognition or statistical methods to evaluate differences in axon length distributions, identifying any increases, decreases, or other changes associated with candidate agent treatment. Additionally, the data processing system can integrate these comparisons with other morphological and functional metrics, such as organelle motility or network complexity, to provide a comprehensive assessment of candidate agent effects relative to controls. By automating data extraction and analysis, the data processing system can efficiently process large datasets, enabling robust and reproducible screening of potential therapeutics for neuronal or metabolic disorders.-66-4896-3340-4800.3Atty Dkt.: 114198-0880

[0298] At operation 3620, the data processing system can identify candidate agents that increase axon length relative to controls. The data processing system can identify candidate agents that increase axon length relative to controls by performing a comprehensive quantitative analysis of axon length metrics derived from high-resolution microscopy image data of neuronal cell cultures. The data processing system may utilize advanced image analysis workflows, such as those implemented in Fiji / ImageJ with TrackMate plugins, to extract and organize axon length measurements for both cultures treated with candidate agents and untreated control cultures. The data processing system can apply statistical methods and pattern recognition algorithms to compare axon length distributions between experimental and control groups, highlighting agents that are associated with statistically significant increases in axon length. Additionally, the data processing system may integrate these findings with other morphological and functional parameters to strengthen the assessment of candidate agent effects. By automating the extraction, comparison, and validation processes, the data processing system can efficiently screen large datasets, facilitating the identification of promising therapeutics that promote axon growth in neuronal or metabolic disorder models.

[0299] The method 3610 offers several technical advantages. For example, by integrating advanced live-cell fluorescence imaging techniques with standardized acquisition protocols, the method ensures reproducibility and quantitative consistency across experiments. The use of specialized image analysis software and machine learning-based segmentation facilitates accurate identification and measurement of axonal morphologies within complex neuronal networks. Additionally, automated workflows for quantifying axon length and comparing metrics to control cultures allow for robust, high-throughput analysis, facilitating the efficient identification of agents that promote axon growth. These features support both early detection and comprehensive evaluation of therapeutic effects, accelerating research and development in neurobiology and drug discovery.

[0300] FIGS. 37A and 37B are block diagrams depicting embodiments of computing devices that can be used in connection with the methods and systems described herein.

[0301] Having discussed specific embodiments of the present solution, it may be helpful to describe aspects of the operating environment as well as associated system components (e.g., hardware elements) in connection with the methods and systems described herein.-67-4896-3340-4800.3Atty Dkt.: 114198-0880

[0302] The systems and methods discussed herein may be deployed as and / or executed on any type and form of computing device, such as a computer, network device or appliance capable of communicating on any type and form of network and performing the operations described herein. FIGS. 37A and 37B depict block diagrams of a computing device 3700 useful for practicing an embodiment of the systems and methods described herein. As shown in FIGS. 37A and 37B, each computing device 3700 includes a central processing unit 3721, and a main memory unit 3722. As shown in FIG. 37A, a computing device 3700 may include a storage device 3728, an installation device 3716, a network interface 3718, an I / O controller 3723, display devices 3724a-3724n, a keyboard 3726 and a pointing device 3727, such as a mouse. The storage device 3728 may include, without limitation, an operating system and / or software. As shown in FIG. 37B, each computing device 3700 may also include additional optional elements, such as a memory port 3703, a bridge 3770, one or more input / output devices 3730a-3730n (generally referred to using reference numeral 3730), and a cache memory 3740 in communication with the central processing unit 3721.

[0303] The central processing unit 3721 is any logic circuitry that responds to and processes instructions fetched from the main memory unit 3722. In many embodiments, the central processing unit 3721 is provided by a microprocessor unit, such as: those manufactured by Intel Corporation of Mountain View, California; those manufactured by International Business Machines of White Plains, New York; or those manufactured by Advanced Micro Devices of Sunnyvale, California. The computing device 3700 may be based on any of these processors, or any other processor capable of operating as described herein.

[0304] Main memory unit 3722 may be one or more memory chips capable of storing data and allowing any storage location to be directly accessed by the microprocessor 3721, such as any type or variant of Static random access memory (SRAM), Dynamic random access memory (DRAM), Ferroelectric RAM (FRAM), NAND Flash, NOR Flash and Solid State Drives (SSD). The main memory 3722 may be based on any of the above described memory chips, or any other available memory chips capable of operating as described herein. In the embodiment shown in FIG. 37 A, the processor 3721 communicates with main memory 3722 via a system bus 3780 (described in more detail below). FIG. 37B depicts an embodiment of a computing device 3700 in which the processor communicates directly with main memory-68-4896-3340-4800.3Atty Dkt.: 114198-08803722 via a memory port 3703. For example, in FIG. 37B the main memory 3722 may be DRDRAM.

[0305] FIG. 37B depicts an embodiment in which the main processor 3721 communicates directly with cache memory 3740 via a secondary bus, sometimes referred to as a backside bus. In other embodiments, the main processor 3721 communicates with cache memory 3740 using the system bus 3780. Cache memory 3740 typically has a faster response time than main memory 3722 and is provided by, for example, SRAM, BSRAM, or EDRAM. In the embodiment shown in FIG. 37B, the processor 3721 communicates with various VO devices 3730 via a local system bus 3780. Various buses may be used to connect the central processing unit 3721 to any of the I / O devices 3730, for example, a VESA VL bus, an ISA bus, an EISA bus, a MicroChannel Architecture (MCA) bus, a PCI bus, a PCI-X bus, a PCI- Express bus, or a NuBus. For embodiments in which the VO device is a video display 3724, the processor 3721 may use an Advanced Graphics Port (AGP) to communicate with the display 3724. FIG. 37B depicts an embodiment of a computer 3700 in which the main processor 3721 may communicate directly with VO device 3730b, for example via HYPERTRANSPORT, RAPIDIO, or INFINIBAND communications technology. FIG. 37B also depicts an embodiment in which local busses and direct communication are mixed: the processor 3721 communicates with I / O device 3730a using a local interconnect bus while communicating with I / O device 3730b directly.

[0306] A wide variety of VO devices 3730a-3730n may be present in the computing device 3700. Input devices include keyboards, mice, trackpads, trackballs, microphones, dials, touch pads, touch screens, and drawing tablets. Output devices include video displays, speakers, inkjet printers, laser printers, projectors and dye-sublimation printers. The I / O devices may be controlled by an I / O controller 3723 as shown in FIG. 37A. The VO controller may control one or more VO devices such as a keyboard 3726 and a pointing device 3727, e.g., a mouse or optical pen. Furthermore, an I / O device may also provide storage and / or an installation device 3716 for the computing device 3700. In still other embodiments, the computing device 3700 may provide USB connections (not shown) to receive handheld USB storage devices such as the USB Flash Drive line of devices manufactured by Twintech Industry, Inc., of Los Alamitos, California.-69-4896-3340-4800.3Atty Dkt.: 114198-0880

[0307] Referring again to FIG. 37A, the computing device 3700 may support any suitable installation device 3716, such as a disk drive, a CD-ROM drive, a CD-R / RW drive, a DVD- ROM drive, a flash memory drive, tape drives of various formats, USB device, hard-drive, a network interface, or any other device suitable for installing software and programs. The computing device 3700 may further include a storage device, such as one or more hard disk drives or redundant arrays of independent disks, for storing an operating system and other related software, and for storing application software programs such as any program or software 3720 for implementing (e.g., configured and / or designed for) the systems and methods described herein. Optionally, any of the installation devices 3716 could also be used as the storage device. Additionally, the operating system and the software can be run from a bootable medium.

[0308] Furthermore, the computing device 3700 may include a network interface 3718 to interface to a network through a variety of connections including, but not limited to, standard telephone lines, LAN or WAN links (e.g., 802.11, Tl, T3, 156kb, X.25, SNA, DECNET), broadband connections (e.g., ISDN, Frame Relay, ATM, Gigabit Ethernet, Ethemet-over- SONET), wireless connections, or some combination of any or all of the above. Connections can be established using a variety of communication protocols (e.g., TCP / IP, IPX, SPX, NetBIOS, Ethernet, ARCNET, SONET, SDH, Fiber Distributed Data Interface (FDDI), RS232, IEEE 802.11, IEEE 802.11a, IEEE 802.11b, IEEE 802.11g, IEEE 802.1 In, IEEE 802.1 lac, IEEE 802.1 lad, CDMA, GSM, WiMax and direct asynchronous connections). In one embodiment, the computing device 3700 communicates with other computing devices 3700’ via any type and / or form of gateway or tunneling protocol such as Secure Socket Layer (SSL) or Transport Layer Security (TLS). The network interface 3718 may include a built-in network adapter, network interface card, PCMCIA network card, card bus network adapter, wireless network adapter, USB network adapter, modem or any other device suitable for interfacing the computing device 3700 to any type of network capable of communication and performing the operations described herein.

[0309] In some implementations, the computing device 3700 may include or be connected to one or more display devices 3724a-3724n. As such, any of the I / O devices 3730a-3730n and / or the I / O controller 3723 may include any type and / or form of suitable hardware, software, or combination of hardware and software to support, enable or provide for the-70-4896-3340-4800.3Atty Dkt.: 114198-0880 connection and use of the display device(s) 3724a-3724n by the computing device 3700. For example, the computing device 3700 may include any type and / or form of video adapter, video card, driver, and / or library to interface, communicate, connect or otherwise use the display device(s) 3724a-3724n. In one embodiment, a video adapter may include multiple connectors to interface to the display device(s) 3724a-3724n. In other embodiments, the computing device 3700 may include multiple video adapters, with each video adapter connected to the display device(s) 3724a-3724n. In some implementations, any portion of the operating system of the computing device 3700 may be configured for using multiple displays 3724a-3724n. One ordinarily skilled in the art will recognize and appreciate the various ways and embodiments that a computing device 3700 may be configured to have one or more display devices 3724a-3724n.

[0310] In further embodiments, an I / O device 3730 may be a bridge between the system bus 3780 and an external communication bus, such as a USB bus, an Apple Desktop Bus, an RS- 232 serial connection, a SCSI bus, a FireWire bus, a FireWire 500 bus, an Ethernet bus, an AppleTalk bus, a Gigabit Ethernet bus, an Asynchronous Transfer Mode bus, a FibreChannel bus, a Serial Attached small computer system interface bus, a USB connection, or a HDMI bus.

[0311] A computing device 3700 of the sort depicted in FIGS. 37A and 37B may operate under the control of an operating system, which control scheduling of tasks and access to system resources. The computing device 3700 can be running any operating system, such as any of the versions of the MICROSOFT WINDOWS operating systems, the different releases of the Unix and Linux operating systems, any version of the MAC OS for Macintosh computers, any embedded operating system, any real-time operating system, any open source operating system, any proprietary operating system, any operating systems for mobile computing devices, or any other operating system capable of running on the computing device and performing the operations described herein. Typical operating systems include, but are not limited to, Android, produced by Google Inc.; WINDOWS 7 and 8, produced by Microsoft Corporation of Redmond, Washington; MAC OS, produced by Apple Computer of Cupertino, California; WebOS, produced by Research In Motion (RIM); OS / 2, produced by International Business Machines of Armonk, New York; and Linux, a freely-available-71-4896-3340-4800.3Atty Dkt.: 114198-0880 operating system distributed by Caldera Corp, of Salt Lake City, Utah, or any type and / or form of a Unix operating system, among others.

[0312] The computer system 3700 can be any workstation, telephone, desktop computer, laptop or notebook computer, server, handheld computer, mobile telephone or other portable telecommunications device, media playing device, a gaming system, mobile computing device, or any other type and / or form of computing, telecommunications or media device that is capable of communication. The computer system 3700 has sufficient processor power and memory capacity to perform the operations described herein.

[0313] In some implementations, the computing device 3700 may have different processors, operating systems, and input devices consistent with the device. For example, in one embodiment, the computing device 3700 is a smart phone, mobile device, tablet or personal digital assistant. In still other embodiments, the computing device 3700 is an Android-based mobile device, an iPhone smart phone manufactured by Apple Computer of Cupertino, California, or a Blackberry or WebOS-based handheld device or smart phone, such as the devices manufactured by Research In Motion Limited. Moreover, the computing device 600 can be any workstation, desktop computer, laptop or notebook computer, server, handheld computer, mobile telephone, any other computer, or other form of computing or telecommunications device that is capable of communication and that has sufficient processor power and memory capacity to perform the operations described herein.

[0314] Although examples of communications systems described above may include devices operating according to an 802.11 standard, it should be understood that embodiments of the systems and methods described can operate according to other standards and use wireless communications devices other than devices configured as devices and APs. For example, multiple-unit communication interfaces associated with cellular networks, satellite communications, vehicle communication networks, and other non-802.11 wireless networks can utilize the systems and methods described herein to achieve improved overall capacity and / or link quality without departing from the scope of the systems and methods described herein.

[0315] It should be noted that certain passages of this disclosure may reference terms such as “first” and “second” in connection with devices, mode of operation, transmit chains,-72-4896-3340-4800.3Atty Dkt.: 114198-0880 antennas, etc., for purposes of identifying or differentiating one from another or from others. These terms are not intended to merely relate entities (e.g., a first device and a second device) temporally or according to a sequence, although in some cases, these entities may include such a relationship. Nor do these terms limit the number of possible entities (e.g., devices) that may operate within a system or environment.

[0316] It should be understood that the systems described above may provide multiple ones of any or each of those components and these components may be provided on either a standalone machine or, in some implementations, on multiple machines in a distributed system. In addition, the systems and methods described above may be provided as one or more computer-readable programs or executable instructions embodied on or in one or more articles of manufacture. The article of manufacture may be a floppy disk, a hard disk, a CD- ROM, a flash memory card, a PROM, a RAM, a ROM, or a magnetic tape. In general, the computer-readable programs may be implemented in any programming language, such as LISP, PERL, C, C++, C#, PROLOG, or in any byte code language such as JAVA. The software programs or executable instructions may be stored on or in one or more articles of manufacture as object code.

[0317] Equivalents

[0318] It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0319] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0320] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such-73-4896-3340-4800.3Atty Dkt.: 114198-0880 terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0321] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0322] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0323] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0324] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0325] Other aspects are set forth within the following claims.-74-4896-3340-4800.3Atty Dkt.: 114198-0880

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Claims

Atty Dkt.: 114198-0880What is claimed is:

1. A method of detecting axon length in a neuronal cell culture, comprising:(a) culturing neuronal cells derived from a subject or an induced pluripotent stem cell line;(b) imaging the neuronal cells to determine axon length; and(c) correlating the axon length to a presence or severity of a neurological or metabolic disorder.

2. A method of monitoring axon length over time in a neuronal cell culture following application of a therapeutic candidate, comprising:(a) culturing neuronal cells derived from a subject or patient population having or at risk for a neuronal disorder or a metabolic disorder;(b) applying the therapeutic candidate to the neuronal cells;(c) imaging the neuronal cells at two or more time points after applying the therapeutic candidate; and(d) determining changes in axon length between the time points, wherein a detectable increase in axon length relative to untreated control cultures is indicative of therapeutic efficacy.

3. A method of screening candidate therapeutics for a neurological or a metabolic disorder using axon length as a phenotypic readout, comprising:(a) generating neuronal cell cultures from subjects diagnosed with the neurological or the metabolic disorder;(b) contacting the neuronal cell cultures with one or more candidate therapeutic agents;(c) imaging the neuronal cell cultures to determine axon length; and(d) comparing axon length in candidate agent-treated cultures to control cultures, wherein an increase in axon length relative to control identifies the candidate agent as having potential therapeutic efficacy.

4. The method of any of claims 1-3, wherein the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

5. The method of any one of claims 1-4, wherein the neuronal cells are motor neurons or sensory neurons differentiated from patient-derived induced pluripotent stem cells.-89-4896-3340-4800.3Atty Dkt.: 114198-08806. The method of any one of claims 1-5, wherein imaging comprises live-cell fluorescence microscopy, transmitted light microscopy, confocal microscopy, superresolution microscopy, or widefield tile imaging.

7. The method of any one of claims 1-6, further comprising quantifying axonal branching or neurite complexity in addition to axon length.

8. The method of any one of claims 2-7, wherein monitoring axon length further comprises measuring axonal transport of organelles selected from mitochondria and lysosomes.

9. The method of claim 2, wherein the therapeutic candidate is selected from a small molecule, antisense oligonucleotide, biologic, gene therapy construct, or neurotrophic factor.

10. The method of any one of claims 3-9, wherein the comparing step comprises statistical analysis of axon length distributions across at least three biological replicates.

11. The method of any one of claims 3-10, wherein the screening further comprises measuring co-migration of mitochondria and lysosomes as a secondary readout of neuronal health.

12. The method of any one of claims 1-11, wherein the neuronal cell cultures are maintained in microfluidic chip devices permitting compartmentalized growth of axons.

13. The method of any one of claims 1-12, wherein the correlation to disorder presence or severity is used to stratify patient populations for clinical trials of the therapeutic candidates.

14. A system for detecting axon length and correlating axon length to a neurological or metabolic disorder, comprising:(a) a neuronal cell culture chamber configured to accommodate cells derived from a subject or induced pluripotent stem cell line;(b) an imaging module configured to acquire images of axons in the neuronal cell culture at high spatial resolution; and(c) an analysis module comprising at least one processor and non-transitory computer- readable instructions configured to:-90-4896-3340-4800.3Atty Dkt.: 114198-0880(i) determine axon length from the images; and(ii) output a correlation of the axon length to a neurological or a metabolic disorder.

15. The system of claim 14, wherein the neuronal cell culture chamber comprises a microfluidic chip having:(a) a soma compartment for neuronal cell bodies; and(b) one or more axon compartments fluidly connected to the soma compartment via microgrooves permitting axonal growth.

16. The system of claim 14 or 15, wherein the imaging module comprises at least one of:(a) a confocal microscope;(b) a super-resolution Airyscan microscope;(c) a widefield tile imaging microscope; or(d) a transmitted light imaging system.

17. The system of any one of claims 14-16, wherein the analysis module is further configured to monitor axon length at multiple time points after application of a therapeutic candidate and to calculate the change in axon length over time.

18. The system of any one of claims 14-17, wherein the analysis module is further configured to compare axon lengths between therapeutic candidate-treated cultures and untreated control cultures to identify candidate agents that increase axon length.

19. The system of any one of claims 14-18, wherein the analysis module is further configured to:(a) detect and quantify axonal branching;(b) analyze neurite complexity; and(c) assess co-migration or transport of axonal organelles selected from mitochondria and lysosomes.

20. The system of any one of claims 14-19, wherein the neuronal cell cultures are motor neurons or sensory neurons differentiated from patient-derived induced pluripotent stem cells.-91-4896-3340-4800.3Atty Dkt.: 114198-088021. The system of any one of claims 14-20, further comprising a therapeutic application module configured to deliver candidate agents to cultured cells and record delivery parameters within the analysis module.

22. The system of any one of claims 14-21, wherein the analysis module outputs both numerical and visualized metrics including kymographs, Sholl analysis plots, and axon length distributions for disease correlation or therapeutic screening.

23. The system of any one of claims 14-22, wherein the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

24. A method of using the system of any one of claims 14-23 to screen for candidate therapeutics, comprising:(a) culturing neuronal cells from a subject diagnosed the neurological or the metabolic disorder in the neuronal cell culture chamber;(b) applying one or more candidate therapeutic agents to the cultures;(c) acquiring high-resolution images of axons over a defined period; and(d) analyzing the images with the analysis module to determine changes in axon length relative to untreated control cultures, wherein an increase in axon length identifies the candidate therapeutic agent as having potential efficacy.

25. The method of claim 24, wherein the method further comprises correlating axon length changes to improvement in organelle transport metrics obtained by the system.

26. A non-transitory computer-readable medium storing instructions that, when executed by at least one processor, cause the processor to perform a method comprising:(a) receiving image data of neuronal cell cultures;(b) processing the image data to segment axons;(c) determining axon length for each segmented axon; and(d) correlating the axon length to one or more neurological or metabolic disorder.

27. The non-transitory computer-readable medium of claim 26, wherein the processing step comprises:(a) skeletonizing segmented axons;-92-4896-3340-4800.3Atty Dkt.: 114198-0880(b) computing branching metrics; and(c) quantifying neurite complexity by Sholl analysis.

28. The non-transitory computer-readable medium of any one of claims 26-27, further comprising instructions that cause the processor to:(a) receive image data from two or more time points before and after application of a therapeutic candidate;(b) compute a change in axon length between the time points; and(c) output the change in axon length as a measure of therapeutic efficacy.

29. The non-transitory computer-readable medium of any one of claims 26-28, wherein the instructions further cause the processor to:(a) measure co-migration of mitochondria and lysosomes in the image data;(b) quantify organelle transport metrics; and(c) correlate changes in axon length with changes in organelle transport metrics.

30. The non-transitory computer-readable medium of any one of claims 26-29, wherein the instructions further cause the processor to:(a) normalize axon length measurements to cell density;(b) apply statistical analyses to compare treated vs. untreated cultures across at least three biological replicates; and(c) generate visualizations selected from kymographs, histograms, and scatter plots of axon length data.

31. The medium of any of claims 26-30, wherein the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Mari e-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

32. A computer-implemented method for screening candidate therapeutics for neuronal disorders or metabolic disorders, comprising:(a) receiving high-resolution microscopy image data of neuronal cell cultures contacted with one or more candidate agents;(b) segmenting axons in the image data;(c) computing axon length metrics;-93-4896-3340-4800.3Atty Dkt.: 114198-0880(d) comparing the axon length metrics to control cultures; and(e) identifying candidate agents that increase axon length relative to controls.

33. The method of claim 32, wherein the method further comprises outputting a ranked list of candidate agents based on magnitude of axon length increase and statistical significance.

34. The method of claim 32 or 33, wherein the neurological or the metabolic disorder is selected from Charcot-Marie-Tooth disease type 2A, Charcot-Marie-Tooth disease type 2B, amyotrophic lateral sclerosis (ALS), or type II diabetes.

35. The method of any one of claims 32-34, wherein the method comprises aggregating patient-derived culture data to stratify patient populations for clinical trials.

36. A method to identify mutations in IFN2 and RAB7A in cells harboring these mutations comprising analyzing the cells for one or more of organelle mobility, morphology, and axonal function.

37. The method of claim 36, wherein organelle mobility is analyzed by a method comprising high spatiotemporal resolution.

38. The method of claim 37, further comprising analyzing the organelle mobility with deep learning-based point-scanning super-resolution microscopy (PSSR) image processing model trained for imaging organelle transport in the cells.

39. The method of any one of claims 36-38, wherein the cells are neurons or induced pluripotent stem cells (iPSCs).

40. The method of any of claims 1-39, wherein the cells are mammalian cells, optionally human cells.-94-4896-3340-4800.3

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