Treatment and detection of inherited neuropathies and associated disorders

Abstract

To provide treatment and detection of inherited neuropathies and associated disorders.SOLUTION: The present disclosure relates to methods of detecting and treating inherited neuropathy. In various aspects, the method comprises detecting the presence of a mutation in a sorbitol dehydrogenase (SORD) gene in a sample from a subject. In various embodiments, the SORD mutation is a DNA variant classified as pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics (ACMG) criteria.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Grant Funding Disclosure This invention was made with government support under Grant Nos. NS065712 and NS075764 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0002] Cross-Reference to Related Applications and Incorporation by Reference of Electronically Filed Materials This application hereby claims priority to U.S. Provisional Patent Application Nos. 62 / 844,370, filed May 7, 2019, and 62 / 987,151, filed March 9, 2020, each of which is incorporated by reference in its entirety.

[0003] The computer-readable nucleotide / amino acid sequence listing submitted herewith and identified as follows is incorporated by reference in its entirety: Filename: 54095A _Seqlisting.txt; Size: 141,930 bytes; Created: May 5, 2020.

[0004] FIELD OF THE INVENTION The present disclosure relates to methods for detecting and treating hereditary neuropathies. [Background technology]

[0005] background Peripheral neuropathies are among the most frequent neurodegenerative diseases, with diabetic neuropathy and genetic origins among the most common mechanisms of action. Regarding hereditary neuropathies (also known as Charcot-Marie-Tooth disease (CMT)), a diagnostic gap of approximately 50% of patients remains. In our understanding, CMT represents a comprehensive concept for clinically and genetically heterogeneous inherited, monogenic, highly phenotypically penetrant conditions affecting peripheral nerves. CMT is classified as demyelinating (CMT1) and axonal (CMT2) types depending on conduction velocity. Distal hereditary motor neuropathy (dHMN) represents a form of CMT2 in which the disease burden falls primarily or exclusively on motor neurons (Rossor, Tomaselli, and Reilly 2016). Similar conditions include ALS4 (juvenile dHMN + brisk reflex as a sign of upper motor neuron involvement). In contrast to CMT1, where over 90% of cases have mutations in known genes, only 20–30% of patients with CMT2 and distal HMN receive a genetic diagnosis (Fridman et al. 2015 ). Summary of the Invention [Means for solving the problem]

[0006] Abstract The present disclosure provides methods for treating and / or detecting hereditary neuropathy. In various aspects, the methods include detecting the presence of a mutation in the sorbitol dehydrogenase (SORD) gene in a sample from a subject. In various embodiments, the SORD mutation is a DNA variant classified as pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria. Optionally, the methods include diagnosing the subject with hereditary neuropathy if the presence of a mutation in the SORD gene is detected. Optionally, the methods include administering to the subject a composition comprising an agent selected from the group consisting of an aldose reductase inhibitor; an aldose reductase antisense oligonucleotide; a polynucleotide encoding a SORD peptide; a SORD peptide; an agent that blocks expression of a mutant SORD gene; and an agent that corrects a mutation in the SORD gene. In various aspects, the method includes administering to the subject one or more of the following: alrestatin, epalrestat, diepalrestat, fidarestat, imirestat, lidrestat, minalrestat, ponalrestat, ranirestat, sulfresin B 11 , sorbinil, tolrestat, zenarestat, or zopolrestat (or a combination thereof). In various aspects, the method includes administering to the subject an aldose reductase antisense oligonucleotide; a polynucleotide encoding a SORD peptide; an agent that blocks expression of a mutant SORD gene; an agent that corrects a mutation in the SORD gene; or a combination of any of the foregoing. In various aspects, the method includes administering to the subject a SORD peptide. Administration of a combination of any of the foregoing is also contemplated. Optionally, the method includes measuring sorbitol levels in a sample from the subject.

[0007] For the treatment of hereditary neuropathy in a subject tested for the presence of a mutation in the sorbitol dehydrogenase (SORD) gene (or for use in the preparation of a medicament for the treatment of hereditary neuropathy), (i) an aldose reductase inhibitor (e.g., alrestatin, epalrestat, diepalrestat, fidarestat, imirestat, lidrestat, minalrestat, ponalrestat, ranirestat, sulfresin B, 11 , sorbinil, tolrestat, zenarestat, and / or zopolrestat); (ii) aldose reductase antisense oligonucleotides, polynucleotides encoding SORD peptides, agents that block expression of a mutant SORD gene, and / or agents that correct a mutation in the SORD gene; and / or (iii) uses of SORD peptides are also provided.

[0008] The present disclosure further provides a method of characterizing a neuropathy in a mammalian subject, the method comprising measuring a level of sorbitol in the subject suffering from the neuropathy, wherein a sorbitol level greater than about 10 g / L indicates that the neuropathy is associated with a mutation in the sorbitol dehydrogenase (SORD) gene. The present disclosure also provides a method of evaluating the effectiveness of a treatment for a hereditary neuropathy in a subject, the method comprising administering to the subject an aldose reductase inhibitor (e.g., alrestatin, epalrestat, diepalrestat, fidarestat, imirestat, lidrestat, minalrestat, ponalrestat, ranirestat, sulfresin B). 11, sorbinil, tolrestat, zenarestat, and / or zopolrestat), aldose reductase antisense oligonucleotide, a polynucleotide encoding a SORD peptide, a SORD peptide, an agent that blocks expression of a mutant SORD gene, and an agent that corrects a mutation in the SORD gene (or any combination of the foregoing); and measuring the level of sorbitol in the subject.

[0009] It is understood that each feature or embodiment, or combination described herein is a non-limiting illustrative example of one of the aspects of the present disclosure and is therefore meant to be combinable with any other feature, embodiment, or combination described herein. For example, when a feature is described with phrases such as "one embodiment," "some embodiments," "various embodiments," or "related embodiments," each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features described herein, without the need to recite every possible combination. Such features or combinations of features fall within any of the aspects of the present invention.

[0010] The headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments, and variations of the invention will be apparent from the detailed description and / or drawings and / or claims. [Brief explanation of the drawings]

[0011] [Figure 1-1]Figures 1A-F. SORD gene and pedigree. Biallelic mutations in SORD cause autosomal recessive dHMN / CMT2. (Figure 1A) Representative pedigree of a dHMN / CMT2 family with biallelic mutations in SORD. Squares represent males, and circles represent females. Diagonal lines are used for deceased individuals. Patients are indicated by solid black shapes. (Figure 1B) Schematic diagram showing all exons, introns, and untranslated regions (UTRs) of SORD based on the NCBI reference sequence: NM_003104.6. Gray and white boxes represent the coding sequence and UTRs of SORD, respectively. Variants identified in the families considered in this study map throughout the coding region of the gene. A nonsense c.757delG; p.(Ala253GlnfsTer27) variant in exon 7 was identified with a particular high frequency. (Figure 1C) Distribution of mutations across SORD protein domains. (Figure 1D) SORD protein ortholog alignment showing that the four missense substitutions identified in the dHMN / CMT2 families in this study are located at highly conserved residues across species, from humans to elephants. (Figures 1E and 1F) Expanded nucleotide sequences of the highly homologous regions in exon 7 of SORD (reverse strand) and SORD2P (forward strand). Nucleotides that differ in SORD from SORD2P, including the deletion in SORD2P in Figure 1C, are indicated by arrows. Representative electropherograms show that the c.757delG; p.(Ala253GlnfsTer27) variant found in SORD in the homozygous state in dHMN / CMT2 patients and in the heterozygous state in available patents (right box, upper plot) is absent in biallelic states from healthy controls (right box, lower plot), but is fixed in SORD2P (left box, lower plot). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0012] [Figure 2]Figure 2A-C. Decreased SORD expression and sorbitol accumulation in patient fibroblasts. (Figure 2A) Schematic diagram of the two-step polyol pathway that converts glucose to fructose. (Figure 2B) Immunoblot showing protein levels of SORD normalized to tubulin using polyclonal antibody ab189248 in healthy controls (n = 4, lanes 1-4), heterozygous carriers of the c.757delG;p.(Ala253GlnfsTer27) variant in SORD (n = 2, lanes 10-11), and patients with a homozygous c.757delG;p.(Ala253GlnfsTer27) alteration (n = 4, lanes 5-8) or a compound heterozygous c.757delG;p.(Ala253GlnfsTer27) variant together with a second nonsense c.895C>T;p.(Arg299Ter) mutation (n = 1, lane 9). (Figure 2C) Intracellular sorbitol levels measured by UPLC and normalized to protein content in healthy controls (n = 5) and patients with biallelic nonsense mutations in SORD (n = 5). Graphs show mean ± SD and data distribution (dots). Two-tailed t-tests were performed to compare SORD-encoded protein (Figure 2B) or sorbitol levels (Figure 2C) across groups. Statistical significance is indicated as *, **, or *** when P values are <0.05, <0.01, or <0.001, respectively. All experiments were independently repeated twice with similar results.

[0013] [Figure 3-1]Figures 3A-F. Loss of Drosophila Sord2 causes age-dependent synaptic degeneration. (Figure 3A) 3D structure of the Drosophila visual system showing the lamina, medulla, and lobula. The xy and xz planes are shown, depicting photoreceptor terminals and lamina neurons. (Figure 3B) The lamina of a yw control fly at 2 DAE. The organized lamina cartridge and columnar photoreceptor neurons are shown in the xy and xz planes, respectively. (Figure 3C) The lamina of Sodh2MB01265 / MB01265 homozygous flies at 2 and 10 DAE. Arrowheads indicate lamina vacuoles. Boxes indicate higher magnification areas of the lamina. BRP intensity is indicated. The dotted line indicates the area of the lamina vacuoles. Scale bar: 30 μm. (Figure 3D) Quantification of vacuole number, size, and BRP intensity. A total of three optic lobes were quantified for each group. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. *P<0.05, **P<0.01, ****P<0.0001. (Figures 3E-3F) Locomotor activity of control flies (yw) and Sodh2MB01265 / MB01265 (Figure 3E) or Sodh1 and Sodh2 pan-neuronal double knockdown (RNAi) (Figure 3F) flies. n=10 in each group. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. ****P<0.0001. [Figure 3-2] Same as above.

[0014] [Figure 4-1]Figure 4A-G. Treatment with the aldose reductase inhibitors epalrestat and ranirestat reduces sorbitol levels and restores function. (Figure 4A) Intracellular sorbitol levels measured by UPLC and normalized to protein content in fibroblasts derived from healthy controls (n = 5, open dots) and patients with biallelic nonsense mutations in SORD (n = 5, square dots) after 3 days of treatment with epalrestat 100 μM, ranirestat 10 μM, or DMSO. (Figure 4B) Sorbitol levels measured by UPLC and normalized to protein concentration from brain / head homogenates from wild-type (yw, open dots), Sodh2MB01265 / MB01265 (closed dots), and neuron-specific knockdown of Sodh1 and Sodh1 by RNAi (square dots) Drosophila at 10 days post-ovo encapsulation. Sodh2 mimic and Sodh1 and Soh2 RNAi Drosophila were fed either 80 μM epalrestat, 80 μM ranirestat, or DMSO. Graphs show mean ± SD. Two-tailed t-tests were performed to compare sorbitol levels. Statistical significance is indicated as *, **, or *** if P values were <0.05, <0.01, or <0.001, respectively, unless otherwise specified. All experiments were repeated twice independently with similar results. (Figure 4C) Locomotor activity of control flies (yw) fed with DMSO, and Sodh2MB01265 / MB01265 flies (n = 10 in each group) fed with DMSO, 80 μM epalrestat, or 80 μM ranirestat. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. *P<0.05, ***P<0.001. (Figures 4D-4F) Optic plate of Sodh2MB01265 / MB01265 homozygous flies at 10 and 40 DAE treated with DMSO (Figure 4D), 80 μM epalrestat (Figure 4E), or 80 μM ranirestat (Figure 4F). Arrowheads indicate optic plate vacuoles. Boxes indicate higher magnification areas of the optic plate. BRP intensity is indicated.Dotted lines indicate the area of optic plate vacuoles. Scale bar: 30 μm. (Figure 4G) Quantification of vacuole number, size, and BRP intensity (Figures 4D-4F). n=3. Data are shown as mean ± sd. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. *P<0.05, **P<0.01, ****P<0.0001. [Figure 4-2] Same as above.

[0015] [Figure 5-1] Figure 5. Pedigree of a family with biallelic mutations in SORD. Boxes represent males and circles represent females. Diagonal lines are used for deceased individuals. Affected individuals are indicated by solid black shapes. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0016] [Figure 6] Figure 6A-B. Double knockdown of Drosophila Sodh1 and Sodh2 results in age-dependent synaptic degeneration. (Figure 6A) Optic plate of Sodh1 and Sodh2 double knockdown homozygous flies at 2 and 10 days after embryogenesis. Arrowheads indicate optic plate vacuoles. Boxes indicate higher magnification areas of the optic plate. BRP intensity is indicated. Dotted lines indicate the area of optic plate vacuoles. Scale bar: 30 μm. (Figure 6B) Quantification of vacuole number, size, and BRP intensity. A total of three optic plate discs were quantified for each group. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. *P<0.05, **P<0.01, ****P<0.0001.

[0017] [Figure 7]Figure 7. Treatment with the aldose reductase inhibitors epalrestat and ranirestat restores locomotor function in Sodh1 and Sodh2 double knockdown flies. Locomotor activity of control flies (yw) fed DMSO (dots, first data point from the left for each DAE indicated) or flies with neuron-specific knockdown of Sodh1 and Sodh2 fed DMSO (squares, second data point from the left for each DAE indicated), 80 μM epalrestat (squares, third data point from the left for each DAE indicated), or 80 μM ranirestat (squares, fourth data point from the left for each DAE indicated). n = 10 in each group. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc multiple comparison test. ***P < 0.001, ****P < 0.0001.

[0018] [Figure 8] Figure 8. Diagram of an exemplary expression vector (pAAV-SORD) encoding the SORD peptide.

[0019] [Figure 9] Figure 9. An exemplary complete AAV vector DNA sequence containing the SORD coding sequence (pAAV-SORD) (SEQ ID NO: 1).

[0020] [Figure 10] Figure 10. SORD primer sequences and thermocycling conditions. PCR: polymerase chain reaction; Fw: forward; Rv: reverse.

[0021] [Figure 11-1] Figure 11. Clinical characteristics of patients with hereditary neuropathy and biallelic mutations in SORD. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0022] [Figure 12] Figure 12. Clinical characteristics of patients affected by hereditary neuropathies and carrying biallelic mutations in SORD. Categorical data are expressed as N (%) if data are available for all individuals, or N / number of individuals studied (%). Continuous variables are expressed as mean ± standard deviation (min-max). CMT, Charcot-Marie-Tooth disease; dHMN, distal hereditary motor neuropathy.

[0023] [Figure 13] Figure 13. Fasting sorbitol levels in serum from 10 unrelated healthy controls and 10 patients with the biallelic p.Ala253GlnfsTer27 mutation in SORD. The graph shows the mean ± sd and data distribution (dots), and p-values of two-tailed t-tests comparing SORD protein and sorbitol levels across groups - *p<0.05, **p<0.01, and ***p<0.001. All experiments were repeated twice independently.

[0024] [Figure 14] Figures 14A-14C. Exemplary vector designs for SORD gene replacement therapy. (Figure 14A) AAV-9 packaged vector design for SORD gene replacement therapy. The CB7 promoter has been shown to be effective in driving high expression, followed by the SORD cDNA (NCBI Reference Sequence: NM_003104.6), a post-transcriptional regulatory element (WPRE) to further enhance expression and target specificity, and a transcription termination poly(A) element, followed by an origin of replication (pUC-ori) and ITR sequences (inverted terminal repeats). (Figure 14B) SORD cDNA sequence. (Figure 14C) SORD polypeptide sequence.

[0025] [Figure 15-1]Figures 15A-15D. Significant knockdown of aldose reductase (AR) (AKR1B1 gene) via antisense oligonucleotide (ASO) (AR 1A, (SEQ ID NO: 22)). The targeted ASO (AR 1A) sequence and the ASO-S mixed sequence (AR-S 1A, (SEQ ID NO: 47)) are shown in Figure 15A. Figure 15B shows modifications to the nucleotide backbone of the ASO. This was performed in SORD patient fibroblasts and control fibroblasts, normalized to β-tubulin, and measured via Western blot (Figures 15C-15D). An additional control used a mixed version of ASO-S (AR-S 1A) displaying random nucleotides (Figure 15C). [Figure 15-2] Same as above.

[0026] [Figure 16-1] Figure 16. Table of antisense oligonucleotide sequences and target sites (exon targets only) in Homo sapiens aldo-keto reductase family 1 member B (AKR1B1). Filter criteria: A) 40%≦GC%≦60%; B) antisense oligo binding energy≦−8 kcal / mol; C) no GGGG in the target sequence. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 16-5] Same as above. [Figure 16-6] Same as above.

[0027] [Figure 17]Figure 17. Table of antisense oligonucleotide (ASO) sequences and target sites (exon targets only) in Homo sapiens aldo-keto reductase family 1 member B (AKR1B1). Filter criteria: A) 40%≦GC%≦60%; B) no GGGG in the target sequence; C) average unpaired probability of target site nucleotides≧0.5; D) for each peak in the accessibility profile above a threshold probability of 0.5, all sites targeted to this same peak are ranked by their average unpaired probability (higher is better), and at most n sites per peak are selected, where n is determined by the maximum ([peak width / site length], 2); E) among sites satisfying criteria A–D, the top 20 unique ones with the highest average unpaired probability are listed.

[0028] [Figure 18-1] Figure 18. Table of antisense oligonucleotide (ASO) sequences and target sites (intron targets only) in Homo sapiens aldo-keto reductase family 1 member B (AKR1B1), hg19_dna range=chr7:134127102-134143944. Filter criteria: A) 40%≦GC%≦60%; B) no GGGG in the target sequence; C) average unpairing probability of target site nucleotides≧0.5; D) for each peak in the accessibility profile above a threshold probability of 0.5, all sites targeted to this same peak are ranked by their average unpairing probability (higher is better), and at most n sites per peak are selected, where n is determined by the maximum ([peak width / site length], 2); E) Among the sites satisfying criteria A–D, the top 20 unique ones with the highest average unpairing probability are listed. [Figure 18-2] Same as above. [Figure 18-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description The present disclosure provides methods for detecting and / or treating hereditary neuropathies and related genetic conditions.

[0030] Inherited (or hereditary) neuropathies include, but are not limited to, Charcot-Marie-Tooth disease (CMT), hereditary motor and sensory neuropathies, hereditary motor neuropathies, distal hereditary motor neuropathy (dHMN), axonal neuropathies, intermediate neuropathies, and amyotrophic lateral sclerosis type ALS4.

[0031] In various aspects, the present disclosure provides methods in which the presence of a mutation in the sorbitol dehydrogenase (SORD) gene is detected in a sample from a subject. The mutation can be detected by examining the DNA sequence of the gene, examining the RNA, or examining a protein with a mutation that results in a loss of function.

[0032] Disclosed herein is the identification of biallelic mutations in the sorbitol dehydrogenase gene (SORD) associated with the most frequent recessive form of CMT. SORD encodes sorbitol dehydrogenase, an enzyme that converts sorbitol to fructose. It belongs to the two-step polyol pathway, previously identified as central to neuronal damage in the hyperglycemic state of diabetes. Forty-two cases of CMT across different ethnicities were identified to harbor a nonsense mutation, c.757delG; p.Ala253GlnfsTer27, in SORD, either in the homozygous or compound heterozygous state. Screening of the p.Ala253GlnfsTer27 alteration in additional case and control sets established this variant as one of the most common pathogenic alleles in men with Mendelian inheritance (MAF=0.003). Patient fibroblast cultures show a complete loss of SORD protein and loss of intracellular sorbitol accumulation, leading to tissue damage. Loss of Sodh1 in Drosophila resulted in synaptic degeneration and progressive motor deficits. Remarkably, reduction of polyol influx by treatment with an aldose reductase inhibitor fully rescued intracellular sorbitol levels in patient fibroblasts and in the Sodh1 Drosophila model. In the latter model, the treatment also completely ameliorated motor and ocular phenotypes. Collectively, these findings demonstrate a key role for the polyol pathway and sorbitol accumulation in inherited neuropathies and establish the molecular basis for a potentially treatable condition in a significant proportion of cases. These findings also represent an example of convergent pathomechanisms in inherited and acquired neuropathies, with broader implications in the field of diabetes.

[0033] Thus, in various aspects of the disclosure, the methods include detecting SORD gene mutations, including: 753delG; p.(Ala253GlnfsTer27), c.757delG; p.Ala253GlnfsTer27, c.28C>T; p.Leu10Phe, c.316_425+165del; p.Cys106Ter, c.329G>C; p.Arg110Pro, c.298C>T; p.Arg100Ter, c.295C>T; p.Arg299Ter, c.964G>A; p.Val322Ile, c.458C>A; p.Ala153Asp; deletion of individual or multiple coding exons or the entire SORD gene via copy number variation; or any protein truncation mutations and / or mutations resulting in a "loss of function" or hypomorphic function of the protein.

[0034] In various aspects, the SORD mutations are detected using DNA sequencing methods such as whole exome sequencing, whole genome sequencing (WGS) and / or next-generation sequencing (NGS), allele-specific oligonucleotides, polymerase chain reaction (PCR), quantitative or real-time PCR (qPCR), multiplex PCR, nested PCR, Amplification Refractory Mutation System (ARMS) PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), protein truncation test (PTT), RFLP, DNA microarray, RNA-seq, CRISPR-based mutation detection (e.g., CRISPR-Chip, Hajian et al., Nature Biomedical Engineering 3, 427-437 (2019)), or other DNA or RNA mutation detection methods suitable for mutation detection.

[0035] In various aspects, the SORD mutation is detected by examining the protein using Western blot (immunoblot), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), antibody-dependent methods (e.g., enzyme-linked immunosorbent assay (ELISA)), protein immunoprecipitation, protein immunostaining, protein chips, or other protein detection methods suitable for detecting mutations.

[0036] Optionally, the method further comprises measuring sorbitol levels in the subject's sample. Methods for measuring sorbitol include, for example, enzyme assays, fluorescence assays, chromatography-based methods, and spectroscopy-based methods. Exemplary methods for measuring sorbitol are provided in the Examples.

[0037] The present disclosure further provides methods for characterizing neuropathies (e.g., hereditary neuropathies) and related conditions involving SORD mutations. In various aspects, the methods include measuring sorbitol levels in a biological sample from a subject suffering from a neuropathy. In various aspects, the methods include detecting an increased level of sorbitol in the biological sample. An "increased level of sorbitol" refers, for example, to a sorbitol level greater than about 10 mg / L. SORD-associated neuropathy results in high levels of sorbitol in patients, as described in the Examples and FIG. 13. Thus, detecting a sorbitol level greater than about 10 mg / L indicates that the neuropathy is a hereditary neuropathy associated with a SORD mutation, thereby allowing a clinician to characterize the neuropathy afflicting the subject. Optionally, the method includes a treatment step comprising administering to the subject an agent selected from the group consisting of an aldose reductase inhibitor; an aldose reductase antisense oligonucleotide; a polynucleotide encoding a SORD peptide; a SORD peptide; an agent that blocks expression of a mutant SORD gene; and an agent that corrects a mutation in the SORD gene.

[0038] In various aspects, the present disclosure provides a method comprising identifying a mutation in the sorbitol dehydrogenase (SORD) gene in a sample from a subject before or after measuring sorbitol levels in the subject. In this regard, the method can be used to confirm a diagnosis of hereditary neuropathy. Similarly, the present disclosure provides a method for identifying a pathogenic SORD mutation, comprising measuring sorbitol levels in a subject containing a mutation in the SORD gene. The presence of increased sorbitol levels (e.g., greater than about 10 mg / L) indicates that the SORD mutation is pathogenic.

[0039] Alternatively (or additionally), the method can be used to evaluate the effectiveness of a treatment for hereditary neuropathy in a subject. In this regard, the method involves administering a treatment to the subject and then measuring sorbitol levels in a biological sample. A decrease in sorbitol levels compared to the sorbitol levels observed with pretreatment (e.g., a reduction in sorbitol levels below about 10 g / L) indicates an improvement in the subject's condition. The materials and methods described herein can also characterize patient compliance in taking medications for the treatment of SORD-associated hereditary neuropathy or monitor the success of candidate therapeutic agents in clinical trials.

[0040] The sample can be any biological sample taken from the subject, including, but not limited to, any tissue, cell, or fluid (e.g., blood, plasma, serum, or urine) that can be analyzed for a trait of interest (e.g., nucleic acid (e.g., SORD mRNA), protein (e.g., SORD protein), or the presence or amount of sorbitol). In various embodiments, the biological sample is plasma, serum, saliva, urine, or a skin sample.

[0041] A "subject," as referred to herein, can be any mammal (e.g., a human). Animals of importance as domesticated companion animals (including dogs and cats); animals of importance in research (including rodents and primates); and large endangered and zoo animals (e.g., primates, cats, giraffes, elephants, rhinos), as well as animals of agricultural importance (e.g., cattle, horses, and swine).

[0042] In various aspects, the method includes treating the subject by administering to the subject a composition comprising one or more aldose reductase inhibitors. In some embodiments, the aldose reductase inhibitor is selected from the group consisting of alrestatin, epalrestat, diepalrestat, fidarestat, imirestat, lidorestat, minalrestat, ponalrestat, ranirestat, sulfresin B, and the like. 11 Aldose reductase inhibitors are sorbinil, tolrestat, zenarestat, or zopolrestat. Aldose reductase inhibitors are reviewed in Expert Opin Ther Pat. 2019;29(3):199-213; Chatzopoulou et al., Expert Opin Ther Pat. 2012;22(11):1303-23 (incorporated by reference in their entireties).

[0043] In some embodiments, enzyme replacement therapy is used and a SORD peptide is administered to the subject. Thus, the therapy supplements SORD peptide levels where endogenous SORD levels are inadequate or absent. An exemplary SORD peptide is provided in SEQ ID NO: 46. The present disclosure contemplates the use of peptides that share at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 46.

[0044] In various embodiments, the method includes administering to the subject a polynucleotide (e.g., an aldose reductase antisense oligonucleotide, a polynucleotide encoding a SORD peptide / protein, an agent that blocks expression of a mutant SORD gene, and / or an agent that corrects a mutation in a SORD gene). Polynucleotides are typically delivered to host cells via an expression vector, which contains the regulatory sequences necessary for delivery and expression, although the use of an expression vector is not required in the context of the present disclosure. In some aspects, the constructs described herein include a promoter (e.g., a cytomegalovirus (CMV) promoter or a CB7 promoter), a protein coding region (optionally with a non-coding (e.g., 3'-UTR) region that facilitates expression), a transcription termination sequence, and / or a regulator element sequence (e.g., a post-transcriptional regulatory element (WPRE), a poly(A) element, an origin of replication (pUC-ori), and / or an ITR sequence (inverted terminal repeat)). In various aspects, the constructs described herein include one or more of the vector features listed in Table 1. Vector features are also reviewed in Powell et al., Discov Med. 2015; 19(102): 49-57 (incorporated by reference in its entirety). For example, the Cre-loxP system can be utilized to express a peptide of interest (e.g., a SORD peptide) in a specific tissue of interest, as needed. The expression vector can be viral-based (e.g., retroviral-, adenoviral-, or adeno-associated viral-based) or a non-viral vector (e.g., a plasmid). Non-vector-based methods (e.g., using naked DNA, DNA complexes, etc.) can also be used. Optionally, the vector is a viral vector (e.g., a lentiviral or baculoviral vector), and in various preferred embodiments, the vector is an adeno-associated viral vector (AAV).The expression vector can be based on any AAV serotype, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, or AAV-13. Polynucleotides can also be delivered via liposomes, nanoparticles, exosomes, microvesicles, hydrodynamic-based gene delivery, or via a "gene gun."

[0045] [Table 1]

[0046] The titer of AAV to be administered in the methods of the present disclosure varies depending, for example, on the particular AAV, the mode of administration, the purpose of treatment, the individual, and the cell type(s) being targeted, and can be determined by methods known in the art. The titer of AAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 or more DNase resistant particles (DRP). Dosages may also be expressed in units of viral genomes (vg).

[0047] In various embodiments, a polynucleotide encoding a SORD peptide is administered to the subject. The amino acid sequence of SORD is provided as SEQ ID NO: 46 (Figure 14C, NCBI Reference Sequence: NP_003095.2). The polynucleotide used in the method optionally encodes the amino acid sequence of SEQ ID NO: 46, or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 46 (which retains SORD function). Optionally, the polynucleotide comprises SEQ ID NO: 45 (Figure 14B), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the polynucleotide sequence of SEQ ID NO: 45 (which encodes SORD). Exemplary expression vectors containing polynucleotides encoding the SORD peptide are illustrated in Figures 8 and 14A. The polynucleotide, in at least one aspect of the present disclosure, comprises the nucleic acid sequence shown in Figure 9 (SEQ ID NO: 1), which corresponds to the sequence of an AAV vector comprising a polynucleotide encoding SORD.

[0048] In various embodiments, the method includes administering to the subject an agent that blocks the expression of a mutant SORD gene. An agent that blocks the expression of a mutant SORD gene refers to an agent that interferes with the expression of the SORD gene such that SORD gene expression and / or SORD protein levels are reduced compared to basal / wild-type levels. "Blocking" the expression of a mutant SORD gene does not require 100% abolition of expression and SORD production; it is recognized that any level of reduced expression of abnormal SORD may be beneficial to the subject. Exemplary agents include, but are not limited to, antisense oligonucleotides (ASOs), short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), or microRNAs (miRNAs).

[0049] In various embodiments, the method comprises administering to the subject an aldose reductase antisense oligonucleotide targeted to the aldose reductase sequence, such that expression of the enzyme is blocked. Aldose reductase, aldo-keto reductase family 1 member B (AKR1B1), is encoded by SEQ ID NO: 48 (NCBI Reference Sequence: NM_001628). The aldose reductase antisense oligonucleotide interferes with the expression of the aldose reductase gene (AKR1B1), such that AKR1B1 gene expression and / or aldose reductase protein levels are reduced compared to basal / wild-type levels. "Blocking" the expression of the aldose reductase gene (AKR1B1 gene) does not require 100% abolition of expression and aldose reductase production; it is recognized that any level of reduced expression of aldose reductase may be beneficial to the subject. For example, in various aspects, the aldose reductase antisense oligonucleotide reduces the expression of aldose reductase. ASO is a single-stranded deoxyribonucleotide that is complementary to an mRNA target sequence. In various aspects, the aldose reductase antisense oligonucleotide targets the exon sequence or intron sequence of the aldose reductase gene.

[0050] In an exemplary method for identifying ASO sequences targeting aldose reductase, the following criteria were used: A) sequences targeting aldose reductase (AKR1B1) were selected that contained ≦40% GC or ≦60% GC content; B) sequences containing GGGG nucleotides were excluded; C) sequences with an average unpairing probability of target site nucleotides ≧0.5 were selected; D) for each peak in the accessibility profile that exceeded a threshold probability of 0.5, all sites targeted to that same peak were ranked by their average unpairing probability (higher is better), and at most n sites per peak were selected, where n is determined by the maximum peak width / site length. Exemplary agents meeting these criteria are provided in Table 2. Further exemplary ASO sequences and filter criteria are shown in Figures 16-18.

[0051] [Table 2]

[0052] In various embodiments, the nucleotide backbone of the ASO sequence is modified into a chimeric or gapmer design to reduce gene expression compared to basal / wild-type levels. In various embodiments, the gapmer design requires the designation of 3-5 nucleotides on each end of the antisense oligonucleotide sequence to have modifications in the ribose sugar moiety that are resistant to RNase H recognition and other nucleases, while all other nucleotides contain RNase H-compatible modifications. RNase H is responsible for cleaving RNA-DNA duplexes (e.g., those formed between aberrant mRNA transcripts and synthetically designed DNA antisense oligonucleotides). In various embodiments, modifications of ASO sequences include, but are not limited to, phosphorothioate (PS)-RNase H recognizable, phosphorodiamidate morpholino (PMO)-RNase H resistant, 2'-O-methyl-RNase H resistant, 2'-O-methoxyethyl (MOE)-RNase H resistant, locked nucleic acid (LNA)-RNase H resistant, ethylene-bridged nucleic acid (ENA)-RNase H resistant, or (S)-constrained ethyl (cEt)-RNase H resistant. Exemplary modifications of ASO sequences are shown in Figures 15A-15B. Modifications to ASO sequences are reviewed in Scoles et al., Neurol Genet. 2019;5(2):e323 (incorporated by reference in its entirety).

[0053] In various aspects, the method uses RNA interference (RNAi) to regulate SORD expression. The RNAi pathway is summarized in Chapter 7, Section 7.3, Duan (ed.) in Muscle Gene Therapy, Springer Science+Business Media, LLC (2010). Suitable agents include, for example, siRNA, miRNA, and shRNA. shRNA / Hairpin vectors are artificial RNA molecules (nucleotides) with tight hairpin turns that can be used to silence target gene expression via RNAi. shRNAs are advantageous mediators of RNAi in that they have relatively slow degradation and turnover rates, but they often require the use of an expression vector. In exemplary aspects, the present disclosure includes the generation and administration of AAV vectors expressing one or more shRNAs targeting SORD. shRNA expression is regulated by the use of various promoters. In various aspects, polymerase II promoters (e.g., U6 and H1) and polymerase III promoters are used. In some aspects, U6 shRNAs are used. It is recognized that RNAi can also be used to downregulate (i.e., block) the expression of aldose reductase (e.g., AKR1B1); thus, the present disclosure contemplates the use of siRNAs, miRNAs, and shRNAs that target aldose reductase intronic or exonic sequences to block the expression of aldose reductase.

[0054] Traditional small / short hairpin RNA (shRNA) sequences are usually transcribed inside the cell nucleus from vectors containing Pol III promoters (e.g., U6). The endogenous U6 promoter usually controls the expression of U6 RNA (a small RNA involved in splicing) and has been well characterized (Kunkel et al., Nature. 322(6074):73-7 (1986); Kunkel et al., Genes Dev. 2(2):196-204 (1988); Paule et al., Nucleic Acids Res. 28(6):1283-98 (2000)). The present disclosure includes both mouse and human U6 or H1 promoters. shRNAs containing sense and antisense sequences from target genes connected by a loop are transported from the nucleus to the cytoplasm, where Dicer processes them into siRNAs.

[0055] In some aspects of the present disclosure, the agent that corrects the mutation in SORD gene is used.The agent that corrects the mutation in SORD gene refers to the agent that can modify the SORD coding sequence or regulatory element and / or the non-coding region associated with SORD gene to achieve the desired change in sequence.In various aspects, genome editing can be used to replace part or all of the SORD gene sequence or change the SORD protein expression level. In various embodiments, the agent may comprise components used in genome editing technologies (e.g., designer zinc fingers, transcription activator-like effector nucleases (TALENs), or CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) systems. An exemplary agent for use in the methods of the disclosure is DNA encoding a Cas9 molecule and / or a gRNA molecule. Cas9 and gRNA may be present in a single expression vector or in separate expression vectors. Adenoviral delivery of the CRISPR / Cas9 system is described in Holkers et al., Nature Methods (2014), 11(10):1051-1057, which is incorporated by reference in its entirety.

[0056] Other publications describing CRISPR systems and Cas9 include: Cong et al. Science (2013) 339:819-23; Jinek et al., Elife. (2013) 2:e00471; Lei et al. Cell (2013) 152: 1173-1183; Gilbert et al. Cell (2013) 154:442-51; Lei et al. Elife (2014) 3:e04766; Perez-Pinela et al. Nat Methods (2013) 10: 973-976; Maider et al. Nature Methods (2013) 10, 977-979; U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; U.S. Patent Publication No. 2014 / 0068797; and International Patent Publication No. WO 2014 / 197568 (all incorporated by reference in their entirety).

[0057] In some embodiments, CRISPR / Cas9 multiplexing involves two or more guide RNAs, as described in CRISPR 101:A Desktop Resource (1 st It can be used to target multiple expressed genomic loci as described in (Edition), Addgene, January 2016, which is incorporated by reference in its entirety.

[0058] The terms "treating" or "treatment" refer to reducing or ameliorating hereditary neuropathy and / or related disorders and / or symptoms associated therewith. These terms include reducing or delaying the frequency of occurrence or recurrence of the neuropathy or symptoms associated therewith (i.e., increasing the duration of remission in patients afflicted with the disorder), as well as reducing the severity of the disorder or any disorders associated therewith. It is recognized, although not excluded, that "treating" or "treatment" of a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0059] The dosage of an active agent (e.g., an aldose reductase inhibitor, an aldose reductase antisense oligonucleotide, a polynucleotide encoding a SORD peptide, a SORD peptide, an agent that blocks expression of a mutant SORD gene, or an agent that corrects a mutation in a SORD gene) will depend on factors such as the route of administration (e.g., local vs. systemic), patient characteristics (e.g., gender, weight, health status, side effects), the nature and extent of the hereditary neuropathy or related disorder, and the particular active agent or combination of active agents selected for administration.

[0060] The active agents described herein are provided in compositions (e.g., pharmaceutically acceptable compositions) that may contain formulation components suitable for administration to a subject and additional therapeutic agents.Appropriate methods for administering pharmaceutically acceptable compositions (e.g., pharmaceutical compositions containing the agents described herein) are well known in the art.In various aspects, more than one route can be used to administer one or more of the agents disclosed herein.Certain routes can provide more immediate and effective responses than other routes. For example, in certain circumstances, it may be desirable to deliver the composition orally; intravenously, intraperitoneally, intracerebrally (intraparenchymal), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, intralesionally, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, enterally, topically, sublingually, urethrally, vaginally, or rectally; by controlled, delayed, sustained, or otherwise modified release systems; by implantable devices; using nanoparticles; or through injection or infusion as a conjugate.

[0061] It is contemplated that two or more active agents described herein can be administered as part of a therapeutic regimen. Alternatively or additionally, one or more of the active agents can be administered with other therapeutic agents as part of a therapeutic regimen. The active agent(s) can be administered as monotherapy or as combination therapy with other treatments administered simultaneously or metronomically. The term "simultaneous" or "simultaneously" refers to the administration of two agents within 6 hours or less (e.g., within 3 hours or 1 hour of each other). In this regard, multiple active (or therapeutic) agents can be administered in the same composition or in separate compositions provided within a short period of time (e.g., within 30 minutes). The term "metronomically" refers to the administration of different agents at different times and with a frequency associated with repeated administration. The active agents do not need to be administered at the same time or by the same route; preferably, in various embodiments, there is an overlap in the time periods during which different active agents exert their therapeutic effects. Further aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative and not limiting. [Example]

[0062] General method family All families provided written informed consent to participate in the study. The study protocol was approved by the sponsoring institution's Institutional Review Board. All patients were clinically evaluated by a neurologist.

[0063] Whole-exome and Sanger sequencing Whole-exome sequencing was performed on index individuals from sporadic and recessive CMT and dHMN families. The SureSelect Human All Exon 50 MB Kit (Agilent) was used for in-solution enrichment, and approximately 120-bp paired-end sequence reads were generated using a HiSeq 2500 instrument (Illumina). Sequence read alignment and variant calling were performed using the Burrows-Wheeler aligner and Freebayers. Final data were uploaded to GENESIS software for analysis. A filtering approach to search for families sharing the same homozygous variant was applied across the whole exome in the database. Sanger sequencing (performed by Eurofins Genomics) confirmed the segregation of SORD variants. Polymerase chain reaction (PCR) was performed on a Veriti Thermocycler (Applied Biosystems), and regions containing targeted mutations were amplified using Platinum Taq (ThermoFisher). The following primers were used to specifically target SORD but not SOR2P (Figure 10).

[0064] Fibroblast culture Fibroblasts were obtained from patients and cultured in Dulbecco's modified Eagle's medium (ThermoFisher) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin (Gibco). Cells were maintained in a humidified incubator at 37°C with 5% CO2. Asynchronous cell cultures were grown to approximately 80% confluence and treated with epalrestat (100 μM), ranirestat (10 μM), or DMSO for 72 hours. The medium containing the drugs or DMSO was changed every 24 hours.

[0065] Western blot Fibroblasts were lysed in RIPA buffer (ThermoFisher) containing protease inhibitors (Roche) and sonicated for 5 minutes using a Bioruptor ultrasonic device (Diagenode). Cell lysates were centrifuged at 13,000 × g for 10 minutes at 4°C, and the supernatant was collected for protein quantification (Pierce BCA Protein Assay Kit). 30 μg of protein sample was mixed with Bolt LDS sample buffer and sample reducing agent (ThermoFisher) and heated to 90°C for 5 minutes. Samples were loaded onto a Bolt 4-12% Bis-Tris Plus minigel and subsequently transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk, incubated with anti-SORD (ab189248, Abcam) antibody for 2 hours, washed with TBS containing 0.01% Tween® 20 (Bio-Rad), and incubated with secondary anti-rabbit antibody (Cell Signaling). The membrane was subsequently incubated with GAPDH primary antibody (Santa Cruz) and secondary anti-mouse antibody (Cell Signaling). Chemiluminescence detection was performed with SuperSignal West Pico PLUS Chemiluminescent Substrate and imaged with FluorChem E (ProteinSimple).

[0066] Sorbitol measurement Fibroblasts were collected and lysed in the absence of proteinase inhibitors as described in the Western blot section. Sorbitol determination in human fibroblast lysates was performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) (Waters Acquity UPLC & TQD Mass Spectrometer - Waters, Milford, MA, USA). Fibroblasts were collected and lysed in the absence of proteinase inhibitors as described in the Western blot section. Proteinase inhibitors contain high concentrations of mannitol (mannitol is an enantiomer of sorbitol), which may interfere with UPLC-MS / MS sorbitol determination. Lysate samples underwent protein precipitation with acetonitrile (1:5), a 10-fold dilution with acetonitrile-water (50 / 50), and cleanup on an Oasis HLB cartridge (10 mg / 1 ml) before injection into the UPLC (3 μL). UPLC conditions: Column, BEH Amide 1.7 μm (2.1 × 100 mm) at 88 °C; Eluent A, 90% acetonitrile-5% water-5% isopropanol; Eluent B, 80% acetonitrile-20% water; Gradient elution: 0 min, 100% A, 3.6 min, 100% B; Flow rate: 0.45 ml / min. Sorbitol retention time was 2.7 min. MS / MS conditions: Interface, electrospray interface in negative ion mode; Multiple Reaction Monitoring acquisition; m / z 180.9 → 88.9 (CV 24, CE 15).

[0067] For fasting sorbitol level testing, blood was collected into serum separator tubes after an overnight fast (the last meal was the night before). Samples were centrifuged at 500g for 10 minutes. Serum was separated and frozen within 1 hour of collection. Sorbitol levels were tested by UPLC using a method modified from Li et al. Biochem Biophys Res Commun. 2009 Oct 2;387(4):778-83. Conditions were as follows: column, BEH Amide 1.7 μm, maintained at 25°C (instead of 45°C); eluent A, 10 mM ammonium acetate pH 10; eluent B, acetonitrile; flow rate, 20 runs of the same gradient at 0.6 ml / min. The retention time of sorbitol was 6.0 min. MS / MS conditions were the same as those for the fibroblast analysis. Serum samples underwent protein precipitation with cold methanol (1:5), 5-fold dilution with acetonitrile-water (50 / 50), and cleanup with Oasis HLB cartridges (10 mg / 1 ml) before injection (3 μL) into the UPLC. A calibration curve was constructed in serum over a sorbitol concentration range of 0.1–20 mg / L.

[0068] Drosophila stocks and genetics Unless otherwise specified, all flies were maintained on cornmeal-molasses-yeast medium at 25°C and 65% humidity with a 12-hour light / 12-hour dark cycle. The following fly strains used in this study were obtained from the Bloomington Drosophila Stock Center: elav C155 -GAL4, GMR-GAL4, Sdh2 MB01265 , UAS-Sdh1RNAi, and UAS-Sdh2RNAi.

[0069] Drug supply Epalrestat or ranirestat was dissolved in dimethyl sulfoxide (DMSO) to achieve a stock concentration of 10 mg / ml and then mixed into 10 ml fly diet at a final concentration of 80 μg / ml. As a control, an equal volume of DMSO was mixed into the fly diet. Vials were allowed to dry at room temperature for 12 hours before feeding.

[0070] Drosophila lifespan assay and negative geotaxis assay For the lifespan assay, 100 newly captive female flies from each group were collected and placed in vials of 20 individuals. Flies were transferred to new vials every two days, and the number of dead flies was counted. Survival data were plotted using a Kaplan-Meier plot, and between-group comparisons were performed using the log-rank test. For the negative geotaxis behavior assay, 10 age-matched female flies were placed in a vial marked with a black horizontal line 8 cm above the bottom. Flies were given 60 min to fully recover from CO2 anesthesia, then gently tapped on the bottom and given 10 s to climb. Flies that crossed the 8 cm line were counted. For each vial, the assay was repeated 10 times, and 10 independent vials per group (a total of 100 flies per group) were tested. To minimize observer expectancy bias, the assay was performed blinded to group assignment.

[0071] Drosophila brain dissection, immunostaining, and confocal microscopy Brain dissection and staining were performed as previously described (Brazill et al., J Vis Exp. 2018; (138)). Briefly, fly brains were dissected in phosphate-buffered saline (PBS, pH 7.4), fixed in 4% formaldehyde for 10 min, and washed three times (15 min each) in PBTX (PBS containing 0.4% v / v Triton X-100). Brains were then incubated overnight at 4°C with gentle shaking with primary mouse anti-BRP antibody (nc82, Developmental Studies Hybridoma Bank) diluted 1:250 in 0.4% PBTX containing 5% normal goat serum. The brains were then incubated overnight at 4°C with a 1:250 dilution of Cy3-conjugated anti-mouse secondary antibody (Rockland) and Cy5-conjugated anti-HRP (Jackson ImmunoLab) with gentle shaking, followed by 4',6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen) staining for 10 minutes at room temperature. Samples were mounted on glass slides with VECTASHIELD Antifade Mounting Medium (Vector Laboratories Inc.). Fly brain slides were imaged using an Olympus IX81 confocal microscope with a 60x oil immersion objective at a scan speed of 8.0 μs / pixel and a spatial resolution of 1024 × 1024 pixels. Images were processed and analyzed using FluoView 10-ASW (Olympus).

[0072] Further aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative and not limiting.

[0073] Example 1: Identification of DNA variants in CMT using GENESIS analysis Hereditary neuropathies (including Charcot-Marie-Tooth disease (CMT)) represent an umbrella term for clinically and genetically heterogeneous conditions affecting peripheral nerves. CMT is classified as demyelinating (CMT1) or axonal (CMT2) depending on conduction velocity. Distal hereditary motor neuropathy (dHMN) represents a form of CMT2 in which the disease burden primarily or exclusively affects motor neurons (Rossor, Tomaselli, and Reilly 2016). In contrast to CMT1, in which over 90% of cases have mutations in known genes, only 20–30% of CMT2 and distal HMN patients receive a genetic diagnosis (Fridman et al. 2015). Because up to 70% of CMT2 and dHMN cases are sporadic, identifying candidate pathogenic genes from whole-exome and genome sequences of a single case becomes more challenging; therefore, large aggregate datasets are needed. Using the aggregation of over 1,100 CMT whole-exome sequencing (WES) and whole-genome sequencing (WGS) data sets available on the GENESIS analysis platform, the largest collection of such high-quality data is now available (Gonzalez et al. 2015). Genes with significant DNA variants present in multiple families were identified, as were individual alleles overrepresented in CMT cases. When a subset of 598 undiagnosed CMT patients was queried for recessive nonsense variants in genes shared by >3 families and with a minor allele frequency <1% in the gnomAD control database, 12 cases were identified from 11 unrelated families with a homozygous c.757delG; p.(Ala253GlnfsTer27) mutation in SORD. Four additional cases from three unrelated families had the heterozygous c.757delG; p.(Ala253GlnfsTer27) variant along with a second variant, c.298C>T; p.(Arg100Ter) in family 2, c.329G>C; p.(Arg110Pro) in family 3, and c.458C>A; p.(Ala153Asp) in family 14, II-1 and II-2 (Figures 1A–D and 5).All mutations represented loss-of-function (LOF) alleles, except for c.329G>C; p.(Arg110Pro). Interestingly, the Arg110Pro change is adjacent to a previously reported Tyr111Phe (equivalent to Tyr110Phe in rats), which has been shown to abolish SORD enzymatic activity and destabilize the protein (Hellgren et al. 2007).

[0074] Interestingly, SORD has a nonfunctional, highly homologous paralog, the pseudogene SORD2P, which is thought to arise from a duplication of SORD within a 0.5 Mb region on chromosome 15 (Carr et al. 2016) (Figure 1E). To specifically amplify SORD but not SORD2P in Sanger confirmation studies, primers were designed taking advantage of nucleotide sequence differences and distinct retrotransposon insertions in both gene regions (Figure 5). Notably, the c.757delG; p.(Ala253GlnfsTer27) mutation in exon 7 of SORD, along with numerous additional exonic indel mutations that prevent effective translation of SORD2P (1000 Genomes Project Consortium et al. 2015; Lek et al. 2016), is anchored within the pseudogene SORP2P in over 95% of control chromosomes. Due to the high similarity of these regions, a nested PCR approach was necessary to obtain specific amplification of exon 7 of SORD and distinguish it from the homologous region in SORD2P. The presence of variants detected by WES was confirmed in all cases by Sanger sequencing, providing segregation data in immediate carrier relatives (Figure 1F and Figure 5).

[0075] We screened an independent set of 103 unexplained CMT2 / dHMN cases by WES at the UCL Institute of Neurology, London, UK. From six unrelated families, we identified nine cases (8.7%) with the homozygous c.757delG; p.(Ala253GlnfsTer27) mutation in SORD. A third independent set of 297 recessive or sporadic CMT2 / dHMN patients was screened by targeted Sanger sequencing of exon 7 of SORD, which, when one c.757delG; p.(Ala253GlnfsTer27) was identified, was extended to other coding exons, revealing 20 additional cases (7%) from 18 families with biallelic mutations in SORD: 16 cases had the homozygous c.757delG; p.(Ala253GlnfsTer27) mutation and 4 cases had c.757delG; p.(Ala253GlnfsTer27) in a compound heterozygous state with a second likely pathogenic variant. The latter included c.964G>A; p.(Val322Ile) in family 29, a 275-bp deletion in exon 4 (c.316_425+165del) in family 30, a novel c.28C>T; p.(Leu10Phe) in family 32, and c.895C>T; p.(Arg299Ter) in family 33. All alterations have a minor allele frequency (MAF) <0.0001 in gnomAD (Lek et al. 2016). The residues affected by the missense mutations are highly conserved across multiple species with GERP scores greater than 3 (Figure 1D). Furthermore, biallelic nonsense variants in SORD were not present in 4,598 index cases with distinct neurological disorders other than CMT in the GENESIS database.

[0076] The allele carrier frequency of the c.757delG; p.(Ala253GlnfsTer27) variant in the normal population is 0.003% based on an allele count of 94 out of 30,872 in the gnomAD genome (Lek et al. 2016). Notably, the gnomAD exome set detected the c.757delG; p.(Ala253GlnfsTer27) variation at a significantly lower rate with a MAF of 0.00008, likely due to failure to pass the random forest filter. GENESIS uses FreeBayes software for variant calling (Gonzalez et al. 2015), which is consistent with the gnomAD genome-based call set (MAF GENESIS = 0.002, 22 out of 9,196), which may have resulted in an allele frequency closer to the previous estimate. Sanger sequencing of 600 healthy controls (including 200 samples of European origin, 100 samples of Turkish origin, and 200 samples of Middle Eastern origin) identified three heterozygous but no homozygous, c.757delG; p.(Ala253GlnfsTer27) alleles (MAF = 0.0025). These calculations support the predicted prevalence of the homozygous c.757delG; p.(Ala253GlnfsTer27) allele alone of approximately 1 in 100,000 individuals, making it the most common individual pathogenic allele in axonal neuropathies and one of the most common alleles in any Mendelian disease.

[0077] A total of 45 individuals affected by hereditary neuropathy from 38 unrelated families were identified in this study as carrying biallelic mutations in SORD (Figures 11 and 12). Notably, 71% of cases were sporadic, with no evidence of family history or consanguinity. Formal clinical diagnoses were axonal CMT in 51% (n = 16) of cases, distal HMN in 40% (n = 18), and intermediate CMT in 9% (n = 4). The mean age at onset of neuropathy was 17 ± 8 years, and difficulty walking was the most common presenting complaint at onset. Delays in motor developmental milestones were rare, but two-thirds of patients reported foot deformities, indicating that the neuropathy likely began early in life. In the initial study, all individuals had limb weakness, but only half exhibited sensory impairment. Weakness was mild in the distal upper extremities and ranged from mild to nearly complete paralysis in the distal lower extremities. Proximal muscles of the upper and lower extremities were typically unaffected. Seven patients had upper extremity tremors, four had mild scoliosis, and two had mild hearing loss. One case had concurrent, possibly unrelated, symptomatic disorders including dysmorphic features, nonprogressive mental retardation since age 3 years, and spastic ataxia with evidence of cerebellar atrophy on brain MRI. None of the patients had cataracts or other organ involvement. According to the CMT neuropathy score, neuropathy was mild in 67% (n = 30), moderate in 31% (n = 14), and severe in one case. Of the patients, 42% (n = 19) required ankle-foot orthoses to support their feet while walking, one patient required unilateral support, and one patient was wheelchair dependent. Detailed nerve conduction studies were available in 42 patients and always demonstrated motor axonal neuropathy, with moderately slowed nerve conduction velocities in 26% (n = 11) and decreased sensory action potentials in 26% (n = 65).

[0078] Example 2: Evaluation of SORD protein expression in human fibroblasts and SORD levels in blood Sorbitol dehydrogenase is a homotetrameric enzyme of 38 kDa subunits that is widely distributed in mammalian tissues (Johansson et al. 2001; Hellgren et al. 2007; Lindstad, Teigen, and Skjeldal 2013). It represents the second enzyme in the two-step polyol pathway, in which glucose is converted to sorbitol, a relatively unmetabolizable sugar, by the enzyme aldose reductase (AR). Sorbitol is then oxidized to fructose by SORD (Figure 2A). To gain further insight into the functional implications of recessive SORD mutations, SORD expression was assessed in fibroblasts derived from five unrelated affected subjects with homozygous c.757delG; p.(Ala253GlnfsTer27) (n = 4) or c.757delG; p.(Ala253GlnfsTer27) and c.895C>T; p.(Arg299Ter) (n = 1) variants, as well as two unaffected carriers of c.757delG; p.(Ala253GlnfsTer27) in the heterozygous state (Figure 2B). SORD protein was absent in all patients, and wild-type levels were reduced in unaffected carriers compared with controls (Figure 2C). Accordingly, intracellular sorbitol concentrations were more than 10-fold higher in patient fibroblasts compared with controls, consistent with loss of SORD enzyme activity (Figure 2C). Fasting sorbitol levels were determined in serum from 10 patients with the homozygous p.Ala253GlnfsTer27 mutation and 10 unrelated controls and were found to be over 100-fold higher (14.82±0.780 vs. 0.046±0.004 mg / L, p<0.0001), confirming the lack of SORD enzyme activity in the patients (FIG. 13). This study also indicates that sorbitol is a useful marker for detecting or characterizing hereditary neuropathies associated with SORD mutations in mammalian subjects.

[0079] Example 3: Investigation of SORD mutations in models of SORD deficiency To further investigate the pathophysiology of SORD mutations in vivo, a Drosophila melanogaster model of SORD deficiency was established. Drosophila has two functional SORD genes (Sodh1 and Sodh2) that share 90% residue identity (Luque et al. 1998). SORD is conserved across distant lineages, and the proteins encoded by Drosophila Sodh1 (NP_001287203.1) and Sodh2 (NCBI Reference Sequence: NP_524311.1) share 75% and 73% identity, respectively, with the human SORD protein (NCBI Reference Sequence: NP_003095.2 (SEQ ID NO: 46)). Mutant alleles of Sodh2 are expressed when the gene is inserted via a transposon Minos-mediated integration cassette (MiMIC) insertion (Sodh2 MB01265 ) disrupted by the homozygous Sodh2 (Sodh2 MB01265 / MB01265 ) mutants can survive for a normal lifespan. To characterize neurodegenerative phenotypes, we used the Drosophila visual system to exploit the highly organized parallel axons of the compound eye, which allows for in vivo detection of fine neuronal and synaptic pathological changes (Bausenwein, Dittrich, and Fischbach 1992). Outer photoreceptor axons traverse the optic lamina cortex and make synaptic connections with monopolar neurons in the optic lamina layer (Figure 3A). In control flies (yw) at 2 days post-eclosion (DAE), the organized optic lamina cartridge of photoreceptor synapses can be visualized in the xy and xz planes, respectively (Figure 3B). Sodh2 MB01265 / MB01265Loss of photoreceptor terminals in the optic lamina of the mutant was observed at 2 days after eclosion (DAE) (Fig. 3C). The phenotype became progressively more severe at 10 DAE, with vacuoles becoming more numerous, larger in size, and distributed throughout the synaptic optic lamina (Fig. 3C, D). These vacuoles showed loss of neuronal membrane (highlighted by HRP labeling) and reduced Bruchpilot (BRP, synaptic active zone cytomatrix protein) labeling, indicating synaptic degeneration (Fig. 3C, D). To validate the findings described herein, a second SORD model was developed using the pan-neuronal driver elav C155 The loss of both Sodh1 and Sodh2 was generated by specific knockdown of the expression of both Sodh1 and Sodh2 in neurons using homozygous Sodh2 (Sodh2 MB01265 / MB01265 ) exhibited age-dependent synaptic degeneration similar to that observed in SORD-deficient mice (Fig. 6). MB01265 / MB01265 We characterized homozygous flies and found that they exhibited a normal lifespan, but their locomotor activity was significantly impaired at later stages (40 DAE) (Figure 3E, F). This indicated a progressive, age-dependent neuromuscular dysfunction reminiscent of hereditary neuropathies. Furthermore, sorbitol levels were measured in the heads of flies at 10 DAE, and, consistent with observations in patient fibroblasts, Sodh2 expression was significantly impaired. MB01265 / MB01265 We observed a significant increase in the Drosophila model (Figure 4B). In summary, a Drosophila model of SORD deficiency was successfully established and recapitulated representative pathological phenotypes in human patients, including (1) normal lifespan, (2) progressive and age-dependent synaptic degeneration and locomotor deficits, and (3) increased sorbitol levels.

[0080] After establishing the mechanism of action and loss of function as a known enzymatic pathway, treatment options for SORD-associated hereditary neuropathies were investigated. Pharmacological inhibition of aldose reductase (an enzyme upstream of SORD) has been shown to be effective in cellular and animal models of diabetes (Kikkawa et al. 1983; Matsumoto et al. 2008; Ramirez and Borja 2008; Hao et al. 2015; Grewal et al. 2016), and controversially, in humans as well (Chalk, Benstead, and Moore 2007; Polydefkis et al. 2015; Sekiguchi et al. 2016). This has previously been shown to represent a successful strategy to reduce toxic sorbitol accumulation in patients with SORD (2019). The effects of two commercially available aldose reductase inhibitors (ARIs), epalrestat and ranirestat, were tested on intracellular sorbitol accumulation in patient fibroblasts lacking functional SORD. Patient and control fibroblasts were grown for 72 hours in the presence or absence of epalrestat (100 μM) or ranirestat (10 μM), and intracellular sorbitol levels were subsequently measured. Both the ARIs, epalrestat and ranirestat, achieved significant reductions in sorbitol to levels comparable to controls (Figure 4A). Furthermore, a Drosophila model of SORD was fed epalrestat and ranirestat starting at 2 DAE. A significant reduction in sorbitol levels was observed in Sodh2 at 10 DAE. MB01265 / MB01265 This was observed in the fly head (Fig. 4B). MB01265 / MB01265 Locomotor activity in flies with neuron-specific knockdown of both Sodh1 and Sodh2 was rescued to the level of control flies (Figure 4C, Figure 7). Furthermore, epalrestat or ranirestat treatment reduced Sodh2 activity. MB01265 / MB01265We rescued age-dependent synaptic defects in mutant flies. In DMSO vehicle-treated flies, the loss of synaptic terminals was highly pronounced at 40 DAE, when the expansion of adjacent vacuoles resulted in much larger, fused vacuoles containing multiple synaptic cartridges (Figure 4D). Remarkably, epalrestat / ranirestat supplementation reduced the number of vacuoles and restored the localization of the synaptic cytomatrix protein BRP at both 10 and 40 DAE (Figure 4E-G).

[0081] In summary, SORD represents a novel recessive gene causing axonal / intermediate, motor-predominant CMT. Genetic data from cohorts and control databases suggest that the predominant pathogenic variant in SORD, c.757delG; p.(Ala253GlnfsTer27) (carrier frequency approximately 3 / 1,000 individuals in the population), may represent one of the most common specific alleles causing recessive Mendelian disorders. Indeed, its frequency in undiagnosed CMT2 and dHMN cases is approximately 10%, which likely explains a significant proportion of diagnostic gaps in inherited axonal neuropathies. Despite their frequency, it is intriguing that mutations in SORD have not been identified as a cause of CMT by previous studies. The presence of a duplication in the human SORD2P gene may have hindered the detection of a functional SORD variant. This is because available annotation programs are highly dependent on the unique mapping of 150- to 300-bp-long reads generated by current next-generation sequencing technologies. Other known pathogenic variants have previously been shown to be masked by the presence of pseudogenes (De Vos et al. 2004). The pathogenicity of SORD mutations is further supported by in vitro data in patient-derived fibroblasts, which showed the absence of SORD protein and intracellular sorbitol accumulation. Two in vivo Drosophila models recapitulated the human phenotype, with progressive synaptic degeneration and motor deficits, SORD deficiency, and increased sorbitol levels.

[0082] The studies described herein demonstrate that loss of enzyme function and subsequent sorbitol accumulation are the mechanism of action for SORD-associated CMT. Previous studies in cellular and animal models of diabetes have shown that increased polyol influx and intracellular sorbitol accumulation parallel increased cellular osmolality, oxidative stress, and decreased NADPH levels, all of which can have deleterious effects on peripheral nerves (Schmidt et al. 2001; Obrosova 2005; Sango et al. 2006). However, previous studies of adult C57BL / LiA mice expressing reduced levels of SORD protein due to an intron splicing mutation did not identify overt neurological defects (Holmes, Duley, and Hilgers 1982; Lee, Chung, and Chung 1995; Ng et al. 1998). Based on patient clinical data and the late-onset phenotype in flies, it will be important to extend these observations to aging C57BL / LiA mice or generate a complete knockout SORD mouse or rat model. This work further elucidates the central role of the polyol pathway in peripheral nerve metabolism and survival under normoglycemic conditions. While the mechanism by which intracellular sorbitol accumulation may lead to selective degeneration of peripheral nerves is unknown, the observation of increased sorbitol levels in patient-derived cells in this study has promising implications both as an adult marker of disease and as a target for future therapeutic intervention, including methods for substrate reduction, gene replacement or correction, and SORD enzyme replacement. Thus, preclinical studies demonstrating the beneficial effects of substrate reduction via ARI application in human-derived cells and Drosophila models are disclosed herein. Epalrestat is currently marketed in several countries for the treatment of diabetic complications (Grewal et al. 2016). Meanwhile, ranirestat has progressed to later stages of clinical trials (Polydefkis et al. 2015; Sekiguchi et al. 2019).

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[0113] Sekiguchi, et al. 2019. “Aldose Reductase Inhibitor Ranirestat Significantly Improves Nerve Conduction Velocity in Diabetic Polyneuropathy: A Randomized Double-Blind Placebo-Controlled Study in Japan.” Journal of Diabetes Investigation 10 (2): 466-74. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating a hereditary neuropathy in a mammalian subject, said method comprising: (a) detecting the presence of a mutation in the sorbitol dehydrogenase (SORD) gene in a sample from the subject; and (b) administering to the subject a polynucleotide encoding a SORD peptide, an aldose reductase antisense oligonucleotide, an agent that blocks expression of a mutant SORD gene, an agent that corrects the mutation in the SORD gene, or any combination thereof; A method that encompasses (Item 2) 2. The method of claim 1, wherein the method comprises administering a polynucleotide encoding the SORD peptide. (Item 3) 2. The method of claim 1, wherein the method comprises administering an agent that corrects the mutation in the SORD gene, wherein the agent is a CRISPR Cas9 protein and one or more guide RNA molecules. (Item 4) 1. A method of treating a hereditary neuropathy in a mammalian subject, said method comprising: (a) detecting the presence of a mutation in the sorbitol dehydrogenase (SORD) gene in a sample from the subject; and (b) administering to the subject alrestatin, epalrestat, diepalrestat, fidarestat, imirestat, lidrestat, minalrestat, ponalrestat, ranirestat, sulfresin B 11 administering sorbinil, tolrestat, zenarestat, or zopolrestat; A method that encompasses (Item 5) 1. A method of treating a hereditary neuropathy in a mammalian subject, said method comprising: (a) detecting the presence of a mutation in the sorbitol dehydrogenase (SORD) gene in a sample from the subject; and (b) administering to said subject a SORD peptide; A method that encompasses (Item 6) The mutations in the SORD gene are: c.753delG; p.Ala253GlnfsTer27, c.329G>C; p.Arg110Pro, c.298C>T; 6. The method according to any one of items 1 to 5, wherein the nucleotide sequence is p.Arg100Ter, or c.458C>A; p.Ala153Asp. (Item 7) 6. The method of any one of items 1 to 5, wherein the mutation in the SORD gene is c.757delG; p.Ala253GlnfsTer27, c.28C>T; p.Leu10Phe, c.316_425+165del; p.Cys106Ter, c.295C>T; p.Arg299Ter, c.964G>A; p.Val322Ile, or a deletion of individual or multiple coding exons or the entire SORD gene. (Item 8) 8. The method according to any one of items 1 to 7, further comprising measuring sorbitol in a sample derived from the subject. (Item 9) A method for characterizing a neuropathy in a mammalian subject, the method comprising measuring a level of sorbitol in a subject suffering from the neuropathy, wherein a sorbitol level greater than about 10 g / L indicates that the neuropathy is associated with a mutation in the sorbitol dehydrogenase (SORD) gene. (Item 10) 1. A method for assessing the effectiveness of a treatment for a hereditary neuropathy in a subject, the method comprising: administering to the subject an agent selected from the group consisting of an aldose reductase inhibitor, an aldose reductase antisense oligonucleotide, a polynucleotide encoding a SORD peptide, a SORD peptide, an agent that blocks expression of a mutant SORD gene, and an agent that corrects a mutation in a SORD gene, or any combination of the foregoing; and measuring the level of sorbitol in the subject; A method that encompasses

Claims

[Claim 1] The invention described in the specification.

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