Dominant-negative SARM1 molecules as a therapeutic strategy for neurodegenerative diseases or disorders

Abstract

To provide compositions useful as inhibitors of SARM1 activity, and pharmaceutical compositions thereof.SOLUTION: A composition comprises a mutant SARM1 molecule, where the mutant SARM1 polypeptide comprises an amino acid substitution at a specific position compared to the wildtype human SARM1 polypeptide with a specific sequence. A pharmaceutical composition comprises the composition, for treating neurodegenerative or neurological diseases or disorders in a subject in need thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 573,967, filed October 18, 2017, and U.S. Provisional Patent Application No. 62 / 644,090, filed March 16, 2018, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Government Rights This invention was made with Government support under Grant Nos. NS087632 and NS091448 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] FIELD OF THEINVENTION The present disclosure relates generally to various compositions and methods useful for inhibiting SARM1 activity and / or treating neurodegenerative or neurological diseases or disorders. In particular, the present disclosure provides nucleic acids encoding dominant-negative SARM1 polypeptides for use in gene therapy. [Background technology]

[0004] background Axonal degeneration is a hallmark of several neurological disorders, including peripheral neuropathy, traumatic brain injury, and neurodegenerative diseases (Gerdts et al., SARM1 activation triggers axon degeneration locally via NAD(+) destruction. Science 348 2016, pp. 453-457, which are incorporated herein by reference in their entirety). In Parkinson's disease and amyotrophic lateral sclerosis, for example, axonal degeneration is an early event preceding the onset of symptoms and widespread neuronal loss (Kurowska et al., 2017;Fischer et al., Axonal degeneration in motor neuron disease Neurodegener. Dis. 4 2007 pp. 431-442; both of which are incorporated herein by reference in their entirety). Summary of the Invention [Problem to be solved by the invention]

[0005] There is therefore a need for the development of novel therapeutic options for neurodegenerative disorders that target the molecular basis of the axonal degeneration program. [Means for solving the problem]

[0006] overview Among various aspects, the present disclosure provides dominant-negative SARM1 molecules for use as therapeutic intervention for a number of neurological disorders involving axonal degeneration or axonopathy.

[0007] In some embodiments, the present invention provides a nucleic acid encoding a dominant negative SARM1 polypeptide to prevent or ameliorate axonal degeneration, axonopathy, and neurological diseases and disorders associated with axonal degeneration. In some embodiments, a nucleic acid encoding a dominant negative SARM1 polypeptide is provided in a gene therapy vector. In some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a dominant negative SARM1 polypeptide. In some embodiments, the present disclosure provides an NAD + Also provided are compositions comprising and methods of using the AAV vectors to inhibit axonal degeneration, including axonal degeneration resulting from the reduction or depletion of AAV.

[0008] In some embodiments, the present invention provides a method for treating a neuropathy or axonopathy associated with axonal degradation. In some such embodiments, the neuropathy or axonopathy associated with axonal degradation is selected from genetic or congenital neuropathy or axonopathy. In some embodiments, the neuropathy or axonopathy associated with axonal degradation is selected from or associated with peripheral neuropathy, glaucoma, traumatic brain injury, Parkinson's disease, Alzheimer's disease, herpes infection, diabetes, amyotrophic lateral sclerosis, demyelinating disease, ischemia or stroke, chemical injury, burn injury, and AIDS. In some embodiments, the neuropathy or axonopathy associated with axonal degradation is selected from Parkinson's disease or non-Parkinson's disease and Alzheimer's disease.

[0009] It has now been found that the compositions of the present disclosure and pharma- ceutically acceptable compositions comprising the compositions of the present disclosure are effective as potent inhibitors of endogenous SARM1 activity through interaction with the dominant negative SARM1 molecules of the present invention. In some embodiments, the dominant negative SARM1 molecules contain at least one mutation in the region corresponding to amino acids 175-200. In some embodiments, the dominant negative SARM1 molecules contain at least one mutation in the region corresponding to amino acids 650-675. In some embodiments, the dominant negative SARM1 molecules contain at least one mutation in the region corresponding to amino acids 675-700. In some embodiments, the dominant negative SARM1 molecules contain at least one mutation in the region corresponding to amino acids 175-200 and at least one mutation in the region corresponding to amino acids 675-700. In some embodiments, the dominant negative SARM1 molecule comprises at least one mutation in a region corresponding to amino acids 175-200, at least one mutation in a region corresponding to amino acids 650-675, and at least one mutation in a region corresponding to amino acids 675-700. In some embodiments, the dominant negative SARM1 molecule comprises an amino acid substitution at a residue corresponding to 193. In some embodiments, the dominant negative SARM1 molecule comprises an amino acid substitution at a residue corresponding to 685. In some embodiments, the dominant negative SARM1 molecule comprises an amino acid substitution at a residue corresponding to 193, and an amino acid substitution at a residue corresponding to 685. In some embodiments, the dominant negative SARM1 molecule results in a degeneration index of about 0.4 or less at 36 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours or more after axotomy.

[0010] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0012] [Figure 1A]Figure 1A, Figure 1B, Figure 1C, Figure 1D and Figure 1E show the identification of SARM1 dominant-negative transgenes. Figure 1A is a schematic of the domain structure of human SARM1. Individual point mutations are shown in red. Dotted segments indicate deleted regions. Abbreviations: delta-TIR-deletion of amino acids (aa) 1-27 and 560-724; mt-mitochondria-binding sequence; ARM-HEAT / armadillo motif; SAM-sterile alpha motif; TIR-Toll-like interleukin receptor domain. Figure 1B shows that axons of wild-type dorsal root ganglion (DRG) neurons expressing the indicated constructs or axons of SARM1-KO DRG neurons expressing an EGFP-vector were sectioned and imaged, at the indicated time points, using high-throughput automated imaging. AxD was quantified using the degeneration index (DI), which ranges from 0 (fully intact) to 1 (fully fragmented). The mean ± standard error (SEM) of three independent experiments is shown, each showing the mean of 5 wells per condition and experiment. DIs of 10–15 images per well were averaged. Data were tested with two-way ANOVA, which showed significant main effects of group: F(6,14) = 25.57; P < 0.0001; time: F(7,98) = 138, P < 0.0001; and interaction: F(42,98) = 8.809; P < 0.0001; post hoc Dunnett's multiple comparison test ****P = 0.0001; vector vs. delta TIR: **P = 0.0013*P = 0.0122; vector vs. K597E **P = 0.0015). Figure 1C, top: Representative brightfield micrographs of wild-type or SARM1-KO axons expressing the indicated constructs, taken 24 h after axotomy. Bottom row: Mitochondrial potential was monitored using red fluorescent tetramethylrhodamine methyl ester (TMRM) in the same axons as shown in the top row. When mitochondrial membrane potential is lost, the red fluorescent signal is no longer visible. Figure 1D shows that axons of SARM1-KO neurons expressing either enzymatically active wild-type (WT) SARM1 or the constructs indicated were transected and AxD was measured over time. Data are shown as mean ± SEM.Two-way ANOVA shows significant main effects of group F(5,12) = 122.5, P < 0.0001; time (F4,48) = 124, P < 0.001 and interaction F(20,48) = 38.94, P < 0.0001; Dunnett's multiple comparisons; ****P = 0.0001; n = 3 independent experiments; averaged 4 wells per experiment. Figure 1E is a representative brightfield image of SARM1-KO axons expressing the constructs shown in (D) 72 hours after transection. (C,E) Scale bar 50 μm. [Figure 1B] Same as above [Figure 1C] Same as above [Figure 1D] Same as above [Figure 1E] Same as above

[0013] [Figure 2A]Figures 2A, 2B, 2C, 2D, 2E and 2F show that SARM1-CDN potently inhibits wild-type SARM1 function. Figure 2A shows degeneration after transection of wild-type DRG neurons expressing EGFP-vector or SARM1-compound dominant negative (SARM1-CDN) and SARM1 KO neurons expressing EGFP-vector. Degeneration index ranges from 0 (completely intact) to 1 (completely fragmented). Data are shown as mean ± SEM and tested by two-way ANOVA, which shows a significant main effect of group F(2,9)=2710, P<0.0001; time(F8,72)=298.7, P<0.0001 and interaction F(16,72)=154, P<0.0001. Dunnett's multiple comparison test, vector vs. SARM1-CDN and SARM1-KO, ****P=0.0001. Figure 2B and 2C show representative bright field (Figure 2B) and TMRM (Figure 2C) images of axons expressing the constructs shown in (Figure 2A) 96 hours after axonal transection. (Figure 2D) HPLC was used to measure NAD+ levels in wild-type and SARM1-KO neurons expressing EGFP-vector or SARM1-CDN from axonal extracts 4 hours after transection and normalized to baseline (=immediately after transection). One-way ANOVA shows a significant main effect F(2,6)=20.01, P=0.0022; post hoc Tukey's multiple comparison test shows vector vs. SARM1-CDN**P=0.0036, vector vs. SARM1-KO**P=0.0039, SARM1-KO vs. SARM1-CDN P=0.9969. Per condition and experiment, results from 4 wells were averaged from n=3 independent experiments. Figure 2E shows that wild-type DRG neurons and SARM1-KO neurons expressing EGFP-vector or SARM1-CDN were treated with 40 nM vincristine or vehicle, and AxD was determined using the degeneration index. Data are shown as mean ± SEM.Two-way ANOVA shows significant main effects of group F(3,8)=259.6; P<0.0001; time F(5,40)=89.28, P<0.0001; and interaction F(15,40)=38.59; P<0.0001; post-hoc Dunnett's multiple comparison test shows wild-type vector vincristine vs. SARM1-CDN vincristine, SARM1-KO vincristine and SARM1-KO vehicle, ****P=0.0001; n=3 independent experiments with 2-4 wells per condition in each experiment. Figure 2F shows representative brightfield (upper row) and TMRM (lower row) images of the constructs shown in (Figure 2D), 96 hours after vincristine administration. Scale bar: 50 µm. [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 2E] Same as above [Figure 2F] Same as above

[0014] [Figure 3A]Figures 3A, 3B, 3C, 3D and 3E show that SARM1-CDN efficiently transduces DRGs in vivo and protects them from AxD. Figure 3A, top: Schematic of AAV vector expressing human SARM1 mutated at K193 and H685 under the control of the neuron-specific human synapsin promoter (Syn-SARM1-CDN-EGFP). Bottom: Schematic of EGFP-vector (Syn-EGFP) used for control experiments. Figure 3B: Mice at postnatal day 11 or 12 (P11 / 12) were intrathecally injected (6x1011vg) with AAV8-Syn-SARM1-CDN-EGFP or EGFP-vector (AAV8-Syn-EGFP). Five weeks later, the right sciatic nerve was cut and 5 days later tissue was collected for analysis. Figure 3C is a representative micrograph taken in situ of (from left to right) the dorsal root ganglion (asterisk) attached to the spinal cord (SC), the left (uninjured) sciatic nerve (arrow) with its branches (arrowhead), and the intercostal nerve expressing green fluorescent protein (white arrowhead) 5.5 weeks after injection of AAV8-Syn-SARM1-CDN-EGFP in the SC-muscle, scale bar: 2 mm. Figure 3D shows representative confocal images of 6 μm thick sections of the dorsal root ganglion after injection of EGFP-vector (left column; Syn-EGFP) or SARM1-CDN (right column; Syn-SARM1-DN-EGFP). Sections were stained with PGP9.5 (red; dorsal root ganglion neurons) and anti-GFP (green; construct expression) and coverslipped with Vectamount containing DAPI (blue; nuclear marker). Figure 3E shows representative confocal images of 6 μm thick sections of the right (transected) sciatic nerve taken 5 days after transection in mice injected with EGFP-vector (Syn-EGFP; left column) or SARM1-CDN (Syn-SARM1-CDN-EGFP; right column). Sections were stained with antibodies against neurofilament 200 and peripherin (red; axonal marker) and green fluorescent protein (green; construct expression) and mounted with Vectashield containing DAPI (blue; nuclear marker). (Figure 3D, Figure 3E) Scale bar: 50 μm. [Figure 3B] Same as above [Figure 3C] Same as above [Figure 3D] Same as above [Figure 3E] Same as above

[0015] [Figure 4A]Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E, Figure 4F and Figure 4G show that SARM1-CDN protects against AxD in vivo with similar efficacy as SARM1-KO. (Figure 4A, Figure 4B) Representative photomicrographs of toluidine blue stained semi-thin transverse sections of the right sural nerve 5 days after sciatic nerve transection from mice injected with vector (A; n=4) or SARM1-CDN (SARM1-CDN) (B; n=5). (A'B') Enlargement of the rectangular area in (Figure 4A) and (Figure 4B). Arrows and arrowheads indicate lipid-laden histiocytes and myelin debris, respectively. (Fig. 4C,D) Representative electron micrographs of the right sural nerve of an EGFP-vector-injected mouse showing complete loss of internal neural structure (Fig. 4C), but maintenance of unmyelinated (asterisk) and myelinated axons after SARM1-CDN injection (Fig. 4D). (C',D') Enlargement of the rectangular area in (Fig. 4C) and (Fig. 4D). (Fig. 4E,F) All axons in transverse sections of the whole sural nerve were counted in wild-type mice injected with vector (n=4 in E; n=3 in F) or SARM1-CDN (n=5 in E, n=3 in F) or in SARM1-KO mice (n=5 in E and F) 5 (Fig. 4E) and 10 (Fig. 4F) days after transection, and expressed as % of the number of intact contralateral axons. One-way ANOVA shows a significant main effect in (Figure 4E) [F(2,11)=97.13, P<0.0001; post hoc Tukey multiple comparison test shows Vector vs. SARM1-CDN****P<0.0001; Vector vs. SARM1-KO****P<0.0001; SARM1-CDN vs. SARM1-KO P=0.5953] and (Figure 4F) [F(2,8)=20.73; P=0.0007; Tukey multiple comparison test shows Vector vs. SARM1-CDN**P=0.0077; Vector vs. SARM1-KO***P=0.0005; SARM1-CDN vs. SARM1-KO P=0.2543]. (Figure 4G) Representative photomicrograph of toluidine blue stained section of sural nerve 10 days after transection. The bottom row shows a magnification of the area indicated in the top image.Scale bars: (A, B) 50 μm, (A', B') 10 μm, (C, D) 5 μm, (C', D') 1 μm, (G, top) 50 μm, (G, bottom) 10 μm. [Figure 4B] Same as above [Figure 4C] Same as above [Figure 4D] Same as above [Figure 4E-F] Same as above [Figure 4G] Same as above

[0016] [Figure 5A] Figure 5A and Figure 5B. Transduction efficiency of DRG neurons (Figure 5A). Representative photomicrographs of DRG neurons transduced with lentivirus expressing SARM1 dominant-negative mutants tagged with venus (green) and counterstained with Hoechst 33342 to label nuclei (blue). (Figure 5B) Mean ± SEM of Hoechst-positive cells expressing SARM1-dominant negative. More than 100 cells were counted per well, and for each construct, data from at least three independent experiments were averaged. SARM1-CDN = SARM1-combined dominant-negative. [Figure 5B] Same as above

[0017] [Figure 6A]Figure 6A and Figure 6B show a comparison of SARM1 dominant-negative transgenes. (Figure 6A, Figure 6B) Axons of wild-type dorsal root ganglion (DRG) neurons expressing the indicated constructs were transected and imaged at the indicated time points using high-throughput automated imaging. AxD was quantified using the degeneration index (DI), which ranges from 0 (fully intact) to 1 (fully fragmented). Means ± standard error (SEM) of three independent experiments are shown, each showing the mean of 4 wells per condition and experiment. DIs of at least 6 images per well were averaged. (Figure 6A) Data were tested with two-way ANOVA, which showed significant main effects of group: F(4,13) = 22.77; P < 0.0001; time: F(5,65) = 48.59, P < 0.0001; and interaction F(20,65) = 15.31; P < 0.0001; post hoc Tukey's multiple comparisons test showed no statistical differences between H685A and H685Y or between K193R and K193A; vector vs all constructs 12-48 hr: ****P < 0.0001; vector vs K193R and K193A 72 hr: ****P < 0.0001; vector vs H685A 72 hr: **P = 0.0040; vector vs H685Y 72 hr: *P = 0.0225. (Figure 6B) Data were tested with a two-way NAOVA showing a significant main effect of group F(2,6) = 228.8, P < 0.0001, time F(5,30) = 189.9, P < 0.0001 and interaction F(10,30) = 54.53, P < 0.0001. Tukey's multiple comparison test, vector vs. SARM1-K193R / H685A and vs. SARM1-K193R / H194A / H685A****P < 0.0001; SARM1 K193R / H685A vs. SARM1-K193R / H194A / H685A - no statistically significant difference. [Figure 6B] Same as above

[0018] [Figure 7A]Figures 7A, 7B, 7C, 7D, 7E and 7F show no morphometric differences between the non-injured sciatic nerve after injection with AAV-vector and the non-injured sciatic nerve after injection with AAV-SARM1 dominant negative. (Figures 7A-7D): Representative photomicrographs of toluidine blue stained semi-thin transverse sections of the non-injured sciatic nerve of a mouse injected with (Figure 7A, 7C) EGFP vector (AAV-Syn-EGFP) or (Figure 7B, 7D) AAV-Syn-SARM1-CDN-EGFP (AAV-SARM1-CDN-EGFP). Figures 7C and 7D are enlargements of A and B, respectively. Nerves from mice injected with AAV8-Syn-EGFP and AAV8-Syn-SARM1-CDN-EGFP show no significant differences in axon size distribution (Figure 7E) or G-ratio, a measure of axon myelination (Figure 7F). Data were subjected to multiple (Figure 7E) or simple (Figure 7F) t-tests (n=3 per group). Scale bars, 20 μm in A and B; 10 μm in C and D. SARM1-CDN=SARM1 compound dominant negative. [Figure 7B] Same as above [Figure 7C] Same as above [Figure 7D] Same as above [Figure 7E] Same as above [Figure 7F] Same as above

[0019] [Figure 8A]Figure 8A, Figure 8B and Figure 8C show that combining SARM1 point mutations results in a strong dominant negative that prevents injury-induced axon degeneration as potently as SARM1 KO. To study whether combining point mutations increases protection from injury-induced axon degeneration, we generated SARM1 constructs with two or three point mutations. K193R / E642A and H685A / E642A do not prevent axon degeneration to a greater extent than K193R or H685A alone, but expressing SARM1 constructs with point mutations at K193R and H685A prevents axotomy-induced axon degeneration for at least 120 hours and to the same extent as SARM1 KO. Figure 8B and Figure 8C show bright field images of axons expressing the constructs shown in A taken at 24 hours (Figure 8B) and 96 hours (Figure 4C). ****P<0.0001; **p<0.01; error bars indicate SEM. [Figure 8B] Same as above [Figure 8C] Same as above

[0020] [Figure 9A]Figures 9A and 9B show that expression of double and triple mutant forms of SARM1 maintains axonal integrity against the chemotherapy agent vincristine. Figure 9A shows that wild-type (WT) DRG neurons expressing the indicated constructs (vector, K193R / E642A, H685A / E642A, K193R / H194A / H685A, K193R / E642A, H685Y) or SARM1 KO DRG expressing empty vector were incubated with 40 nM vincristine and axonal degeneration was assessed at the indicated time points. In WT DRG expressing empty vector, axons degenerated by 24 hours and were completely fragmented 48 hours after vincristine administration. In contrast, axons of WT DRG expressing double and triple point mutations of SARM1 as well as axons of SARM1 KO neurons are protected from vincristine-induced axonal degeneration for at least 120 hours. Figure 9B shows bright field photographs of the indicated constructs evaluated in A taken 120 hours after vincristine administration. ****P<0.0001; error bars indicate ±SEM. [Figure 9B] Same as above

[0021] [Figure 10] Figure 10 shows that many amino acid substitutions at K193 generate strong dominant-negative SARM1 constructs. SARM1 transgenes were generated with the indicated amino acid substitutions (K193A, K193E, K193Q, K193M, and K193R as above). When expressed in wild-type axons, all the indicated mutant forms are strong dominant-negative and provide long-lasting axonal protection after axotomy. Thus, mutation of K193 to a wide variety of amino acid types generates strong dominant-negative SARM1 transgenes. Note that K193A is particularly strong.

[0022] [Figure 11A]Figure 11A, Figure 11B and Figure 11C show the potency of individual point mutants to act as dominant negatives. Figure 11A, Wild type CD1 DRG neurons were infected with viruses carrying wild type Sarm1 (WT), E189K, H190A or C199S mutations. Five days later, axons were cut and the severity of axon degeneration (ADI) was monitored. E189K and H190A showed significant protection compared to WT infection, while the C199S mutation did not confer any protection. Figure 11B, Wild type CD1 DRG neurons were infected with viruses carrying wild type Sarm1 (WT), R570A mutations. Five days later, axons were cut and the severity of axon degeneration (ADI) was monitored. R570A showed significant protection compared to WT. Figure 11C illustrates the axon degeneration inhibition ability of neurons expressing the indicated SARM1 constructs. Axons expressing SARM1 E569K, D627K, K628D or C629S degenerated as rapidly as wild type at 24 hours, indicating that they do not act as dominant negatives. [Figure 11B] Same as above [Figure 11C] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Reference to various embodiments does not limit the scope of the invention. The figures shown herein are presented for illustrative purposes only and are not intended to be limitations on various embodiments according to the invention.

[0024] Detailed Description The present disclosure generally provides methods and compositions for preventing or ameliorating diseases and disorders caused by axonal degeneration.In particular, provided herein are dominant-negative SARM1 molecules for use as therapeutic interventions for many neurological disorders involving axonal degeneration or axonopathy.

[0025] Axonal degeneration (AxD) is an early potential initiating event in some of the most prevalent neurological disorders, including peripheral neuropathy, traumatic brain injury, Parkinson's disease, and glaucoma (Howell et al., 2007, 2013;Johnson et al., 2013;Cashman and Hoeke, 2015;Tagliaferro and Burke, 2016;Bellucci et al., 2017). Although AxD is central to many neurological disorders, there are currently no treatments that effectively target axonal destruction.

[0026] Considerable progress has been made in elucidating the mechanism of AxD. The discovery of Wallerian delayed degeneration mice, which carry a spontaneous mutation that significantly delays AxD (Lunn et al., 1989), revealed that axons distal to the transection do not degenerate passively, but instead degenerate through the activation of a genetically encoded AxD program. Recently, two unbiased large-scale forward genetic screens, one in invertebrates and one in mammals, independently identified sterile alpha and TIR motif containing protein 1 (SARM1) as the central executioner of this endogenous AxD program (Osterloh et al., 2012; Gerdts et al., 2013). Genetic deletion of SARM1 significantly preserves the integrity of Drosophila olfactory bulb distal axons >50 days after transection and mouse sciatic nerve distal segments >2 weeks after transection. SARM1 is not only necessary but also sufficient for AxD (Gerdts et al., 2015): activation of SARM1 in healthy axons leads to AxD even in the absence of injury, thus making SARM1 a fundamental executer of the AxD program (Gerdts et al., 2016).

[0027] Genetic deletion of SARM1 protects axons from degeneration not only after transection but also in several models of neurological disease, including peripheral neuropathy (Geisler et al., 2016; Turkiew et al., 2017) and traumatic brain injury (Henninger et al., 2016; Ziogas and Koliatsos, 2018). This axonal protection is associated with greatly improved functional outcomes, suggesting that targeting SARM1 is a viable strategy for treating neurological diseases characterized by early AxD. Importantly, SARM1 is expressed primarily in neurons, and SARM1 knockout mice have normal life spans and no obvious behavioral abnormalities, suggesting that targeting SARM1 may be well tolerated. Unfortunately, there are currently no known drugs that inhibit SARM1 activity.

[0028] SARM1 is a multidomain protein consisting of an autoinhibited N-terminus, a tandem SAM domain, and an executer TIR NADase domain that mediates constitutive homomultimerization (Gerdts et al., 2013, 2016;Essuman et al., 2017). Upon injury, release of the N-terminal inhibition activates the intrinsic NADase enzyme, thereby allowing TIR-TIR interactions that cleave the essential metabolic cofactor NAD+ and trigger AxD (Gerdts et al., 2016;Essuman et al., 2017). Because SARM1 exists as a homomultimer, coexpression of mutant SARM1 with wild-type SARM1 can act as a dominant negative that blocks wild-type SARM1 function (Gerdts et al., 2013). SARM1 lacking the TIR domain inhibits wild-type SARM1 function, presumably by disrupting the TIR-TIR interaction that activates the enzyme. Furthermore, we previously identified highly conserved residues in the TIR domain that are required for the release of N-terminal autoinhibition and thus damage-induced activation of SARM1 (Summers et al., 2016). Expression of this SARM1 mutant form (SARM1-K597E) slows AxD in vitro by inhibiting wild-type SARM1 function. Expressing these SARM1 dominant-negative mutant forms in wild-type neurons inhibits pathological AxD, but neither blocks axon loss as effectively as in the absence of SARM1.

[0029] The components used to prepare the disclosed compositions and the compositions themselves used within the methods disclosed herein are disclosed. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it will be understood that each of the various individual and collective combinations, and specific mention of the permutations of these compounds may not be expressly disclosed, but each is specifically contemplated and described herein. For example, when a particular compound is disclosed and described, and several modifications that can be made to some molecules of the compound are described, any and all possible combinations and permutations of the compounds and modifications are specifically contemplated, unless otherwise indicated. Thus, when a class of molecules A, B, and C is disclosed, and a class of molecules D, E, and F and an example of a combination molecule, AD, are disclosed, each is intended to mean that the combinations, AE, AF, BD, BE, BF, CD, CE, and CF, individually and collectively, are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups AE, BF and CE will be considered as disclosed. This concept applies to all aspects of this application, including, but not limited to, the steps in the method of making and using the disclosed compositions. Thus, when there are various additional steps that can be performed, it will be understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the disclosed method.

[0030] Various aspects of the invention are described in further detail in the following sections.

[0031] (I) Composition (a) Dominant negative SARM1 molecule One aspect of the present disclosure provides dominant negative SARM1 polypeptide and the nucleic acid that codes for it.As used herein, the term " dominant negative SARM1 molecule " is used to include both dominant negative SARM1 polypeptide and the nucleic acid that codes for dominant negative SARM1 polypeptide.Exemplary dominant negative polypeptide and coding nucleic acid are described below.

[0032] As used herein, the term "dominant negative SARM1" refers to a mutated SARM1 polypeptide or a nucleic acid encoding the same that can interact with and inhibit the function of wild-type SARM1 polypeptide. In some embodiments, administration of a dominant negative SARM1 molecule inhibits the function of endogenous SARM1, which acts as a central executor of the endogenous AxD program. In particular, the terms "mutant", "mutated" or "mutation", when referring to SARM1, are intended to include any polypeptide or representative thereof (e.g., truncation or fragment) that differs from its corresponding wild-type polypeptide by having at least one amino acid substitution, addition or deletion. In some embodiments, a dominant negative SARM1 comprises one or more amino acid substitutions. In an exemplary embodiment, the amino acid substitution is an arginine substitution.

[0033] As used herein, "SARM1" includes both "SARM1 protein" and "SARM1 analogs". Unless otherwise indicated, "protein" includes proteins, protein domains, polypeptides or peptides and any fragments thereof. Human wild-type SARM1 protein has the amino acid sequence set forth in SEQ ID NO: 1. Homologs can be identified by comparing amino acid sequences, for example, manually or using known homology-based search algorithms, such as those commonly known and referred to as BLAST, FASTA and Smith-Waterman. Local sequence alignment programs, such as BLAST, can be used to search databases of sequences to find similar sequences and summary expectation values ​​(E-values) used to measure sequence-based similarity. Because the protein hit with the highest E-value for a particular organism is not necessarily the ortholog or the only ortholog, reciprocal queries are used in the present invention to screen hit sequences with significant E-values ​​for identification of orthologs. Cross-query requires searching for significant hits against a database of amino acid sequences from reference organisms that are similar to the sequence of the query protein. If the best hit of the cross-query is the query protein itself or a protein encoded by a duplicated gene after speciation, the hit may be an ortholog. In some embodiments, the dominant negative SARM1 is human dominant negative SARM1, mouse dominant negative SARM1, zebrafish dominant negative SARM1, chimpanzee dominant negative SARM1, rhesus dominant negative SARM1, dog dominant negative SARM1, cat dominant negative SARM1, rat dominant negative SARM1, chicken dominant negative SARM1, fruit fly dominant negative SARM1, mosquito dominant negative SARM1, C. elegans dominant negative SARM1 or frog dominant negative SARM1.

[0034] In certain embodiments, the dominant negative SARM1 has at least one mutation in a region corresponding to amino acids 1-25, at least one mutation in a region corresponding to amino acids 25-50, at least one mutation in a region corresponding to amino acids 50-75, at least one mutation in a region corresponding to amino acids 75-100, at least one mutation in a region corresponding to amino acids 100-125, at least one mutation in a region corresponding to amino acids 125-150, at least one mutation in a region corresponding to amino acids 150-175, at least one mutation in a region corresponding to amino acids 175-200, at least one mutation in a region corresponding to amino acids 200-225, at least one mutation in a region corresponding to amino acids 225-250, at least one mutation in a region corresponding to amino acids 250-275, at least one mutation in a region corresponding to amino acids 275-300, at least one mutation in a region corresponding to amino acids 300-325, at least one mutation in a region corresponding to amino acids 325-350, at least one mutation in a region corresponding to amino acids 325-350, at least one mutation in a region corresponding to amino acids 330-340, at least one mutation in a region corresponding to amino acids 340-350, at least one mutation in a region corresponding to amino acids 350-360, at least one mutation in a region corresponding to amino acids 360-370, at least one mutation in a region corresponding to amino acids 370-380, at least one mutation in a region corresponding to amino acids 380-390, at least one mutation in a region corresponding to amino acids 390-400, at least one mutation in a region corresponding to amino acids 400-410, at least one mutation in a region corresponding to amino acids 420-430, at least one mutation in a region corresponding to amino acids 440-450, at least one mutation in a region corresponding at least one mutation in the region corresponding to amino acids 350-375, at least one mutation in the region corresponding to amino acids 375-400, at least one mutation in the region corresponding to amino acids 400-425, at least one mutation in the region corresponding to amino acids 425-450, at least one mutation in the region corresponding to amino acids 450-475, at least one mutation in the region corresponding to amino acids 475-500, at least one mutation in the region corresponding to amino acids 500-525, at least one mutation in the region corresponding to amino acids 525-550, at least one mutation in the region corresponding to amino acids 550-575, at least one mutation in the region corresponding to amino acids 575-600, at least one mutation in the region corresponding to amino acids 600-625, at least one mutation in the region corresponding to amino acids 625-650, at least one mutation in the region corresponding to amino acids 650-675, at least one mutation in the region corresponding to amino acids 675-700,or a combination thereof. In some embodiments, the location of the mutation in the dominant negative SARM1 is determined by sequence alignment with SEQ ID NO:1. In another aspect, the dominant negative SARM1 comprises at least one mutation in a region corresponding to amino acids 625-650, with the amino acid corresponding to 642 not mutated. In one aspect, the dominant negative SARM1 comprises an amino acid substitution at a residue corresponding to 193. In another aspect, the dominant negative SARM1 comprises an amino acid substitution at a residue corresponding to 685. In yet another aspect, the dominant negative SARM1 comprises an amino acid substitution at a residue corresponding to 193, and an amino acid substitution at a residue corresponding to 685. In yet still another aspect, the dominant negative SARM1 consists essentially of an amino acid substitution at a residue corresponding to 193 and an amino acid substitution at a residue corresponding to 685. In another aspect, the dominant negative SARM1 consists of an amino acid substitution at a residue corresponding to 193 and an amino acid substitution at a residue corresponding to 685.

[0035] In some embodiments, the dominant negative SARM1 comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90 or 95% homology to SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 10, 13 or 14. In one embodiment, the dominant negative SARM1 comprises an amino acid sequence having at least about 95, 96, 97, 98 or 99% sequence homology to SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 10, 13 or 14. As used herein, a "homologous" amino acid sequence refers to an amino acid sequence that differs from a reference amino acid sequence only by one or more (e.g., 1, 2, 3, 4 or 5) conservative amino acid substitutions, or one or more (e.g., 1, 2, 3, 4 or 5) non-conservative amino acid substitutions, deletions or additions. A homologous amino acid sequence includes a peptide sequence that is identical or substantially identical to a reference amino acid sequence. "Substantially identical amino acid sequence" means a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to a reference amino acid sequence, and that differs from the reference sequence, if at all, preferably by the majority of conservative amino acid substitutions.

[0036] Conservative amino acid substitutions typically involve substitutions between amino acids of the same class, including, for example, (a) amino acids with uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine, (b) amino acids with basic side chains, such as lysine, arginine, and histidine, (c) amino acids with acidic side chains, such as aspartic acid and glutamic acid, and (d) amino acids with nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

[0037] As used herein, the term "inhibit SARM1 activity" refers to the inhibition, neutralization or reduction of certain activities of endogenous SARM1. These specific attributes include the activity of SARM1 as a central regulator of axonal degradation. Inhibiting SARM1 activity refers to a measurable change in endogenous SARM1 activity in a sample containing a dominant negative SARM1 or a dominant negative SARM1 composition, when compared to an equivalent sample containing endogenous SARM1 activity in the absence of a provided dominant negative SARM1 or a dominant negative SARM1 composition. In some embodiments, a dominant negative SARM1 or a dominant negative SARM1 composition "inhibits" SARM1 activity by inhibiting, slowing or reducing SARM1-mediated axonal degeneration. In some embodiments, SARM1 inhibition results in a reduction in degeneration index, when compared to a control. In some embodiments, the dominant negative SARM1 or dominant negative SARM1 composition provides a degeneration index provided at about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1, about 0.5 or about 0.01. In some embodiments, the dominant negative SARM1 molecule provides a degeneration index of about 0.4 or less at 36 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours or more after axotomy. In some embodiments, the dominant negative SARM1 or dominant negative SARM1 composition "inhibits" SARM1 activity by at least 10%, 20%, 25%, 50%, 75% or more when compared to a control.

[0038] The method for determining the degeneration index resulting from the expression of dominant-negative SARM1 molecules is as follows. For the generation of lentiviral particles containing a SARM1 expression transgene, the following plasmids are transfected into HEK293t cells: pcDNA expressing vesicular stomatitis virus glycoprotein, PspAX2 lentiviral packaging plasmid, and FUGW plasmid containing the SARM1 open reading frame downstream of the human ubiquitin promoter. Two days after transfection, the medium supernatant containing the lentiviral particles is collected and stored at -80 °C until use in primary neuronal cultures.

[0039] For functional analysis of SARM1 dominant-negative molecules, primary embryonic dorsal root ganglion (DRG) neurons are isolated from embryonic day 13.5 mouse embryos. DRG neurons are maintained in neurobasal medium supplemented with L-glutamine, 2% (vol / vol) B27 supplement, 50 ng / mL NGF and 1 μM 5-fluoro-2' deoxyuridine + 1 μM uridine to induce mitotic cell death. DRG neurons are seeded on plates precoated with poly-D-lysine and laminin. At day in vitro (DIV) 1, DRGs were transduced with lentiviruses containing expression transgenes encoding wild-type SARM1 or dominant-negative SARM1 molecules. At DIV 7, axons are cut with a razor blade and distal axons are visualized under a bright-field microscope at the indicated time points after axotomy.

[0040] Axonal degeneration was quantified from brightfield images using an ImageJ macro (Sasaki, 2009) that defines the ratio of the area of ​​fragmented axons to the total area of ​​axons, expressed as the degeneration index (DI). This metric ranges from 0 (fully intact) to 1 (perfectly fragmented), with values ​​above 0.5 corresponding to extensive axonal degeneration. Measure 10 images per well as technical replicates and average 4–6 wells per condition. Perform at least three independent experiments.

[0041] The dominant negative SARM1 polypeptide may optionally include an additional functional domain (e.g., cell-penetrating) or at least one tag to enhance the effectiveness or targeting of the dominant negative molecule to cells. In some embodiments, the dominant negative protein further includes at least one additional domain. In one embodiment, the cell-penetrating domain may be a cell-penetrating peptide sequence derived from HIV-1 TAT protein. In a non-limiting example, the at least one tag is a StrepTag, a polyhistidine tag, an antibody epitope (e.g., derived from myc), or the like, or a combination thereof. The additional domain or at least one tag may be located at the N-terminus, C-terminus, or at an internal position of the protein. The additional domain or tag may be linked to the dominant negative SARM1 by a linker domain, which may include an enzyme cleavage site to release the cell-penetrating domain from the dominant negative SARM1 upon entry into the cell. Preferably, the cleavage enzyme is an enzyme that is abundant in neurons.

[0042] Another aspect of the disclosure provides a nucleic acid encoding any of the above dominant negative molecules. The nucleic acid may be DNA or RNA. In one embodiment, the DNA may be present in a vector. The nucleic acid sequence encoding the dominant negative molecule of the present invention may be operably linked to an expression control sequence. "Operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended manner. An expression control sequence operably linked to a coding sequence is achieved under conditions compatible with the expression control sequence. As used herein, an expression control sequence refers to a nucleic acid sequence that regulates the expression of a nucleic acid sequence to which it is operably linked. An expression control sequence is operably linked to a nucleic acid sequence if the expression control sequence controls and regulates the transcription and optionally the translation of the nucleic acid sequence. Thus, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (i.e., ATG) in front of the gene encoding the protein, splicing signals for introns and to maintain the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The term "control sequences" is intended to include, at a minimum, components whose presence can affect expression, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Expression control sequences can include promoters.

[0043] In some embodiments, the dominant negative SARM1 comprises a nucleic acid sequence having at least 60, 65, 70, 75, 80, 85, 90 or 95% homology to SEQ ID NO: 15. In one embodiment, the dominant negative SARM1 comprises a nucleic acid sequence having at least about 95, 96, 97, 98 or 99% sequence homology to SEQ ID NO:15.

[0044] In one aspect, the present disclosure provides a vector comprising a nucleic acid sequence encoding a dominant negative SARM1 polypeptide. In one aspect, the present disclosure is based at least in part on the ability of an adeno-associated virus (AAV) vector to be safely administered to humans and provide sustained expression of a therapeutic transgene. The present invention provides an adeno-associated virus (AAV) vector comprising, essentially consisting of, or consisting of a nucleic acid sequence encoding a dominant negative SARM1 polypeptide. When an AAV vector essentially consists of a nucleic acid sequence encoding a dominant negative SARM1 polypeptide, it can comprise additional components that do not substantially affect the AAV vector (e.g., genetic elements such as poly(A) sequences or restriction enzyme sites that facilitate the operation of the vector in vitro). When an AAV vector consists of a nucleic acid sequence encoding a dominant negative SARM1 polypeptide, the AAV vector does not comprise any additional components (i.e., components that are not endogenous to AAV and are not required to cause expression of the nucleic acid sequence and thereby provide a dominant negative SARM1).

[0045] Adeno-associated viruses are members of the Parvoviridae family and contain a linear, single-stranded DNA genome of less than about 5,000 nucleotides. AAV requires co-infection with a helper virus (i.e., adenovirus or herpes virus) or expression of a helper gene for efficient replication. AAV vectors used for administration of therapeutic nucleic acids typically have approximately 96% of the parent genome removed, leaving only the terminal repeats (ITRs) that contain recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects due to the expression of viral genes. Furthermore, if necessary, delivery of specific AAV proteins to producer cells allows the integration of AAV vectors containing AAV ITRs into specific regions of the cell genome (see, e.g., U.S. Patent Nos. 6,342,390 and 6,821,511). Host cells containing an integrated AAV genome do not show changes in cell growth or morphology (see, e.g., U.S. Patent No. 4,797,368).

[0046] AAV ITRs are flanked by unique coding nucleotide sequences for the nonstructural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self-complementary and are organized such that energetically stable intramolecular duplexes forming T-shaped hairpins can form. These hairpin structures serve as initiation points for viral DNA replication by acting as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. The Rep78 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication to allow degradation of the AAV termini [see, e.g., Im et al., Cell, 61:447-57 (1990)]. These proteins also regulate transcription from the endogenous AAV promoter and promoters in the helper virus [see, e.g., Pereira et al., J. Virol., 71:1079-1088 (1997)]. Other Rep proteins modify the function of Rep78 and Rep68. The cap gene encodes the capsid proteins, VP1, VP2 and VP3. The cap gene is transcribed from the p40 promoter. In certain embodiments, AAV comprises a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter directing cell-specific expression (e.g., neuronal) operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus (e.g., dominant negative SARM1). Typically, the AAV and B19 coding regions are removed, resulting in a safe, non-cytotoxic vector. The AAV ITRs, or variants thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression.U.S. Patent No. 6,261,834 is incorporated herein by reference in its entirety for material regarding AAV vectors.

[0047] As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype.In non-limiting examples, AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and their mutant forms.AAV vectors may have one or more of AAV wild-type genes, preferably the whole or part of rep and / or cap genes, removed, but retain functional adjacent ITR sequences.Despite high homology, different serotypes have tropism for different tissues.

[0048] AAV vectors, as disclosed herein, can be produced using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or non-human primate tissues [reviewed, for example, in Wu et al., Molecular Therapy, 14(3): 316-327 (2006)]. In general, AAV serotypes have significant homology of genome sequences at the nucleic acid and amino acid sequence levels, so that different serotypes have the same set of genetic functions, essentially produce physically and functionally equivalent virions, and replicate and assemble by virtually identical mechanisms. AAV serotypes 1-6 and 7-9 are defined as "true" serotypes in that they do not cross-react efficiently with neutralizing sera specific for all other extant and characterized serotypes. In contrast, AAV serotypes 6, 10 (also referred to as Rh10) and 11 are considered "variant" serotypes because they do not conform to the definition of a "true" serotype. AAV serotype 2 (AAV2) is widely used in gene therapy applications due to its lack of pathogenicity, broad infectivity and ability to establish long-term transgene expression [see, e.g., Carter, BJ, Hum. Gene Ther., 16: 541 -550 (2005); and Wu et al., supra]. The genomic sequences of various AAV serotypes and comparisons thereof are disclosed, for example, in GenBank accession numbers U89790, J01901, AF043303 and AF085716; Chiorini et al., J. Virol., 71: 6823-33 (1997); Srivastava et al., J. Virol., 45: 555-64 (1983); Chiorini et al., J. Virol., 73: 1309-1319 (1999); Rutledge et al., J. Virol., 72: 309-319 (1998); and Wu et al., J. Virol., 74: 8635-47 (2000).

[0049] The AAV rep and ITR sequences are particularly conserved across most AAV serotypes. For example, the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4 and AAV6 are reportedly about 89-93% identical [see Bantel-Schaal et al., J. Virol., 73(2): 939-947 (1999)]. AAV serotypes 2, 3A, 3B and 6 are reported to have about 82% overall nucleotide sequence identity at the genomic level (Bantel-Schaal et al., supra). Furthermore, the rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally replace) corresponding sequences from other serotypes during production of AAV particles in mammalian cells.

[0050] In general, the cap protein and related cap protein coding sequences that determine the cellular tropicity of AAV particles are not significantly conserved across different AAV serotypes compared to the Rep gene. Given the ability of Rep and ITR sequences to complement the corresponding sequences of other serotypes, AAV vectors can contain a mixture of serotypes, and thus can be "chimeric" or "pseudotyped" AAV vectors. Chimeric AAV vectors typically contain AAV capsid proteins from two or more (e.g., two, three, four, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors contain one or more ITRs of one AAV serotype packaged in the capsid of another AAV serotype. Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Patent No. 6,723,551; Flotte, Mol. Ther., 13(1): 1-2 (2006); Gao et al., J. Virol., 78: 6381-6388 (2004); Gao et al., Proc. Natl. Acad. Sci. USA, 99: 11854-11859 (2002); De et al., Mol. Ther., 13: 67-76 (2006); and Gao et al., Mol. Ther., 13: 77-87 (2006).

[0051] In one embodiment, the AAV vector is generated using an AAV that infects humans (e.g., AAV2). Alternatively, the AAV vector is generated using an AAV that infects non-human primates, such as great apes (e.g., chimpanzees), Old World monkeys (e.g., macaques) and New World monkeys (e.g., marmosets). Preferably, the AAV vector is generated using an AAV that infects non-human primates, pseudotyped with an AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed, for example, in Cearley et al., Molecular Therapy, 13: 528-537 (2006). In one embodiment, an AAV vector can be generated that comprises a capsid protein derived from an AAV that infects rhesus monkeys, pseudotyped with AAV2 inverted terminal repeats (ITRs). In a particularly preferred embodiment, the AAV vector of the present invention comprises a capsid protein from AAV10 (also referred to as "AAVrh.10"), which infects rhesus macaques, pseudotyped with the AAV2 ITRs (see, e.g., Watanabe et al., Gene Ther., 17(8): 1042-1051 (2010); and Mao et al., Hum. Gene Therapy, 22: 1525-1535 (2011)).

[0052] AAV vectors, as disclosed herein, comprise a nucleic acid sequence encoding a dominant negative SARM1 polypeptide. "Nucleic acid sequence" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which may be single-stranded or double-stranded and may contain non-natural or altered nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. The terms include, as equivalents, either analogs of RNA or DNA made of nucleotide analogs, as well as modified polynucleotides, such as, but not limited to, methylated and / or capped polynucleotides.

[0053] In some embodiments, the vector containing the nucleic acid sequence encoding the dominant negative SARM1 can be a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a viral vector or a bacteriophage. The vector can lead to the replication of the dominant negative SARM1 nucleic acid, the expression of the dominant negative SARM1 polypeptide, or the integration of the dominant negative SARM1 nucleic acid into the chromosome of the host cell. The choice of vector depends on the desired purpose. Certain cloning vectors are useful for cloning, mutating and manipulating the dominant negative SARM1 nucleic acid. Other vectors are useful for the expression of the dominant negative SARM1 polypeptide, and can express the polypeptide in large amounts for purification purposes, or express the dominant negative SARM1 polypeptide in a temporal or tissue-specific manner, such as expressing the dominant negative SARM1 only in neurons. The vector can also be selected based on the host cell, for example, to facilitate expression in bacteria, mammalian cells, insect cells, fish cells (e.g., zebrafish) and / or amphibian cells. The choice of vector to be compatible with the host cells will be apparent to one skilled in the art, and host cell types are discussed below. Many vectors or vector systems are commercially available, for example the pET bacterial expression system (Invitrogen (商標) , Carlsbad Calif.).

[0054] The vector disclosed herein can be a viral vector or a non-viral vector. For example, the disclosed vector can be a viral vector. In particular, the disclosed vector can be an adenoviral vector. There are several compositions and methods that can be used to deliver nucleic acid to cells either in vitro or in vivo. These methods and compositions can be broadly divided into two classes: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acid can be delivered by several direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acid, phage nucleic acid, phage, cosmids, or through the transfer of genetic material into cells or carriers, such as cationic liposomes. Suitable means of transfection, including viral vectors, chemical transfectants or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, by Wolff, JA, et al., Science, 247, 1465-1468, (1990); and Wolff, JA Nature, 352, 815-818, (1991). Such methods are well known in the art and can be easily adapted for use with the compositions and methods described herein. In some cases, the methods are modified to work specifically with large DNA molecules. Furthermore, these methods can be used to target certain neurodegenerative diseases or disorders and cell populations using the targeting properties of the carrier.

[0055] A vector can include various components, including, but not limited to, an origin of replication, one or more marker or selection genes (e.g., GFP, neo), promoters, enhancers, terminators, polyadenylation sequences, repressors or activators. Such elements are provided in the vector so as to be operably linked to the coding region of the nucleic acid encoding the dominant negative SARM1, thereby promoting expression in the intended host cell. Cloning and expression vectors can include an origin of replication that allows the vector to replicate in the host cell. A vector can also include a selection marker, for example, to confer resistance to drugs or to complement growth deficiencies. Examples of drug resistance markers include, but are not limited to, ampicillin, tetracycline, neomycin or methotrexate. Examples of other marker genes can be fluorescent polypeptides, such as one of the members of the fluorescent family of proteins, for example, GFP, YFP, BFP, RFP, etc. These markers can be contained on the same vector as the gene of interest or can be on a separate vector and co-transfected with the vector containing the gene of interest.

[0056] The vector can include a promoter suitable for the expression of dominant negative SARM1 in mammalian cells, and the promoter can be operably linked to cause inducible or constitutive expression of the dominant negative SARM1 peptide. Exemplary inducible promoters include, for example, metallothionine promoter or ecdysone-responsive promoter. Exemplary constitutive promoters include, for example, viral promoters from cytomegalovirus (CMV), Rous sarcoma virus (RSV), Simian virus 40 (SV40), avian sarcoma virus, beta-actin promoter and heat shock promoter. The promoter can be selected for its tissue specificity. Some promoters are only expressed in certain tissues, and one of these promoters can be used when it is desired to express the polypeptide of interest only in selected tissues. For example, the synapsin 1 gene promoter was used in a recombinant adenovirus vector system to express a therapeutic protein only in neuronal cells [Kugler et al., Mol Cell Neurosci. (2001) 17(1):78-96]. The choice of promoter will be apparent to one skilled in the art for the desired host cell system.

[0057] The vector encoding dominant negative SARM1 can be a viral vector. Examples of viral vectors include retroviral vectors, such as adenovirus, simian virus 40 (SV40), cytomegalovirus (CMV), Moloney murine leukemia virus (MoMuLv), Rous sarcoma virus (RSV), lentivirus, herpes virus, poxvirus and vaccinia virus. Viral vectors can be used to promote expression in target cells, for example, for the generation of dominant negative SARM1, or for use in therapy (for example, for the purpose of delivering dominant negative SARM1 to patients by expression from vector). When used for therapy, the vector encoding dominant negative SARM1 (for example, viral vector) can be administered directly to patients via a suitable route, or can be administered using an ex vivo strategy that uses the patient's cells (autologous) or allogeneic cells, which are suitable for administration to the patient to be treated.

[0058] As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acid, e.g., a nucleic acid sequence that can code for one or more of the disclosed peptides, into a cell without degradation and contains a promoter that results in the expression of the gene in the cell to which it is delivered. In some embodiments, the nucleic acid sequence disclosed herein is derived from either a virus or a retrovirus. Viral vectors are, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Sindbis and other RNA viruses, including those viruses with an HIV backbone. Also preferred are any virus families that have the properties of these viruses that make them suitable for use as vectors. Retroviruses include mouse Maloney leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors can carry a larger genetic payload, i.e., transgene or marker gene, than other viral vectors, and for this reason are commonly used vectors. However, they are less useful in non-proliferating cells. Adenoviral vectors are relatively stable, easy to handle, have high titers, can be delivered in aerosol formulations, and can transfect non-dividing cells. Poxvirus vectors are large, have several sites for inserting genes, are heat stable, and can be stored at room temperature. As with the above, viral vectors can be formulated in pharmaceutical compositions. In addition, viral vectors can be formulated for direct delivery to the central nervous system, outside the blood / brain barrier, inside the blood / brain barrier, or any combination thereof. Viral vectors can be formulated for administration by intrathecal, intravenous, or intracranial injection. Viral vectors can be in the form of isolated viral particles.

[0059] Retroviral vectors are generally described by Verma, IM, Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. for Microbiology, pp. 229-232, Washington, (1985), which is incorporated herein by reference in its entirety. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO90 / 02806 and WO89 / 07136; and Mulligan, [Science 260:926-932 (1993)]. These teachings are incorporated herein by reference in their entirety for teaching methods for using retroviral vectors for gene therapy.

[0060] Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.In addition, the disclosed nucleic acid sequences can be delivered to target cells by non-nucleic acid-based systems.For example, the disclosed polynucleotides can be delivered by electroporation, or by lipofection, or by calcium phosphate precipitation.The delivery mechanism selected depends in part on the type of cells to be targeted and whether delivery is, for example, in vivo or in vitro.

[0061] Thus, in addition to the disclosed expression vector, the composition can include lipids such as liposomes, for example cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. The liposomes can further include proteins, if necessary, to facilitate targeting to specific cells. The administration of the composition comprising the peptide and cationic liposomes can be administered to the blood, to a target organ, or inhaled into the airways to target cells of the airways. For example, the composition comprising the peptide or nucleic acid sequence described herein and cationic liposomes can be administered to lung cells of a subject. For liposomes, see, for example, Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Patent No. 4,897,355. Additionally, the compound can be administered as a component of a microcapsule that can be targeted to a particular cell type, such as macrophages, or the compound can be administered as a component of a microcapsule where the diffusion of the compound from the microcapsule or delivery of the compound is designed for a particular rate or dosage.

[0062] Host cells modified to cause expression of the dominant negative SARM1 peptide disclosed herein are also contemplated. Such host cells can be modified to cause expression of the dominant negative SARM1 peptide from either episomal or genomically integrated nucleic acid. Such host cells can be generated by any suitable method, for example, electroporation, transfection or transformation with a vector encoding the dominant negative SARM1 peptide. Host cells can be selected according to the desired use (e.g., mammalian cell expression) and can be modified to cause expression of the dominant negative SARM1 according to methods well known in the art. Techniques for introducing vectors into host cells and subsequently culturing host cells are well known in the art.

[0063] A host cell (e.g., a mammalian host cell) suitable for replicating and expressing the vector containing dominant negative SARM1 is provided, and the cell may be stably or transiently transfected and / or stably or transiently express dominant negative SARM1. Such dominant negative SARM1-expressing mammalian cells are used, for example, to produce dominant negative SARM1 polypeptides. The production of dominant negative SARM1 in mammalian cells may result in post-translational modification of dominant negative SARM1 and / or heterologous amino acids that may be fused [e.g., glycosylation, cleavage of signal peptide (if present)]. Furthermore, a mammalian cell line can be selected for use in replicating, packaging, and producing high-titer viral particles that contain the desired dominant negative SARM1 or nucleic acid encoding dominant negative SARM1. Such a virus containing dominant negative SARM1 can then be used to deliver the nucleic acid encoding dominant negative SARM1 and dominant negative SARM1 peptide to a subject in need thereof.

[0064] Exemplary host cells include bacteria, yeast, mammalian cells (e.g., human cells or cell lines), insect cells, and the like. Examples of bacterial host cells include Escherichia coli (E. coli) and other bacteria that can be used to clone, manipulate and generate dominant negative SARM1 nucleic acids or generate dominant negative SARM1 polypeptides. Examples of mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, HEK293 cells, human cervical carcinoma cells (Hela), canine kidney cells (MDCK), human hepatocytes (HepG2), baby hamster kidney cells (BHK) and monkey kidney cells (CV1).

[0065] (b) A composition comprising a dominant-negative SARM1 The present disclosure also provides pharmaceutical compositions. The pharmaceutical compositions include a dominant negative SARM1 as an active ingredient and at least one pharma- ceutically acceptable excipient. In some embodiments, the present disclosure provides compositions that include, consist essentially of, or consist of the AAV vectors described above and a pharma- ceutically acceptable (e.g., physiologically acceptable) carrier. When the composition consists essentially of the AAV vectors of the present invention and a pharma- ceutically acceptable carrier, it may include additional components that do not substantially affect the composition (e.g., auxiliary agents, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.). When the composition consists of the AAV vectors of the present invention and a pharma- ceutically acceptable carrier, the composition does not include any additional components. Any suitable carrier may be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier is determined in part by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition may be appropriately sterilized, except for the AAV vectors described herein. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).

[0066] Suitable formulations for the composition include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers and bacteriostatic agents) and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above. Preferably, the carrier is a buffered saline solution. More preferably, the AAV vector is administered in a composition formulated to protect the AAV vector from damage prior to administration. For example, the composition may be formulated to reduce loss of the AAV vector in the equipment, such as glassware, syringes or needles, used to prepare, store or administer the AAV vector. The composition may be formulated to reduce the light sensitivity and / or temperature sensitivity of the AAV vector. For this purpose, the composition preferably comprises a pharma- ceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.The use of such a composition will extend the shelf life of AAV vector, facilitate administration, and increase the efficiency of the method of the present invention.Formulations for AAV vector-containing compositions are further described, for example, in Wright et al., Curr. Opin. Drug Discov. Devel., 6(2): 174-178 (2003) and Wright et al., Molecular Therapy, 12: 171-178 (2005).

[0067] The composition can also be formulated to enhance transduction efficiency. Moreover, one skilled in the art will recognize that the AAV vector can be present in the composition with other therapeutic or biologically active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the AAV vector. Thus, in addition to the therapies described herein, other therapies known to be effective in treating diseases, disorders, or conditions can also be provided to the subject. In some embodiments, the additional drug or therapeutic agent can be a small molecule, a polypeptide, a nucleic acid, a cell or a portion thereof, an antibody, or the like. In some embodiments, the administration of the dominant negative SARM1 can be administered before, simultaneously with, or after the administration of the additional drug or therapeutic agent.

[0068] The pharma- ceutically acceptable excipient may be a diluent, binder, filler, buffer, pH adjuster, disintegrant, dispersant, preservative, lubricant, taste-masking agent, flavoring agent, or coloring agent. The amounts and types of excipients utilized to form a pharmaceutical composition may be selected in accordance with known principles of pharmacy.

[0069] (i) Diluent In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., mixed esters of acetate and butyrate), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, corn starch, phosphorylated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasive brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), tribasic calcium phosphate, calcium carbonate, and magnesium carbonate.

[0070] (ii) Binder In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

[0071] (iii) Filler In another embodiment, the excipient may be a filler.Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds and polyvinylpyrrolidone.As non-limiting examples, the filler may be dibasic and tribasic calcium sulfate, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol or sorbitol.

[0072] (iv) Buffer In yet another embodiment, the excipient may be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphate, carbonate, citrate, Tris buffers and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

[0073] (v) pH adjuster In various embodiments, the excipient may be a pH adjusting agent. By way of non-limiting example, the pH adjusting agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0074] (vi) disintegrant In further embodiments, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches, such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums, such as agar, guar, carob, karaya, pectin and tragacanth. Suitable non-limiting examples of effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0075] (vii) Dispersants In yet another embodiment, the excipient may be a dispersant or dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0076] (viii) excipients In another alternative embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E or retinyl palmitate, citric acid, sodium citrate; chelating agents, such as EDTA or EGTA; and antimicrobial agents, such as parabens, chlorobutanol or phenol.

[0077] (ix) Lubricants In a further embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals, such as talc or silica; and fats, such as vegetable stearin, magnesium stearate, or stearic acid.

[0078] (x) Flavoring agent In yet another embodiment, the excipient may be a flavoring agent, including cellulose ethers, polyethylene glycols, polyvinyl alcohols, copolymers of polyvinyl alcohol and polyethylene glycol, mono- or triglycerides, acrylic polymers, mixtures of acrylic polymers and cellulose ethers, cellulose acetate phthalates, and combinations thereof.

[0079] (xi) Flavoring agents In an alternative embodiment, the excipient may be a flavoring agent, which may be selected from synthetic flavor oils and flavoring aromatic compounds and / or natural oils extracted from plants, leaves, flowers, fruits, and combinations thereof.

[0080] (xii) Coloring agent In yet further embodiments, the excipient may be a colorant. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C) or topical drug and cosmetic colors (Ext. D&C).

[0081] The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or less, or about 1% or less of the total weight of the composition.

[0082] The agents and compositions described herein can be formulated by any conventional method using one or more pharma- ceutically acceptable carriers or excipients, for example, as described in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of the bioactive agent described herein, which may be present in purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject.

[0083] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, e.g., a human. Thus, a "formulation" can include a pharma- ceutically acceptable excipient, including a diluent or carrier.

[0084] As used herein, the term "pharmaceutical acceptable" may describe a substance or ingredient that does not cause unacceptable loss of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutical acceptable ingredients may be those that have monographs in the United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF") or more recent editions, and those listed in the FDA's continuously updated Inactive Ingredient Search online database. Other useful ingredients that are not listed in USP / NF etc. may also be used.

[0085] As used herein, the term "pharmaceutical acceptable excipient" can include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents or absorption retardants.The use of such media and agents for pharmaceutical active substances is well known in the art [see generally Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)].Except where any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into the composition.

[0086] A "stable" formulation or composition can refer to a composition that has sufficient stability to permit storage at a convenient temperature, e.g., between about 0° C. and about 60° C., for a commercially reasonable period of time, e.g., at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0087] The formulation must be suitable for the mode of administration.The agent useful with the present disclosure can be formulated by known methods for administering to a subject using several routes, including but not limited to parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ocular, buccal and rectal.Each agent can also be administered in combination with one or more additional agents, or with other biologically active or biologically inactive agents.Such biologically active or inactive agents can be in fluid or mechanical communication with the agent, or can be bound to the agent by ionic, covalent, van der Waals, hydrophobic, hydrophilic or other physical forces.

[0088] Controlled release (or sustained release) preparations can be formulated to extend the activity of the drug and reduce the frequency of administration. Controlled release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the drug, and thus affect the occurrence of side effects. Controlled release preparations can be designed to initially release an amount of drug that produces a desired therapeutic effect, and gradually and continuously release another amount of drug to maintain the level of therapeutic effect over an extended period of time. To maintain a near constant level of drug in the body, the drug can be released from the dosage form at a rate that will replace the amount of drug metabolized or excreted from the body. Controlled release of the drug can be stimulated by various inducers, such as changes in pH, changes in temperature, enzymes, water, or other physiological conditions or molecules. The composition can be administered in or on a device that allows for controlled or sustained release, such as a sponge, a biocompatible mesh structure, a mechanical reservoir, or a mechanical implant. Implants (see, e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No. 4,863,457), e.g., implantable devices, e.g., implants or devices comprised of mechanical reservoirs or polymeric compositions, are particularly useful for administering AAV vectors. The compositions can also be administered in the form of sustained release formulations (see, e.g., U.S. Pat. No. 5,378,475), including, e.g., gelfoam, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphates, e.g., bis-2-hydroxyethyl-terephthalate (BHET) and / or polylactic-glycolic acid.

[0089] (c) Administration (i) Dosage form The composition can be formulated into various dosage forms and can be administered by several different means that will deliver a therapeutically effective amount of the active ingredient. Such compositions can be administered orally (e.g., inhalation), parenterally, or topically, optionally in dosage unit formulations that contain conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles. Topical administration can also include transdermal administration, such as the use of transdermal patches or iontophoresis devices. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, or intrasternal injection or infusion techniques. Drug formulations are discussed, for example, in Gennaro, AR, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, HA and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, NY (1980). In certain embodiments, the composition can be a dietary supplement, or the composition can be a cosmetic.

[0090] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets and granules. In such solid dosage forms, the active ingredient is usually combined with one or more pharma- ceutically acceptable excipients, examples of which are detailed above. Oral preparations can also be administered as aqueous suspensions, elixirs or syrups. For these, the active ingredient can be combined with various sweeteners or flavorings, coloring agents, emulsifying and / or suspending agents if necessary, and diluents, such as water, ethanol, glycerin, and combinations thereof. For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0091] For parenteral administration (including subcutaneous, intraocular, intradermal, intravenous, intramuscular, intraarticular and intraperitoneal), the preparation may be an aqueous or oily solution. Aqueous solutions may contain a sterile diluent, such as water, saline, pharma- ceutically acceptable polyol, such as glycerol, propylene glycol or other synthetic solvent; antibacterial and / or antifungal agents, such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate or phosphate; and / or agents for adjusting osmolality, such as sodium chloride, dextrose or polyalcohols, such as mannitol or sorbitol. The pH of aqueous solutions may be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. Oily solution or suspension can further contain sesame oil, peanut oil, olive oil or mineral oil.The composition can be provided in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid, for example water for injection, immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0092] For topical (e.g., transdermal or transmucosal) administration, a penetrant appropriate for the barrier to be permeated is generally included in the preparation. Pharmaceutical compositions suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient can be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eye include eye drops that are dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the mouth include lozenges, pastilles, and mouthwashes. Transmucosal administration can be accomplished using nasal sprays, aerosol sprays, tablets or suppositories, while transdermal administration may be via ointments, salves, gels, patches or creams as generally known in the art.

[0093] In certain embodiments, the composition comprising the dominant negative SARM1 is encapsulated in a suitable vehicle to aid in the delivery of the compound to target cells, to enhance the stability of the composition, or to minimize the potential toxicity of the composition.As those skilled in the art will recognize, a variety of vehicles are suitable for delivering the composition of the present invention.Non-limiting examples of suitable structured fluid delivery systems can include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems.Methods for incorporating compositions into delivery vehicles are known in the art.

[0094] In general, a composition comprising a safe and effective amount of dominant negative SARM1 is, for example, an amount that will cause a desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, a composition comprising an effective amount of dominant negative SARM1 as described herein can substantially inhibit endogenous SARM1 activity and treat related diseases. In some embodiments, an effective amount is an amount that can inhibit, delay or reduce SARM1-mediated axon degeneration. In some embodiments, a dominant negative SARM1 or a dominant negative SARM1 composition produces a degeneration index of about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1, about 0.5 or about 0.01. In some embodiments, a composition comprising a dominant negative SARM1 molecule produces a degeneration index of about 0.4 or less at 36 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours or more after axotomy. In some embodiments, the compositions provided herein contain and / or deliver an amount of dominant-negative SARM1 that, when administered to a subject in an appropriate dosing regimen, is effective to measurably inhibit endogenous SARM1 activity in a subject, and / or to treat a disease or disorder associated with axonal degeneration, and / or to reduce susceptibility, morbidity and mortality associated with a disease or disorder associated with axonal degeneration.

[0095] The amount of the compositions described herein that can be combined with pharma- ceutical acceptable carriers to produce a single dosage form varies according to the recipient treated and the specific mode of administration.It will be understood by those skilled in the art that the unit content of the drug contained in each individual dose of each dosage form does not necessarily constitute a therapeutically effective amount by itself, since the required therapeutically effective amount can be reached by administering several individual doses.

[0096] Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50, and it is generally understood in the art that a larger therapeutic index is optimal.

[0097] The specific therapeutically effective amount level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition used; the duration of treatment; drugs used in combination with or concomitantly with the specific compound used; and factors well known in the medical arts (see, for example, Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start the dose of the composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.If necessary, the effective daily dose can be divided into multiple doses for administration.Thus, a single dose composition can contain such amounts or submultiples thereof to make up a daily dose.However, it should be understood that the total daily use of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0098] Also, each of the conditions, diseases, disorders and conditions described herein, as well as others, can benefit from the compositions and methods described herein. In general, treating a condition, disease, disorder or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted or susceptible to the condition, disease, disorder or condition, but has not yet experienced or exhibited clinical or subclinical symptoms thereof. Treating can also include inhibiting the condition, disease, disorder or condition, e.g., arresting or reducing the occurrence of the disease or at least one clinical or subclinical symptom thereof. Additionally, treating can include alleviating the disease, e.g., causing the regression of the condition, disease, disorder or condition, or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated can be either statistically significant or at least perceptible by the subject or the physician.

[0099] The administration of the composition comprising dominant negative SARM1 can occur as a single event or over a time course of treatment.For example, the composition comprising dominant negative SARM1 can be administered daily, weekly, biweekly or monthly.For the treatment of acute conditions, the time course of treatment is usually at least several days.For certain conditions, the treatment can be extended from several days to several weeks.For example, the treatment can be extended for more than one, two or three weeks.For more chronic conditions, the treatment can be extended for several weeks to several months or even for more than one year.

[0100] (II) Method The present disclosure encompasses a method for measurably inhibiting the activity of endogenously expressed SARM1 polypeptide. The present disclosure provides a method for inhibiting SARM1 activity in vitro, in vivo, in situ or ex vivo. In general, the method comprises administering an effective amount of a dominant negative SARM1 or a composition comprising a dominant negative SARM1 to downregulate the activity of endogenously expressed SARM1 polypeptide. In some embodiments, a dominant negative SARM1 or a composition comprising a dominant negative SARM1 is administered to a biological sample. As used herein, the term "biological sample" includes, but is not limited to, tissue, cell, cell culture or extract thereof; biopsy material obtained from a subject or extract thereof; and blood, saliva, urine, feces, semen, tears or other bodily fluids or extract thereof. In some embodiments, a dominant negative SARM1 or a composition comprising a dominant negative SARM1 is administered to a subject in need thereof. Suitable dominant negative SARM1 molecules or compositions comprising dominant negative SARM1 are those disclosed herein, for example, those described in Section I.

[0101] In one aspect of the disclosure, a method is provided for inhibiting endogenously expressed SARM1 activity, thereby treating a neurodegenerative or neurological disease in a subject in need thereof. In general, the method comprises administering a therapeutically effective amount of a composition comprising a dominant negative SARM1. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a composition comprising an AAV comprising a nucleic acid sequence encoding a dominant negative SARM1. The dominant negative SARM1 molecules described herein are useful for reducing the susceptibility, morbidity and / or mortality associated with various axon degeneration-mediated diseases and disorders. As used herein, the terms "treatment", "treat" and "treating" refer to ameliorating a disease or disorder or one or more symptoms thereof, alleviating a disease or disorder or one or more symptoms thereof, delaying the onset of a disease or disorder or one or more symptoms thereof, or inhibiting the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, the treatment can be administered after one or more symptoms have occurred. In other embodiments, the treatment can be administered in the absence of symptoms. For example, treatment can be administered to susceptible individuals prior to the onset of symptoms (e.g., taking into account a history of the condition and / or taking into account genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, e.g., to prevent or delay recurrence.

[0102] For predisposition to neurodegenerative or neurological disease, dominant negative SARM1 can be administered prophylactically.The effective amount of dominant negative SARM1 that will prevent or slow the progression of neurodegenerative or neurological disease is known as "prophylactically effective amount".The prophylactically effective amount depends on factors such as body weight, age, route of administration and the severity of predisposition.The dosage may be lower than or the same as the effective amount used to treat the diagnosed neurodegenerative or neurological disease.

[0103] In one aspect, administration of a dominant negative SARM1 or a composition comprising a dominant negative SARM1 transiently prevents axonal degeneration in a subject in need thereof. In some embodiments, the dominant negative SARM1 or a composition comprising a dominant negative SARM1 is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more before or after axonal injury or damage. In some embodiments, administration of a dominant negative SARM1 or a composition comprising a dominant negative SARM1 results in about 50% normal fiber content, about 60% normal fiber content, about 70% normal fiber content, about 80% normal fiber content, about 90% normal fiber content, or about 95% or more normal fiber content.

[0104] In some embodiments, the neurodegenerative or neurological disease or disorder is associated with axonal degeneration, axonal injury, axonopathy, demyelinating disease, central pontine myelinolysis, nerve injury disease or disorder, metabolic disease, mitochondrial disease, metabolic axonal degeneration, leukoencephalopathy, or leukodystrophy. In some embodiments, the neurodegenerative or neurological disease or disorder is associated with axonal injury due to diabetic peripheral neuropathy, hereditary neuropathy, acute angle glaucoma, spinal cord injury, stroke, multiple sclerosis, progressive multifocal leukoencephalopathy, congenital hypomyelination, encephalomyelitis, acute disseminated encephalomyelitis, central pontine myelinolysis, or leukodystrophy. myelolysis, osmotic hyponatremia, hypoxic demyelination, ischemic demyelination, adrenoleukodystrophy, Alexander disease, Niemann-Pick disease, Pelizaeus-Merzbacher disease, periventricular leukomalacia, globoid cell leukodystrophy (Krabbe disease), Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Huntington's disease, Alzheimer's disease, Parkinson's disease, Tay-Sachs disease, Gaucher disease, Hurler syndrome, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy (chemotherapy-induced neuropathy; CIPN), neuropathy, acute ischemic optic neuropathy, vitamin B12 deficiency, isolated vitamin E deficiency syndrome syndrome, Bassen-Kornzweig syndrome, glaucoma, Leber's hereditary optic atrophy, Leber's congenital amaurosis, neuromyelitis optica, metachromatic leukodystrophy, acute hemorrhagic leukoencephalitis, trigeminal neuralgia, Bell's palsy, cerebral ischemia, multiple system atrophy, traumatic glaucoma, tropical spastic paraparesis human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy, West Nile virus encephalopathy, La Crosse virus encephalitis, Bunyaviral encephalitis, childhood viral encephalitis, essential tremor, Charcot-Marie-Tooth disease, motor neuron disease, spinal muscular atrophy (SMA), hereditary sensory and autonomic neuropathy (HSAN), adrenomyeloneuropathy, progressive supranuclear palsy (PSP), Friedrich's ataxia, hereditary ataxia, noise-induced hearing loss, and congenital hearing loss.

[0105] According to another embodiment, the present invention provides a composition comprising a dominant negative SARM1 molecule and a pharma- ceutically acceptable carrier, adjuvant or vehicle. In some embodiments, the amount of dominant negative SARM1 provided by the therapeutic composition of the present invention is effective to measurably inhibit endogenous SARM1 activity and / or treat a neurodegenerative or neurological disease in a biological sample or subject. In some embodiments, the compositions provided herein comprise and / or deliver a composition comprising an amount of a dominant negative SARM1 molecule effective to measurably inhibit SARM1 activity in a biological sample. In some embodiments, the compositions provided herein comprise and / or deliver an amount of a dominant negative SARM1 molecule effective to measurably inhibit such SARM1 activity and / or treat a neurodegenerative or neurological disease and / or reduce axonal degradation, morbidity and mortality associated with a neurodegenerative or neurological disease or disorder in a subject when administered to a subject in an appropriate dosing regimen. In certain embodiments, the compositions of the present invention are formulated for administration to a subject in need of such a composition. In some embodiments, a composition of the invention is formulated for injectable administration to a subject.

[0106] The methods described herein are generally performed on a subject in need thereof. The subject may be a rodent, a human, a livestock animal, a companion animal, or a zoo animal. In one embodiment, the subject may be a rodent, such as a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals can include pigs, cows, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals can include pets, such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoo animal. As used herein, "zoo animal" refers to an animal that can be found in a zoo. Such animals can include non-human primates, big cats, wolves, and bears. In a preferred embodiment, the subject is a human.

[0107] (III) Kit Kits are also provided. Such kits may include the agents or compositions described herein and, in certain embodiments, may include instructions for administration. Such kits may facilitate the implementation of the methods described herein. When supplied as a kit, different components of the composition may be packaged in separate containers and may be mixed immediately before use. Components include, but are not limited to, compositions and pharmaceutical formulations comprising the dominant negative SARM1 molecules described herein. Such packaging of components may, if necessary, be provided separately in a pack or dispenser device that may contain one or more unit dosage forms comprising the composition. The pack may, for example, comprise metal or plastic foil, such as a blister pack. Such packaging of components may also allow for long-term storage separately, in some cases, without loss of activity of the components.

[0108] The kit may also contain reagents in separate containers, such as sterile water or saline, which are added to the separately packaged lyophilized active ingredient. For example, a sealed glass ampoule may contain the lyophilized ingredient, and in another ampoule, sterile water, sterile saline, or sterile, each of which is packaged with a neutral non-reactive gas, such as nitrogen. The ampoules may be made of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include bottles made of similar materials as the ampoules and envelopes that may consist of a foil-lined interior, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that, when removed, allows the components to mix. The removable membrane may be glass, plastic, rubber, and the like.

[0109] In certain embodiments, the kit may be supplied with instructional materials. The instructions may be printed on paper or other substrate and / or may be supplied as an electronically readable medium, such as a floppy disk, a mini CD-ROM, a CD-ROM, a DVD-ROM, a Zip disk, a videotape, an audiotape, etc. The detailed instructions need not physically accompany the kit, but instead may direct the user to an internet website designated by the kit manufacturer or distributor.

[0110] The compositions and methods described herein that utilize molecular biology protocols can follow a variety of standard techniques known in the art [e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253].

[0111] definition It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" can include plural referents unless the content clearly dictates otherwise. Furthermore, all units, prefixes, and symbols can be denoted in their SI acceptable form.

[0112] Numerical ranges described herein are inclusive of the numbers that define the range, and include each integer within the defined range. Throughout this disclosure, various aspects of the invention are provided in a range format. It should be understood that the description of range formats is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges, fractions, and individual numbers within that range. For example, description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, as well as decimals and fractions, such as 1.2, 3.8, 1 and 1 / 2, and 4 and 3 / 4. This applies regardless of the breadth of the range.

[0113] As used herein, the term "about" refers to the variation in quantity that may occur through typical measurement techniques and equipment for any quantifiable variable, including, but not limited to, mass, dose, time, distance, wavelength, frequency, voltage, current, and electromagnetic field. Furthermore, given the solid and liquid handling procedures used in the real world, there are certain inadvertent errors and variations that may be due to differences in manufacture, source, or purity of the components used to make the composition or perform the method, etc. For example, the present disclosure provides the following embodiments. [1] A composition comprising a SARM1 molecule, wherein the SARM1 has at least one amino acid change that prevents axonal fragmentation following injury or damage. [2] the SARM1 molecule is a region corresponding to amino acids 1 to 25, a region corresponding to amino acids 25 to 50, a region corresponding to amino acids 50 to 75, a region corresponding to amino acids 75 to 100, a region corresponding to amino acids 100 to 125, a region corresponding to amino acids 125 to 150, a region corresponding to amino acids 150 to 175, a region corresponding to amino acids 175 to 200, a region corresponding to amino acids 200 to 225, a region corresponding to amino acids 225 to 250, a region corresponding to amino acids 250 to 275, a region corresponding to amino acids 275 to 300, a region corresponding to amino acids 300 to 325, a region corresponding to amino acids 325 to 350, a region corresponding to amino acids 350 to 375, a region corresponding to amino acids 375 to 400 2. The composition according to claim 1, comprising at least one mutation in a region corresponding to amino acids 400 to 425, in a region corresponding to amino acids 425 to 450, in a region corresponding to amino acids 450 to 475, in a region corresponding to amino acids 475 to 500, in a region corresponding to amino acids 500 to 525, in a region corresponding to amino acids 525 to 550, in a region corresponding to amino acids 550 to 575, in a region corresponding to amino acids 575 to 600, in a region corresponding to amino acids 600 to 625, in a region corresponding to amino acids 625 to 650, in a region corresponding to amino acids 650 to 675, in a region corresponding to amino acids 675 to 700, in a region corresponding to amino acids 700 to 724, or in a combination thereof. [3] The composition described in 1, wherein the SARM1 molecule contains at least one mutation in the region corresponding to amino acids 175 to 200. [4] The composition described in 1, wherein the SARM1 molecule contains at least one mutation in the region corresponding to amino acids 650 to 675. [5] The composition described in 1, wherein the SARM1 molecule contains at least one mutation in the region corresponding to amino acids 675-700. [6] The composition described in 3, wherein the SARM1 molecule further contains at least one mutation in the region corresponding to amino acids 675-700. [7] The composition described in 6, wherein the SARM1 molecule further contains at least one mutation in the region corresponding to amino acids 650 to 675. [8] The composition of claim 1, 3, 6 or 7, wherein the SARM1 molecule comprises an amino acid substitution at the residue corresponding to 193. [9] The composition of any one of claims 1 to 7, wherein the SARM1 molecule comprises an amino acid substitution at the residue corresponding to 685.

[10] The composition of claim 1, wherein the SARM1 molecule comprises an amino acid substitution at a residue corresponding to 193 and an amino acid substitution at a residue corresponding to 685.

[11] 3. The composition according to claim 2, wherein at least one mutation in the region corresponding to 575-600 is at a residue corresponding to 597.

[12] 3. The composition according to claim 2, wherein at least one mutation in the region corresponding to 550-575 is at a residue corresponding to 570.

[13] 3. The composition according to claim 2, wherein at least one mutation in the region corresponding to 175-200 is at a residue corresponding to 189 or 190.

[14] 14. The composition according to any one of 1 to 13 above, which is a pharmaceutical composition.

[15] A nucleic acid comprising a nucleic acid sequence encoding the SARM1 polypeptide according to any one of items 1 to 7 or 10 to 13.

[16] A vector comprising the nucleic acid according to 15 above.

[17] 17. The vector according to claim 16, which is a viral vector, for example a retrovirus, lentivirus or adenovirus.

[18] 17. The vector according to claim 16, which is an adeno-associated virus.

[19] 19. A pharmaceutical composition comprising the viral vector described in 17 or 18.

[20] A pharmaceutical composition comprising an isolated viral particle having a genome including an open reading frame encoding the dominant negative SARM1 described in 1 to 7 or 10 to 13 above. [twenty one] 21. The pharmaceutical composition according to claim 20, wherein the isolated viral particle is an adeno-associated virus virion. [twenty two] An adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a dominant negative SARM1, wherein the dominant negative SARM1 comprises amino acid substitutions at positions 193 and 685 as determined by sequence alignment with SEQ ID NO:1. [twenty three] 23. The AAV vector according to claim 22, which is produced using a human or non-human adeno-associated virus. [twenty four] A composition comprising the AAV vector described in 22 and a pharma- ceutical acceptable carrier. [twenty five] A method for inhibiting the activity of endogenously expressed SARM1 and / or treating a neurodegenerative or neurological disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a composition described in any one of items 1 to 7, 10 to 13, or 15 to 24.

[26] 26. The method of claim 25, wherein the neurodegenerative or neurological disease or disorder is associated with axonal damage resulting from axonal degeneration, axonal injury, axonopathy, demyelinating disease, central pontine myelinolysis, neuronal injury disease or disorder, metabolic disease, mitochondrial disease, metabolic axonal degeneration, leukoencephalopathy, or leukodystrophy.

[27] Neurodegenerative or neurological diseases or disorders include spinal cord injury, stroke, multiple sclerosis, progressive multifocal leukoencephalopathy, congenital dysmyelination, encephalomyelitis, acute disseminated encephalomyelitis, central pontine myelinolysis, osmotic hyponatremia, hypoxic demyelination, ischemic demyelination, adrenoleukodystrophy, Alexander disease, Niemann-Pick disease, Pelizaeus-Merzbacher disease, periventricular leukomalacia, globoid cell leukodystrophy (Krabbe disease), Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Huntington's disease, Alzheimer's disease, Parkinson's disease, Tay-Sachs disease, Gaucher disease, Hurler syndrome, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy (chemotherapy-induced neuropathy; CIPN), neuropathy, acute ischemic optic neuropathy, vitamin B12 deficiency, isolated vitamin B12 27. The method of claim 26, wherein the inflammatory bowel disease is selected from the group consisting of E deficiency syndrome, Bassen-Kornzweig syndrome, glaucoma, Leber's hereditary optic atrophy, Leber's congenital amaurosis, neuromyelitis optica, metachromatic leukodystrophy, acute hemorrhagic leukoencephalitis, trigeminal neuralgia, Bell's palsy, cerebral ischemia, multiple system atrophy, traumatic glaucoma, tropical spastic paraparesis human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy, West Nile virus encephalopathy, La Crosse virus encephalitis, Bunyaviral encephalitis, childhood viral encephalitis, essential tremor, Charcot-Marie-Tooth disease, motor neuron disease, spinal muscular atrophy (SMA), hereditary sensory and autonomic neuropathy (HSAN), adrenomyeloneuropathy, progressive supranuclear palsy (PSP), Friedreich's ataxia, hereditary ataxia, noise-induced hearing loss, frontotemporal dementia, acute angle glaucoma, and congenital hearing loss.

[28] A method for treating axonal damage in a subject in need thereof, comprising administering to the subject an effective amount of a composition described in any one of items 1 to 7, 10 to 13, or 15 to 24.

[29] A method for inhibiting SARM1 activity, comprising contacting SARM1 with a dominant-negative SARM1 molecule described in any one of 1 to 13 above.

[30] 28. The method of claim 25 or 27, wherein the composition is administered to the subject once during the treatment period.

[31] 28. The method of claim 25 or 27, wherein the composition is administered to the subject two or more times during the treatment period.

[32] 28. The method according to claim 25 or 27, wherein the subject is a human.

[33] 28. The method of claim 25 or 27, wherein the subject is selected from the group consisting of rodents, livestock animals, companion animals or zoo animals.

[34] 33. The method of claim 32, wherein the livestock animal is selected from the group consisting of pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal.

[35] 33. The method of claim 32, wherein the companion animal is selected from the group consisting of dogs, cats, rabbits and birds. EXAMPLES

[0114] The following examples are included to illustrate various embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples illustrate techniques that the inventors have found to work well in implementing the present invention, and therefore may be considered to constitute preferred modes for its implementation. However, those skilled in the art should recognize that in light of the present disclosure, many changes can be made in specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0115] Herein, we introduced point mutations into human SARM1 and expressed this construct in wild-type neurons with the goal of identifying a SARM1 dominant negative that potently inhibits SARM1 function. We unexpectedly found several SARM1 single mutant forms that strongly inhibit AxD in vitro. Surprisingly, combining the best two of these results in a dominant negative that potently inhibits SARM1 enzymatic function and protects axons in a cellular model of axotomy and neuropathy as robustly as SARM1 knockout neurons. Using adeno-associated virus-mediated expression of this optimized construct in adult wild-type mice and sciatic nerve transection as a model of the most severe AxD, we show axonal preservation similar to that observed in SARM1 knockout mice. Thus, a novel strategy is provided to effectively and persistently inhibit SARM1 function in vivo. AAV-mediated expression of SARM1-dominant negative represents a therapeutic option for blocking pathological AxD and improving the functional outcome of neurological disorders and other diseases that may be characterized by acute and chronic axonal degeneration. Unlike conventional gene therapy that aims to treat a single genetic disorder, gene therapy targeting SARM1 has the potential to treat a wide range of diseases that are characterized by a common pathological process, namely axonal loss.

[0116] Example 1 - Identification of dominant-negative SARM1 mutants that inhibit axon degeneration To develop potent SARM1 dominant-negatives, individual point mutations were introduced into highly conserved regions of the N-terminus and TIR domain (Figure 1A) and we tested whether lentivirus-mediated expression of these constructs reduced AxD. All constructs were expressed well in cultured dorsal root ganglion neurons (Figure 5). Axons were cut and cell bodies were removed to avoid axonal regrowth into the injury site. Distal axon fragmentation was assessed over time using high-throughput automated imaging and an automated AxD index (Sasaki et al., 2009). Axon fragmentation begins 6 h after transection and is complete by 24 h (Figure 1B, Figure 1C). Expression of the previously discovered dominant-negatives SARM1-K597E (Summers et al., 2016) and SARM1-deltaTIR (Gerdts et al., 2013) in wild-type DRG neurons delays AxD until 36 hours after axotomy (Figure 1B, Figure 1C). We recently identified glutamate E642 as a key catalytic residue within the active site of TIR NADase (Essuman et al., 2017). To assess whether blocking enzyme function would result in a strong dominant-negative, we expressed SARM1-E642A in wild-type neurons. Although SARM1-E642A is not functional when expressed in SARM1-KO neurons (Essuman et al., 2017), expression of SARM1-E642A in wild-type neurons leads to axon fragmentation with the same kinetics as in wild-type axons expressing EGFP-vector (Figure 1B). Surprisingly, although the SARM1-E642A mutant is not functional, it does not act as a dominant-negative, suggesting that this mutant is unable to disrupt the TIR-TIR interaction that allows activation of wild-type TIR NADase. In contrast, introducing a point mutation at another highly conserved position in the TIR domain, residue H685 (Figure 1A), results in a dominant-negative that potently protects axons for 72 hours after axotomy (Figure 1B, Figure 1C).Mutation of H685 to either Y or A resulted in constructs with indistinguishable dominant-negative potency, suggesting that it is the loss of H, rather than conversion to any particular amino acid, that produces the dominant-negative effect (Figure 6). Therefore, this histidine may be required for TIR-TIR interaction involved in SARM1 activity. The strongest dominant-negative effect was observed when the N-terminal lysine at 193 was mutated (Figure 1A). This lysine is located within a highly conserved region at the N-terminus that we hypothesize may be required for injury-induced activation of SARM1. Expression of SARM1-K193R in wild-type DRG strongly protects axons for 72 hours after axotomy (Figure 1B, Figure 1C). Similar to H685, mutating K193 to either R or A generated constructs with indistinguishable dominant-negative activity, suggesting that it is the loss of the lysine that blocks damage-induced activation of SARM1 (Fig. 6 ).

[0117] As a second indicator of neuronal health, we examined mitochondrial potential in transected axons using the fluorescent mitochondrial membrane indicator TMRM. Activation of SARM1 causes a decrease in mitochondrial membrane potential (Summers et al., 2016), as indicated by the disappearance of red fluorescence. TMRM fluorescence is no longer observed 24 hours after axotomy in wild-type DRG neurons expressing EGFP-vector or SARM1-E642A (Figure 1C). In contrast, TMRM-positive mitochondria are preserved in wild-type DRG expressing a SARM1 dominant-negative mutation and in SARM1-KO DRG (Figure 1C), indicating that morphologically intact transected axons remain metabolically active.

[0118] To assess whether these SARM1 mutants have a pro-degeneration function by themselves, we expressed either wild-type SARM1 or SARM1 mutants in cultured SARM1-KO DRG neurons (FIG. 1A) and assessed AxD after axotomy. Although the axons of SARM1-KO neurons are completely intact for at least 72 hours after axotomy (FIG. 1D, FIG. 1E), reintroduction of enzymatically active wild-type SARM1 promotes rapid AxD after transection (FIG. 1D, FIG. 1E). In contrast, expression of dominant-negative mutants and SARM1-E642A in SARM1-KO DRG does not induce AxD for at least 72 hours after transection (FIG. 1D, FIG. 1E), indicating that the assessed SARM1 mutants do not have pro-degeneration capabilities.

[0119] Example 2 - The combination of the SARM1-K193R and SARM1-H685A mutations blocks wild-type SARM1 enzymatic activity and potently protects against axonal degeneration in cellular models of axotomy and neuropathy. Expression of SARM1-K193R (FIG. 1B, FIG. 1C) and SARM1-H685A (FIG. 6A) strongly delayed AxD after axotomy, but the protection was not as robust as deletion of SARM1 (FIG. 1B, FIG. 1C). Therefore, we evaluated whether a SARM1 molecule with mutations at both K193 and H685 would provide even more potent dominant-negative activity. We generated a construct with both mutations and a third mutation, H194A, which we term SARM1-compound dominant-negative (SARM1-CDN). Subsequent analysis showed that the addition of H194A did not affect the potency of SARM1-CDN (FIG. 6B), but the remaining analysis uses SARM1-CDN because it was generated first and provided such a strong dominant-negative effect. Expression of SARM1-CDN completely prevents AxD (Figure 2A,B) and preserves TMRM-positive mitochondria for at least 96 hours after axotomy (Figure 2C). Upon activation, wild-type SARM1 rapidly degrades NAD+ (Gerdts et al., 2015;Sasaki et al., 2016;Essuman et al., 2017), which leads to local metabolic failure (Gerdts et al., 2015;Yang et al., 2015) and subsequent AxD. To assess whether SARM1-CDN also blocks this molecular activity of SARM1, we measured axonal NAD+ levels in healthy and injured axons. As expected, 4 hours after transection, NAD+ is nearly depleted in wild-type neurons expressing the control vector (Figure 2D). In contrast, NAD+ is preserved in wild-type neurons expressing ARM1-CDN and in SARM1-KO neurons (Figure 2D). Thus, expression of SARM1-CDN in wild-type neurons potently blocks the enzymatic function of SARM1 and its pro-degeneration activity.

[0120] Loss of SARM1 inhibits AxD in models of disease including traumatic brain injury (Henninger et al., 2016;Ziogas and Koliatsos, 2018) and chemotherapy-induced peripheral neuropathy (Geisler et al., 2016;Turkiew et al., 2017). To examine the effect of SARM1-CDN in chemotherapy-induced peripheral neuropathy, we treated wild-type DRGs with vincristine and observed complete fragmentation of axons 48 hours after drug administration (Figure 2E). In contrast, axons of both SARM1-KO neurons and wild-type neurons expressing a dominant-negative mutant form of SARM1 were morphologically intact and retained TMRM-positive mitochondria for at least 96 hours after administration of vincristine (Figure 2E, Figure 2F). Collectively, these data indicate that expression of SARM1-CDN in wild-type neurons potently inhibits SARM1 function in response to diverse insults in vitro.

[0121] Example 3 - SARM1-CDN is strongly expressed in dorsal root ganglion neurons and peripheral nerves upon AAV-mediated gene transfer We next analyzed any axonal protective qualities of SARM1-CDN in vivo. As a proof of concept, we utilized sciatic nerve transection as a model of severe pathological AxD, as we believe it is the most robust test of efficacy. SARM1-CDN fused to EGFP (Figure 3A) or EGFP alone was cloned into an AAV8 vector, which was expressed under the neuron-specific synapsin (Syn) promoter (Figure 3A). Five weeks after intrathecal administration of AAV8-Syn-SARM1-CDN-EGFP (AAV-SARM1-CDN) or control EGFP virus (Figure 3B), robust EGFP labeling of DRG and peripheral nerves, including the sciatic nerve (Figure 3C, Figure 3D) and intercostal nerve (Figure 3C), was observed. DRG was efficiently transduced as judged by staining with the neuronal marker PGP9.5 (Figure 3D).

[0122] Before assessing the role of SARM1-CDN in blocking injury-induced axon degeneration, we first assessed whether expression of AAV-SARM1-CDN induces axonal morphological changes in the absence of injury. Toluidine blue-stained semi-thin transverse sections of uninjured sciatic nerves from mice injected with dominant-negative or control EGFP viruses were analyzed. Axons in sciatic nerves from AAV-SARM1 CDN-injected and AAV-EGFP-injected mice appeared morphologically intact (Figure 7A-D). There were no differences in axonal size distribution (Figure 7E) and G ratio (Figure 7F) between the two treatment groups. Thus, expression of SARM1-CDN has no detectable effect on axons in the absence of injury.

[0123] Example 4 - SARM1-CDN potently protects against axonal degeneration in vivo Five days after sciatic nerve transection, there are almost no axons in the distal nerve segments of mice injected with control virus, as evidenced by the absence of EGFP fluorescence and loss of neurofilament and peripherin staining (Figure 3E). In contrast, EGFP fluorescence and neurofilament and peripherin staining are readily observed in nerves from mice injected with AAV-SARM1-CDN (Figure 3E), indicating that dominant-negative expressing axons are protected from degeneration.

[0124] The severity of axonal loss after nerve transection and the extent of protection afforded by the dominant negative are better appreciated using plastic-embedded thin sections. Five days after transection, signs of extensive Wallerian degeneration, including Schwann cells with myelin debris, lipid-laden histiocytes, and axonal remnants with dark cytoplasm (Fig. 4A, A', C, C'), are evident in transverse sections of the sural nerve, a sensory branch of the sciatic nerve distal to the transection. In contrast, in mice injected with AAV-SARM1-CDN, myelinated axons are very well preserved, with normal shape, myelin thickness, and internal structure (Fig. 4B, B', D, D'). Using electron microscopy, it becomes clear that this protection extends to unmyelinated axons as well (Fig. 4D, D'). When comparing the number of myelinated axons in the injured and non-injured sides, mice injected with EGFP-vector show a reduction of about 99% (ipsilateral 5±0.9 axons / nerve; contralateral 446±57 axons / nerve; n=4 mice; Figure 4E), but no significant axonal loss in mice expressing SARM1-CDN (transection site: 389±13 axons / nerve; contralateral 396±28 axons, n=5 mice, Figure 4E) or in SARM1-KO mice (Figure 4E). Even 10 days after transection, distal axons are present in mice injected with SARM1-CDN (Figure 4G). While no axons were detected in the sural nerve in the vector-treated group at that time point (0.3±0.3 axons; n=3 mice, Figure 4F, G), 160±40 axons per sural nerve were identified in the AAV-SARM1-CDN group (n=3 mice), which is about 59±15% of the number of axons present in the uninjured nerve (Figure 4F). This preservation was comparable to SARM1-KO mice (Figure 4F, G), where 79±9% of axons remained distal to the injury site (Figure 4F, G).

[0125] The above examples establish AAV-mediated neuron-specific expression of SARM1-dominant negative as a powerful tool to potently inhibit AxD in vivo. These findings show that SARM1 acts neuron-autonomously and can be effectively targeted in adult wild-type mice. These findings translate recent dramatic progress in defining the molecular mechanisms of axon degeneration into novel therapeutic strategies to block pathological AxD in neurological disorders.

[0126] SARM1 is a multimer, and mutant SARM1 assembles with wild-type SARM1 (Gerdts et al., 2013). Thus, the relative dominant-negative potency of different SARM1 mutants may provide mechanistic clues to the function of SARM1, although differences in expression and / or localization may also contribute. Mutation of residues K193 and H685 of SARM1 generated highly potent dominant-negative transgenes, indicating that K193 at the N-terminus and H685 in the TIR domain may be essential for SARM1 activation. Interestingly, a mutation in the active site of the TIR domain (E642A) completely suppresses SARM1 function when re-expressed in SARM1 null neurons, but has no dominant-negative effect when expressed in wild-type neurons. This suggests that each enzymatic active site in the complex functions independently. However, our data (Gerdts et al., 2013) and the large literature on other TIR domain proteins (Narayanan and Park, 2015) indicate that TIR dimer interactions are required for TIR domain activation. We suggest that the E642 residue is required for the intrinsic enzymatic activity of the TIR domain, whereas the H685 residue is required for activation of the enzymatic activity in the nearby interacting TIR domain and therefore may act as a dominant negative when mutated. Furthermore, N-terminal residues such as K193 appear to be required for damage-induced activation of SARM1 through the release of autoinhibitory interactions with the TIR domain. As the effects of the H685A and K193R mutations are additive, their inhibitory mechanisms may be different.

[0127] Many common neurodegenerative diseases are characterized by early AxD (Burke and O'Malley, 2013;Johnson et al., 2013;Howell et al., 2013;Yin et al., 2016). Peripheral neuropathy is the most common neurodegenerative disease affecting more than 20 million people in the United States alone. Many neuropathies are caused by degeneration of long axons, and as yet, there are no specific therapies to block this degeneration. Furthermore, in some of the most widespread diseases of the central nervous system, such as Parkinson's disease, traumatic brain injury, and glaucoma, AxD precedes neuronal degeneration (Tagliaferro and Burke, 2016;Caminiti et al., 2017;Fazio et al., 2018;O'Keeffe and Sullivan, 2018). Studies using wallerian degeneration delayed (wlds) mice, which carry a spontaneous mutation that significantly delays AxD (Lunn et al., 1989), and SARM1-KO mice, show that inhibition of the axon destruction program leads to greatly improved functional outcomes (Wang et al., 2001, 2002;Sajadi et al., 2004;Meyer zu Horste et al., 2011;Geisler et al., 2016;Henninger et al., 2016;Fernandes et al., 2018). The active component of the WLDs fusion protein is NMNAT1 (Araki et al., 2004), which inhibits SARM1 (Gilley et al., 2015;Sasaki et al., 2016). However, expression of WLDs is not as effective as genetic deletion of SARM1 in protecting axons and maintaining synapses in aged mice ( Conforti et al., 2014 ; Gilley et al., 2017 ), suggesting that direct targeting of SARM1 would provide better inhibition of pathological AxD.Here, we show that viral delivery of SARM1-K193R / H685A induces long-lasting axonal protection after sciatic nerve transection, the most rapid and aggressive inducer of AxD, providing a template for gene therapy treatment of slow axonal loss in chronic neurodegenerative diseases.

[0128] AAV-mediated gene delivery has been safely used in patients in clinical trials and has shown promising results in neurological diseases, such as spinal muscular atrophy (Mendell et al., 2017;Deverman et al., 2018;Sumner and Crawford, 2018). AAV effectively transduces neurons, is non-pathogenic, and supports long-term sustained expression after a single delivery (Mittermeyer et al., 2012;Hwu et al., 2012). Thus, AAV-mediated SARM1-CDN expression, if proven safe in humans, may be useful in treating chronic neurodegenerative diseases, such as hereditary and idiopathic neuropathies and Parkinson's disease, as well as acquired neuropathies, such as chemotherapy-induced neuropathy. It is tempting to speculate that in the treatment of chemotherapy-induced neuropathy, a single injection of AAV-SARM1-CDN may be sufficient to provide axonal protection for the duration of chemotherapy treatment.

[0129] Example 5 - Efficacy of various SARM1 mutations To study whether combining point mutations increases protection from injury-induced axon degeneration, SARM1 constructs with two or three point mutations were generated. Although K193R / E642A and H685A / E642A do not prevent axon degeneration to a greater extent than K193R or H685A alone, expressing a SARM1 construct with point mutations at K193R and H685A prevents axotomy-induced axon degeneration for at least 120 hours and to the same extent as SARM1 KO. Combining SARM1 point mutations results in a potent dominant negative that prevents injury-induced axon degeneration as potently as SARM1 KO (Figure 8A). To study the efficacy of dominant-negative SARM1 molecules in protecting axonal integrity against chemotherapeutic agents, wild-type (WT) DRG neurons expressing the indicated constructs (vector, K193R / E642A, H685A / E642A, K193R / H194A / H685A, K193R / E642A, H685A) or SARM1 KO DRG expressing empty vector were incubated with 40 nM vincristine and axonal degeneration was assessed at the indicated time points (Figure 9A). In WT DRG expressing empty vector, axons degenerate by 24 h and are completely fragmented by 48 h after vincristine administration. In contrast, axons of WT and SARM1 KO neurons expressing SARM1 double and triple point mutations are protected from vincristine-induced axonal degeneration for at least 120 h. Expression of double and triple mutant forms of SARM1 maintains axonal integrity in response to the chemotherapeutic agent vincristine. Furthermore, many amino acid substitutions at K193 generate potent dominant-negative SARM1 constructs. SARM1 transgenes were generated with the amino acid substitutions shown (K193A, K193E, K193Q, K193M, and K193R as above). When expressed in wild-type axons, all of the mutant forms shown are potent dominant-negative, resulting in long-lasting axonal protection after axotomy. Thus, mutation of K193 to a wide variety of amino acid types generates potent dominant-negative SARM1 transgenes.Note that K193A is particularly potent (Figure 10).

[0130] Mutations near 193 were also tested. Viruses carrying wild-type Sarm1 (WT), E189K, H190A or C199S mutations were infected into wild-type CD1 DRG neurons (Figure 11A). Five days later, axons were cut and the severity of axon degeneration (ADI) was monitored. E189K and H190A showed significant protection compared to WT infection, whereas the C199S mutation did not confer any protection. Viruses carrying wild-type Sarm1 (WT), R570A mutation were infected into wild-type CD1 DRG neurons (Figure 11B). Five days later, axons were cut and the severity of axon degeneration (ADI) was monitored. R570A showed significant protection compared to WT. Finally, neurons expressing SARM1 E569K, D627K, K628D or C629S constructs were tested for their ability to inhibit axon degeneration. Axons expressing SARM1 E569K, D627K, K628D or C629S degenerated as rapidly as wild type at 24 hours, indicating that they do not act as dominant negatives (FIG. 11C).

[0131] Materials and Methods for the Examples All procedures were performed in accordance with the guidelines mandated by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Washington University of Saint Louis Medical School Animal Care and Use Committee (protocols #20170030 and 20150043). This manuscript was prepared in compliance with the ARRIVE guidelines. Pregnant C57BI / 6NTac mice were purchased from Taconic (Rensselaer, NY), and pregnant CD1 mice were purchased from Charles River (Wilmington, MA). SARM1 knockout mice were a gift from Marco Colonna (Szretter et al., 2009) and bred in our colony. Male and female mice were used for all experiments. Chemicals were purchased from Sigma Aldrich (Saint Louis, MO) unless otherwise indicated.

[0132] Dorsal root ganglion culture Dorsal root ganglia (DRG) were dissociated from embryonic day 13.5 (E13.5) or E14.5 CD-1 (Charles River) or from E13.5 SARM1 knockout mouse embryos in Dulbecco's modified Eagle's medium. DRG neurons were dissociated in 0.05% trypsin-EDTA at 37°C, resuspended in Neurobasal medium (Invitrogen) containing 2% B27 (Invitrogen), 50 ng / mL nerve growth factor (Harlan Laboratories), 1 μM 5-fluoro-2-deoxyuridine and 1 μM uridine, and plated in 48-well plates coated with poly-D-lysine and laminin. The next day (DIV 1), concentrated lentivirus was added at a final dilution of 50-fold. All experiments with direct comparisons were performed on the same plate to minimize variability.

[0133] Lentiviral constructs and infection Mammalian expression constructs were derived from FCIV lentiviral vectors (Araki et al., 2004) containing a ubiquitin promoter and a Venus marker. Venus-tagged SARM1 fusion proteins were generated by inserting SARM1 cDNA in frame with Venus using an Ala-Thr-Thr linker between the C-terminus of SARM1 and Venus. SARM1 mutant constructs were generated by megaprime PCR method. SARM1 deletion mutants (delta TIR) lacking residues 2–27 and 561–724 were subcloned into FCIV using the InFusion system. Control vectors contained enhanced green fluorescent protein (EGFP) under the control of the venus or ubiquitin promoter (EGFP vector). Successful insertion of clones was confirmed by sequencing. Lentiviral particles were generated by co-transfection of the lentiviral expression vector FCIV with the lentiviral packaging plasmids psPAX2 and vesicular stomatitis virus glycoprotein into HEK293T cells as described previously (Araki et al., 2004). Lentiviral particles were collected 48 h post-transfection and concentrated to a final concentration of 1–10 × 107 infectious particles / ml using a Lenti-X concentrator (Clontech). Lentiviral expression of the constructs was confirmed by positive fluorescent signals in the cell bodies and ranged from 92.2 ± 7.6% (SARM1-deltaTIR) to 98.8 ± 0.7% (SARM1-E642A; Figure 6) of infected DRGs.

[0134] Axotomy and vincristine DRG neurons were cultured in 48-well microtiter plates and cell bodies were isolated to allow imaging of axons by automated microscopy. At DIV 8, DRG axons were manually cut near the somae with a 3 mm wide flat blade under microscope guidance. Cell bodies were removed to prevent axonal regeneration. Vincristine (40 nM in DMSO) or vehicle (DMSO) was added at DIV 8 and left in the wells until the end of the experiment.

[0135] Imaging and quantification of axonal degeneration Fifteen to twenty bright-field images per well of live axons were acquired at the indicated time points using an Operetta high-content imaging system (PerkinElmer) with a 20× objective. Using an ImageJ-based script (Sasaki et al., 2009), axonal degeneration was quantified based on axonal morphology and reported as a “degeneration index” (DI), ranging from 0 (fully intact) to 1 (fully fragmented). Values ​​above 0.5 indicate extensive axonal degeneration. All images were inspected closely and fields with no axons or inadequate imaging were excluded. Ten to fifteen images per well were measured as technical replicates. All experiments were performed at least three times and three to five wells per condition were averaged for each experiment. To assess mitochondrial potential, tetramethylrhodamine methyl ester (TMRM; ThermoFisher Scientific) was added and axons were imaged 30 min later using an inverted Olympus CKX41 microscope and a Nikon DS-QiIMC camera. Exposure times were kept constant between constructs. Bright-field images were obtained with phase contrast using the same microscope and camera.

[0136] Quantification of NAD+ by HPLC CD1 E13.5 DRG and SARM1 KO E13.5 DRG were plated as spot cultures in 24-well plates coated with poly-D-lysine and laminin. Neurons were transduced with concentrated viruses expressing Ubquitin-EGFP or Ubquitin-SARM1-CDN-Venus on DIV 1. On DIV 6, axons were cut and cell bodies removed as described. Immediately (=0 hours time point) or 4 hours after axotomy, axons were washed with 0.9% cold saline and lysed by addition of 0.5 M perchloric acid. Extracts were centrifuged and supernatants were collected, neutralized with 3 M K2CO3, and diluted in potassium phosphate buffer. NAD+ was assayed by HPLC on an LC-18T HPLC column (Supelco) at a flow rate of 1 ml / min. Elution peaks were matched to NAD+ standards. Four wells were averaged per condition, and three independent experiments were performed.

[0137] AAV constructs and virus injections An AAV vector expressing EGFP under the control of the human synapsin promoter was obtained from Addgene (a gift from Bryan Roth; Addgene #50465) and used as an EGFP-vector control (Addgene virus prep #50465-AAV8). pAAV-hSyn-EGFP (Addgene #50465) was cut with BamHI and NcoI, and SARM1-K193R / H194A / H685A (SARM1-CDN) was inserted between the synapsin promoter and EGFP sequences using the inFusion (Clontech) system. AAV8-hSYN-SARM1-CDN-EGFP was generated by the viral vector core of the Hope Center for Neurological Diseases at Washington University Saint Louis. Viral particles were purified by iodixanol gradient ultracentrifugation, and viral titers were measured by dot blot. Under light anesthesia with avertin, 6x1011 viral genomes (vg) were injected intrathecally at L6 / S1 into male (n=7) and female (n=10) C57BI / 6 mice (Taconic) at postnatal day 11 or 12. Two female mice injected with EGFP-vector subsequently died, whereas none of the mice injected with the SARM1 dominant-negative construct died.

[0138] Sciatic nerve transection and tissue collection Five weeks after virus injection, one group of mice (n=9) was anesthetized with isoflurane and the right sciatic nerve was transected, with the nerve ends bent away from each other to prevent reconnection. Five days after nerve transection, mice were anesthetized with avertin, and the sciatic and sural nerves were transected and then transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline. Spinal cords and dorsal root ganglia were transected, cryoprotected overnight in 30% sucrose, and frozen in OCT (Tissue Tec) in liquid 2-methylbutane cooled on dry ice. Eight weeks after virus injection, the sciatic nerve of a second group of mice (n=6) was transected, and tissue was collected 10 days later, as described above. For comparison, age-matched (11 weeks) SARM1 knockout mice (n=10; 5 males / 5 females) underwent sciatic nerve transection and the sural nerves were dissected and removed 5 days (n=5 mice; 2 females / 3 males) or 10 days (n=5 mice, 3 females / 2 males) after transection.

[0139] Toluidine blue staining and axon quantification Sural and sciatic nerves were fixed overnight by immersion in freshly prepared 3% glutaraldehyde in 0.1 M PBS and processed as recently described (Geisler et al., 2016). Transverse sections (400 nm thick) were cut using a Leica EM UC7 ultramicrotome, which were stained with 1% toluidine blue (Fisher Scientific). Sural nerve sections were imaged using a 63× oil immersion objective on a Leica DMI 4000B microscope equipped with a Leica DFC 7000-T camera. Photomicrographs were stitched using Leica software, and all axons per transverse section were counted with ImageJ. To determine the axon size distribution and G-ratio of the sciatic nerve, four non-overlapping areas per transverse section were imaged using a 100× oil objective on a Zeiss Axioskop and photographed with a Hitachi camera. Photographs were analyzed using a customized semi-automated binary image analysis method (Hunter et al., 2007). Three nerves were analyzed per treatment group, and results from four fields per nerve were averaged. All analyses were performed by a blinded observer.

[0140] Electron microscope Selected blocks of the sural nerve were used to cut 90-nm-thin sections, which were stained with uranyl acetate and lead citrate and viewed under a JEOL JEM 1400 TEM.

[0141] immunohistochemistry Six-μm-thick sections of dorsal root ganglia and sciatic nerves were cut on a cryostat (Leica CM1860), mounted on slides, and processed as recently described (Geisler et al., 2016). Experiments with direct comparisons between groups were performed in parallel to minimize variability. Primary antibodies included rabbit anti-protein gene product 9.5 (1:1000, EMD Millipore#AB1761-1-I), rabbit anti-peripherin (1:250, EMD Millipore#AB1530), rabbit anti-neurofilament 200 (1:1000, Sigma Aldrich#N4142) and mouse anti-green fluorescent protein conjugated to alexa-fluor488 (1:250, ThermoFisherScientific#A-21311). The secondary antibody was Alexa Fluor594-conjugated goat anti-rabbit (Invitrogen) at 1:500 dilution. Sections were coverslipped with Vectashield (Vector laboratories) containing DAPI to allow visualization of nuclei. Sciatic nerves and DRGs were imaged in confocal mode on a Leica DMI 4000B using 40× and 20× immersion oil objectives, respectively. PGP9.5- and GFP-positive DRG neurons were counted in one stitched confocal slice of the whole DRG in Image J. One section each of two different DRGs per animal was counted and values ​​were averaged. After injection of AAV8-Syn-SARM1-CDN-EGFP, 85±0.4% of PGP9.5-positive DRG neurons expressed GFP.

[0142] statistical analysis Data are reported as mean ± standard error of the mean (SEM) unless otherwise stated. Between-group comparisons were performed with one-way or two-way ANOVA, as appropriate. Two-tailed significance tests were used throughout, and P<0.05 was considered statistically significant. All statistics were calculated using Prism software.

[0143] References Araki, T., Y. Sasaki, and J. Milbrandt. 2004. Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science. 305:1010-1013. doi:10.1126 / science.1098014. Bellucci, A., A. Antonini, M. Pizzi, and P. Spano. 2017. The End Is the Beginning: Parkinson's Disease in the Light of Brain Imaging. Front Aging Neurosci. 9:330. doi:10.3389 / fnagi.2017.00330. Burke, RE, and K. O'Malley. 2013. Axon degeneration in Parkinson's disease. Exp. Neurol. 246:72-83. doi:10.1016 / j.expneurol.2012.01.011. Caminiti, S.P., L. Presotto, D. Baroncini, V. Garibotto, R.M. Moresco, L. Gianolli, M.A. Volonte, A. Antonini, and D. Perani. 2017. Axonal damage and loss of connectivity in nigrostriatal and mesolimbic dopamine pathways in early Parkinson's disease. Neuroimage Clin. 14:734-740. doi:10.1016 / j.nicl.2017.03.011. Cashman, C.R., and A. Hoke. 2015. Mechanisms of distal axonal degeneration in peripheral neuropathies. Neurosci. Lett. 596:33-50. doi:10.1016 / j.neulet.2015.01.048. Conforti, L., J. Gilley, and M.P. Coleman. 2014. Wallerian degeneration: an emerging axon death pathway linking injury and disease. Nature Reviews Neuroscience. 15:394-409. doi:10.1038 / nrn3680. Deverman, B.E., B.M. Ravina, K.S. Bankiewicz, S.M. Paul, and D.W.Y. Sah. 2018. Gene therapy for neurological disorders: progress and prospects. Nat Rev Drug Discov. doi:10.1038 / nrd.2018.110. Essuman, K., D.W. Summers, Y. Sasaki, X. Mao, A. DiAntonio, and J. Milbrandt. 2017. The SARM1 Toll / Interleukin-1 Receptor Domain Possesses Intrinsic NAD+ Cleavage Activity that Promotes Pathological Axonal Degeneration. Neuron. 93:1334-1343.e5. doi:10.1016 / j.neuron.2017.02.022. Fazio, P., P. Svenningsson, Z. Cselenyi, C. Halldin, L. Farde, and A. Varrone. 2018. Nigrostriatal dopamine transporter availability in early Parkinson's disease. Movement Disorders. 33:592-599. doi:10.1002 / mds.27316. Fernandes, K.A., K.L. Mitchell, A. Patel, O.J. Marola, P. Shrager, D.J. Zack, R.T. Libby, and D.S. Welsbie. 2018. Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury. Exp. Eye Res. 171:54-61. doi:10.1016 / j.exer.2018.03.007. Geisler, S., R.A. Doan, A. Strickland, X. Huang, J. Milbrandt, and A. DiAntonio. 2016. Prevention of vincristine-induced peripheral neuropathy by genetic deletion of SARM1 in mice. Brain. 139:3092-3108. doi:10.1093 / brain / aww251. Gerdts, J., E.J. Brace, Y. Sasaki, A. DiAntonio, and J. Milbrandt. 2015. SARM1 activation triggers axon degeneration locally via NAD+ destruction. Science. 348:453-457. doi:10.1126 / science.1258366. Gerdts, J., D.W. Summers, J. Milbrandt, and A. DiAntonio. 2016. Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism. Neuron. 89:449-460. doi:10.1016 / j.neuron.2015.12.023. Gerdts, J., D.W. Summers, Y. Sasaki, A. DiAntonio, and J. Milbrandt. 2013. Sarm1-mediated axon degeneration requires both SAM and TIR interactions. J. Neurosci. 33:13569-13580. doi:10.1523 / JNEUROSCI.1197-13.2013. Gilley, J., G. Orsomando, I. Nascimento-Ferreira, and M.P. Coleman. 2015. Absence of SARM1 rescues development and survival of NMNAT2-deficient axons. Cell Rep. 10:1974-1981. doi:10.1016 / j.celrep.2015.02.060. Gilley, J., R.R. Ribchester, and M.P. Coleman. 2017. Sarm1 Deletion, but Not WldS, Confers Lifelong Rescue in a Mouse Model of Severe Axonopathy. Cell Rep. 21:10-16. doi:10.1016 / j.celrep.2017.09.027. Henninger, N., J. Bouley, E.M. Sikoglu, J. An, C.M. Moore, J.A. King, R. Bowser, M.R. Freeman, and R.H. Brown. 2016. Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1. Brain. 139:1094-1105. doi:10.1093 / brain / aww001. Howell, G.R., R.T. Libby, T.C. Jakobs, R.S. Smith, F.C. Phalan, J.W. Barter, J.M. Barbay, J.K. Marchant, N. Mahesh, V. Porciatti, A.V. Whitmore, R.H. Masland, and S.W.M. John. 2007. Axons of retinal ganglion cells are insulted in the optic nerve early in DBA / 2J glaucoma. J. Cell Biol. 179:1523-1537. doi:10.1083 / jcb.200706181. Howell, G.R., I. Soto, R.T. Libby, and S.W.M. John. 2013. Intrinsic axonal degeneration pathways are critical for glaucomatous damage. Exp. Neurol. 246:54-61. doi:10.1016 / j.expneurol.2012.01.014. Hunter, D.A., A. Moradzadeh, E.L. Whitlock, M.J. Brenner, T.M. Myckatyn, C.H. Wei, T.H.H. Tung, and S.E. Mackinnon. 2007. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. Journal of Neuroscience Methods. 166:116-124. doi:10.1016 / j.jneumeth.2007.06.018. Hwu, W.-L., S. Muramatsu, S.-H. Tseng, K.-Y. Tzen, N.-C. Lee, Y.-H. Chien, R.O. Snyder, B.J. Byrne, C.-H. Tai, and R.-M. Wu. 2012. Gene therapy for aromatic L-amino acid decarboxylase deficiency. Sci Transl Med. 4:134ra61. doi:10.1126 / scitranslmed.3003640. Johnson, V.E., W. Stewart, and D.H. Smith. 2013. Axonal pathology in traumatic brain injury. Exp. Neurol. 246:35-43. doi:10.1016 / j.expneurol.2012.01.013. Lunn, E.R., V.H. Perry, M.C. Brown, H. Rosen, and S. Gordon. 1989. Absence of Wallerian Degeneration does not Hinder Regeneration in Peripheral Nerve. Eur. J. Neurosci. 1:27-33. Mendell, J.R., S. Al-Zaidy, R. Shell, W.D. Arnold, L.R. Rodino-Klapac, T.W. Prior, L. Lowes, L. Alfano, K. Berry, K. Church, J.T. Kissel, S. Nagendran, J. L'Italien, D.M. Sproule, C. Wells, J.A. Cardenas, M.D. Heitzer, A. Kaspar, S. Corcoran, L. Braun, S. Likhite, C. Miranda, K. Meyer, K.D. Foust, A.H.M. Burghes, and B.K. Kaspar. 2017. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 377:1713-1722. doi:10.1056 / NEJMoa1706198. Meyer zu Horste, G., T.A. Miesbach, J.I. Muller, R. Fledrich, R.M. Stassart, B.C. Kieseier, M.P. Coleman, and M.W. Sereda. 2011. The Wlds transgene reduces axon loss in a Charcot-Marie-Tooth disease 1A rat model and nicotinamide delays posttraumatic axonal degeneration. Neurobiol. Dis. 42:1-8. doi:10.1016 / j.nbd.2010.12.006. Mittermeyer, G., C.W. Christine, K.H. Rosenbluth, S.L. Baker, P. Starr, P. Larson, P.L. Kaplan, J. Forsayeth, M.J. Aminoff, and K.S. Bankiewicz. 2012. Long-Term Evaluation of a Phase 1 Study of AADC Gene Therapy for Parkinson's Disease. Hum Gene Ther. 23:377-381. doi:10.1089 / hum.2011.220. Narayanan, K.B., and H.H. Park. 2015. Toll / interleukin-1 receptor (TIR) domainmediated cellular signaling pathways. Apoptosis. 20:196-209. doi:10.1007 / s10495-014-1073-1. O'Keeffe, G.W., and A.M. Sullivan. 2018. Evidence for dopaminergic axonal degeneration as an early pathological process in Parkinson's disease. Parkinsonism Relat. Disord. doi:10.1016 / j.parkreldis.2018.06.025. Osterloh, J.M., J. Yang, T.M. Rooney, A.N. Fox, R. Adalbert, E.H. Powell, A.E. Sheehan, M.A. Avery, R. Hackett, M.A. Logan, J.M. MacDonald, J.S. Ziegenfuss, S. Milde, Y.-J. Hou, C. Nathan, A. Ding, R.H. Brown, L. Conforti, M. Coleman, M. Tessier-Lavigne, S. Zuchner, and M.R. Freeman. 2012. dSarm / Sarm1 is required for activation of an injury-induced axon death pathway. Science. 337:481-484. doi:10.1126 / science.1223899. Sajadi, A., B.L. Schneider, and P. Aebischer. 2004. Wlds-mediated protection of dopaminergic fibers in an animal model of Parkinson disease. Curr. Biol. 14:326-330. doi:10.1016 / j.cub.2004.01.053. Sasaki, Y., T. Nakagawa, X. Mao, A. DiAntonio, and J. Milbrandt. 2016. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion. Elife. 5. doi:10.7554 / eLife.19749. Sasaki, Y., B.P.S. Vohra, F.E. Lund, and J. Milbrandt. 2009. Nicotinamide Mononucleotide Adenylyl Transferase-Mediated Axonal Protection Requires Enzymatic Activity But Not Increased Levels of Neuronal Nicotinamide Adenine Dinucleotide. J. Neurosci. 29:5525-5535. doi:10.1523 / JNEUROSCI.5469-08.2009. Summers, D.W., D.A. Gibson, A. DiAntonio, and J. Milbrandt. 2016. SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation. Proc. Natl. Acad. Sci. U.S.A. 113:E6271-E6280. doi:10.1073 / pnas.1601506113. Sumner, C.J., and T.O. Crawford. 2018. Two breakthrough gene-targeted treatments for spinal muscular atrophy: challenges remain. Journal of Clinical Investigation. 128:3219-3227. doi:10.1172 / JCI121658. Szretter, K.J., M.A. Samuel, S. Gilfillan, A. Fuchs, M. Colonna, and M.S. Diamond. 2009. The immune adaptor molecule SARM modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts West Nile Virus pathogenesis. J. Virol. 83:9329-9338. doi:10.1128 / JVI.00836-09. Tagliaferro, P., and R.E. Burke. 2016. Retrograde Axonal Degeneration in Parkinson Disease. J Parkinsons Dis. 6:1-15. doi:10.3233 / JPD-150769. Turkiew, E., D. Falconer, N. Reed, and A. Hoke. 2017. Deletion of Sarm1 gene is neuroprotective in two models of peripheral neuropathy. J. Peripher. Nerv. Syst. 22:162-171. doi:10.1111 / jns.12219. Wang, M.S., A.A. Davis, D.G. Culver, and J.D. Glass. 2002. WldS mice are resistant to paclitaxel (taxol) neuropathy. Ann. Neurol. 52:442-447. doi:10.1002 / ana.10300. Wang, M.S., G. Fang, D.G. Culver, A.A. Davis, M.M. Rich, and J.D. Glass. 2001. The WldS protein protects against axonal degeneration: a model of gene therapy for peripheral neuropathy. Ann. Neurol. 50:773-779. Yang, J., Z. Wu, N. Renier, D.J. Simon, K. Uryu, D.S. Park, P.A. Greer, C. Tournier, R.J. Davis, and M. Tessier-Lavigne. 2015. Pathological axonal death through a MAPK cascade that triggers a local energy deficit. Cell. 160:161-176. doi:10.1016 / j.cell.2014.11.053. Yin, T.C., J.R. Voorhees, R.M. Genova, K.C. Davis, A.M. Madison, J.K. Britt, C.J. Cintron-Perez, L. McDaniel, M.M. Harper, and A.A. Pieper. 2016. Acute Axonal Degeneration Drives Development of Cognitive, Motor, and Visual Deficits after Blast-Mediated Traumatic Brain Injury in Mice. eNeuro. 3. doi:10.1523 / ENEURO.0220-16.2016. Ziogas, N.K., and V.E. Koliatsos. 2018. Primary Traumatic Axonopathy in Mice Subjected to Impact Acceleration: A Reappraisal of Pathology and Mechanisms with High-Resolution Anatomical Methods. J. Neurosci. 38:4031-4047. doi:10.1523 / JNEUROSCI.2343-17.2018.

Claims

1. 1. A composition comprising a mutant SARM1 polypeptide having an amino acid sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:1 and having an amino acid substitution at position K193 compared to a wild-type human sterile alpha and TIR motif containing protein 1 (SARM1) polypeptide having the sequence set forth in SEQ ID NO:1, wherein the mutant SARM1 polypeptide has dominant negative SARM1 activity, and: the amino acid substitutions K193R, H194A, and H685A, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; the amino acid substitutions K193R and H685A, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; or the amino acid substitutions K193A, K193E, K193Q, K193M, or K193R, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; A composition comprising:

2. 1. A composition comprising a vector comprising a nucleic acid encoding a mutant SARM1 polypeptide comprising an amino acid sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:1 and having an amino acid substitution at position K193 compared to a wild-type human sterile alpha and TIR motif-containing protein 1 (SARM1) polypeptide having the sequence set forth in SEQ ID NO:1, wherein the mutant SARM1 polypeptide has dominant negative SARM1 activity, and: the amino acid substitutions K193R, H194A, and H685A, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; the amino acid substitutions K193R and H685A, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; or the amino acid substitutions K193A, K193E, K193Q, K193M, or K193R, as compared to the wild-type human SARM1 polypeptide of SEQ ID NO:1; A composition comprising:

3. The composition of claim 2 , wherein the vector is a viral vector.

4. The composition of claim 3 , wherein the viral vector is a retroviral, lentiviral, adenoviral or adeno-associated viral vector.

5. The composition of any one of claims 1 to 4, wherein the mutant SARM1 polypeptide comprises the amino acid substitutions K193R, H194A and H685A compared to the wild-type human SARM1 polypeptide of SEQ ID NO:

1.

6. The composition of any one of claims 1 to 4, wherein the mutant SARM1 polypeptide comprises the amino acid substitutions K193R and H685A compared to the wild-type human SARM1 polypeptide of SEQ ID NO:

1.

7. 5. The composition of any one of claims 1 to 4, wherein the mutant SARM1 polypeptide comprises the amino acid substitutions K193A, K193E, K193Q, K193M or K193R compared to the wild-type human SARM1 polypeptide of SEQ ID NO:

1.

8. A pharmaceutical composition comprising the composition of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. 9. The pharmaceutical composition of claim 8 for treating a neurodegenerative or neurological disease or disorder in a subject in need thereof.

10. The neurodegenerative or neurological disease or disorder is spinal cord injury, stroke, multiple sclerosis, progressive multifocal alveolar nephropathy, or Interstitial encephalopathy, congenital hypomyelination, encephalomyelitis, acute disseminated encephalomyelitis, central pontine myelin degradation, osmotic hyponatremia, hypoxic demyelination, ischemic demyelination, adrenoleukodystrophy, Alexander disease, Niemann-Pick disease, Pelizaeus-Merzbacher disease, periventricular leukomalacia, globoid cell leukodystrophy (Krabbe disease), Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Huntington's disease, Alzheimer's disease, Parkinson's disease, Tay-Sachs disease, Gaucher disease, Hurler syndrome, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy (chemotherapy-induced neuropathy; CIPN), neuropathy, acute ischemic optic neuropathy, vitamin B12 deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome 10. The pharmaceutical composition of claim 9, wherein the therapeutic agent is selected from the group consisting of glaucoma, Leber's hereditary optic neuropathy, Leber's congenital amaurosis, neuromyelitis optica, metachromatic leukodystrophy, acute hemorrhagic leukoencephalitis, trigeminal neuralgia, Bell's palsy, cerebral ischemia, multiple system atrophy, traumatic glaucoma, tropical spastic paraparesis human T-lymphotropic virus 1 (HTLV-1) associated myelopathy, West Nile virus encephalopathy, La Crosse virus encephalitis, Bunyaviral encephalitis, childhood viral encephalitis, essential tremor, Charcot-Marie-Tooth disease, motor neuron disease, spinal muscular atrophy (SMA), hereditary sensory and autonomic neuropathy (HSAN), adrenomyeloneuropathy, progressive supranuclear palsy (PSP), Friedreich's ataxia, hereditary ataxia, noise-induced hearing loss, frontotemporal dementia, acute angle glaucoma, and congenital hearing loss.

11. The pharmaceutical composition according to any one of claims 9 to 10, wherein the subject is a human.

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