Methods and Compositions for Treating Neuropathies

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(11) EP 1 755 391 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 38/45 (2006.01) A01N 43/04 (2006.01) 11.11.2015 Bulletin 2015/46 A61K 31/70 (2006.01) C07H 19/00 (2006.01) C07H 19/22 (2006.01) (21) Application number: 05790283.5 (86) International application number: (22) Date of filing: 03.06.2005 PCT/US2005/019524

(87) International publication number: WO 2006/001982 (05.01.2006 Gazette 2006/01)

(54) METHODS AND COMPOSITIONS FOR TREATING NEUROPATHIES VERFAHREN UND ZUSAMMENSETZUNGEN ZUR BEHANDLUNG VON NERVENLEIDEN METHODES ET COMPOSITIONS DE TRAITEMENT DE NEUROPATHIES

(84) Designated Contracting States: • MACK T.G.A. ET AL.: "WALLERIAN AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DEGENERATION OF INJURED AXONS AND HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR SYNAPSES IS DELAYED BY A UBE4B/NMNAT CHIMERIC GENE" NATURE NEUROSCIENCE, (30) Priority: 04.06.2004 US 577233 P NATURE AMERICA, INC, US, vol. 4, no. 12, 1 04.01.2005 US 641330 P December 2001 (2001-12-01), pages 1199-1206, XP008017577 ISSN: 1097-6256 (43) Date of publication of application: • SAMSAM M. ET AL.: "The Wld Mutation Delays 28.02.2007 Bulletin 2007/09 Robust Loss of Motor and Sensory Axons in a Genetic Model for Myelin-Related Axonopathy" (73) Proprietor: Washington University JOURNAL OF NEUROSCIENCE, NEW YORK, NY, St. Louis, MO 63130 (US) US, vol. 23, no. 7, 1 April 2003 (2003-04-01), pages 2833-2839, XP003001698 ISSN: 0270-6474 (72) Inventors: • MIN SHENG WANG ET AL.: "THE WLDS PROTEIN • MILBRANDT, Jeffrey PROTECTS AGAINST AXONAL St. Louis, MO 63105 (US) DEGENERATION: A MODEL OF GENE THERAPY • ARAKI, Toshiyuki FOR PERIPHERAL NEUROPATHY" ANNALS OF Tokyo 187-0031 (JP) NEUROLOGY, JOHN WILEY AND SONS, • SASAKI, Yo BOSTON, US, vol. 50, no. 6, 1 December 2001 St. Louis, MO 63144 (US) (2001-12-01), pages 773-779, XP008017595 ISSN: 0364-5134 (74) Representative: Smaggasgale, Gillian Helen • FERRI A. ET AL.: "Inhibiting Axon Degeneration WP Thompson and Synapse Loss Attenuates Apoptosis and 55 Drury Lane Disease Progression in a Mouse Model of London WC2B 5SQ (GB) Motoneuron Disease" CURRENT BIOLOGY, CURRENT SCIENCE, GB, vol. 13, no. 8, 15 April (56) References cited: 2003 (2003-04-15) , pages 669-673, XP004545215 FR-A1- 2 842 424 ISSN: 0960-9822

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• FERRI A. ET AL.: "THE NEUROPROTECTIVE • ARAKI ET AL.: ’Increased Nuclear NAD PROTEIN WLDS CAN DELAY Biosynthesis and SIRT1 Activation Prevent NEURODEGENERATION IN A MOUSE MODEL Axonal Degeneration’ SCIENCE vol. 305, 2004, WITH MOTOR NEURON DISEASE" ABSTRACTS pages 1010 - 1013, XP003001697 OF THE ANNUAL MEETING OF THE SOCIETY • SAMSAM ET AL.: ’The Wld Mutation Delays FOR NEUROSCIENCE, SOCIETY FOR Robust Loss of Motor and Sensory Axons in a NEUROSCIENCE,WASHINGTON, DC, US, vol. 32, Genetic Model for Myelin-Related Axonopathy’ 1 November 2002 (2002-11-01), XP009128321 THE JOURNALOF NEUROSCIENCE vol. 23, 2003, ISSN: 0190-5295 pages 2833 - 2839, XP003001698 • GARAVAGLIA S. ET AL.: "Structure of human • MAGNI G ET AL: "Enzymology of NAD+ NMN adenylyltransferase: A key nuclear enzyme homeostasis in man", CMLS CELLULAR AND for NAD homeostasis" JOURNAL OF MOLECULAR LIFE SCIENCES, BIRKHAUSER BIOLOGICAL CHEMISTRY 20020308 AMERICAN VERLAG, HEIDELBERG, DE, vol. 61, no. 1, 1 SOCIETY FOR BIOCHEMISTRY AND January 2004 (2004-01-01), pages 19-34, MOLECULAR BIOLOGY INC. US, vol. 277, no. 10, XP002445101, ISSN: 1420-682X, DOI: 8 March 2002 (2002-03-08), pages 8524-8530, 10.1007/S00018-003-3161-1 XP002563937 • FRYE R.A.: "Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 5 JUL 2000, vol. 273, no. 2, 5 July 2000 (2000-07-05), pages 793-798, XP002563938 ISSN: 0006-291X

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Description NAD such as a nicotinamide mononucleotide adenylyltransferase (NMNAT) or a nucleic acid encoding a nico- FIELD tinamide mononucleotide adenylyltransferase. The nicotinamide mononucleotide adenylyltransferase can be an [0001] This invention relates to the use of an agent for 5 NMNAT1 protein. treating or preventing axonopathies. [0006] In an embodiment, the agent can be resveratrol. [0007] Such agent can be NAD or NADH, nicotinamide BACKGROUND mononucleotide, nicotinic acid mononucleotide or nicotinamide riboside or derivatives thereof; or an enzyme [0002] Axon degeneration occurs in a variety of neu- 10 that generates NAD such as a nicotinamide mononucle- rodegenerative diseases such as Parkinson’s and Alzhe- otide adenylyltransferase or a nucleic acid encoding an imer’s diseases as well as upon traumatic, toxic or enzyme that generates NAD such as a nucleic acid en- ischemicinjury to neurons. Such diseases and conditions coding a nicotinamide mononucleotide adenylyltrans- are associated with axonopathies including axonal dys- ferase or an agent that increases expression of a nucleic function. One example of axonopathy is Wallerian de- 15 acid encoding an enzyme in a pathway that generates generation (Waller, Philos Trans R. soc. Lond. NAD or an agent that increases activity and/or stability 140:423-429,1850), whichoccurs when the distal portion of an enzyme in a pathway that generates NAD or an of the axon is severed from the cell body. The severed agent that increases NAD activity. The nicotinamide axon rapidly succumbs to degeneration. Axonopathy mononucleotide adenylyltransferase can be an NMNAT1 can, therefore, be a critical feature of neuropathic dis- 20 protein or an NMNAT3 protein. eases and conditions and axonal deficits can be an im- [0008] In the present invention, the neuropathy asso- portant component of the patient’s disability. ciated with axonal degradation can be any of a number of neuropathies such as, for example, those that are he- SUMMARY reditary or congenital or associated with Parkinson’s dis- 25 ease, Alzheimer’s disease, Herpes infection, diabetes, [0003] Accordingly, the present inventors have suc- amyotrophic lateral sclerosis, a demyelinating disease, ceeded in discovering that axonal degeneration can be ischemia or stroke, chemical injury, thermal injury, AIDS diminished or prevented by increasing NAD activity in and the like. In addition, neurodegenerative diseases not diseased and/or injured neurons. It is believed that the mentioned above as well as a subset of the above men- increased NAD activity can act to increase sirtuin activity 30 tioned diseases can also be treated with the agent of which then produces a decrease in axonal degeneration present invention. Such subsets of diseases can include of injured neuronal cells. Thus, one approach to prevent- Parkinson’sdisease ornon-Parkinson’s diseases, Alzhe- ing axonal degeneration can be by activating sirtuin mol- imer’s disease or non-Alzheimer’s diseases and so forth. ecules, i.e. SIRT1 in injured mammalian axons. The activation of SIRT1 can be through direct action on the35 BRIEF DESCRIPTION OF THE DRAWINGS SIRT1 molecule or by increasing the supply of nicotinamide adenine dinucleotide (NAD) which acts as a sub- [0009] Figure 1 illustrates that NMNAT1 activity of the strate for the histone/protein deacetylase activity of Wlds fusion protein produces a delayed degeneration of SIRT1. The activation of SIRT1 results in a decrease in injured axons showing: severity of axonal degeneration or a prevention of axonal 40 degeneration. It is also believed possible that the in- A) in vitro Wallerian degeneration in lentivirus-infect- crease in NAD activity could act through other mecha- ed dorsal root ganglia (DRG) neuronal explant cul- nisms not involving sirtuin. Thus, increasing NAD activity, tures expressing Wlds protein or EGFP wherein tu- which may act through increasing SIRT1 activity or bulin βIII-immunoreactive neurites are shown before through one or more other mechanisms or both can di- 45 transectionand 12,24, 48, and 72 hr aftertransection minish or prevent axonal degeneration in injured mam- (Scale Bar=1mm and the "*" denotes the location of malian axons. the cell bodies prior to removal; and [0004] Thus, in various embodiments, the present in- B) in vitro Wallerian degeneration in lentivirus-infect- vention is directed to the use of an agent for treatment ed DRG neurons expressing EGFP only, Wlds pro- or prevention of an axonopathy in a mammal in need50 tein, Ufd2a portion (70 residues) of Wlds protein thereof, wherein the agent is NaMN, NMN, NmR, a nu- fused to EGFP (Ufd2a(1-70)-EGFP), cleic acid encoding an NMNAT or resveratrol. Ufd2a(1-70)-EGFP with C-terminal nuclear localiza- [0005] The agent can increase SIRT1 activity through tion signal, NMNAT1 portion of Wlds protein fused increasing NAD activity. It is believed that increasing to EGFP, dominant-negative Ufd2a NAD activity can increase sirtuin activity because NAD 55 (Ufd2a(P1140A)), or Ufd2a siRNA construct in which can actas a substrate ofSTRT1. Suchagents can include representative images of neurites and quantitative NAD or NADH, a precursor of NAD, an intermediate in analysis data of remaining neurite numbers (per- the NAD salvage pathway or a substance that generates centage of remaining neurites relative to pre-

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transection 6 S.D.) at the indicated time-point with dicated time points and the "*" indicates significant each construct (bottom left) are shown and the "*" axonal protection compared to control (p<0.0001). indicates significant difference (p<0.0001) with EGFP-infected neurons; also showing EGFP signal [0012] Figure 4 illustrates that NAD-dependent Axonal before transection confirming transgene expression 5 Protection is mediated by SIRT1 activation showing: (bottom row; Scale bar =50mm) and immunoblot analysis confirming protein expression by lentiviral A)In vitro Walleriandegeneration using DRGexplant gene transfer and siRNA downregulation of Ufd2a culturespreincubated with 1 mM NADalone (control) protein (bottom right panels). or in the presence of either 100 mM Sirtinol (a Sir2 10 inhibitor) or 20 mM 3-aminobenzimide (3AB, a PARP [0010] Figure 2 illustrates that increased NAD supply inhibitor); protects axons from degeneration after injury showing: B) invitro Wallerian degeneration using DRG explant cultures incubated with resveratrol (10, 50 or 100 A) Enzymatic activity of wild type and mutant Wlds mM); and and NMNAT1 proteins in which lysates were pre- 15 C) left: in vitro Wallerian degeneration using DRG pared from HEK293 cells expressing the indicated explant cultures infected with lentivirus expressing protein were assayed for NAD production using nico- siRNA specific for each member of the SIRT family tinamide mononucleotide as a substrate and the (SIRT1-7) wherein the bar chart shows the quanti- amount of NAD generated in 1 h was converted to tative analysis of the number of remaining neurites NADH, quantified by fluorescence intensity, and nor- 20 (percentage of remaining neurites relative to pre- malized to total protein concentration showing that transection 6 S.D.) at indicated time-point for each both mutants have essentially no enzymatic activity; condition and the "*" indicates points significantly dif- and ferent than control (<0.0001); B) In vitro Wallerian degeneration in lentivirus-infect- middle table: The effectiveness of each SIRT siRNA ed DRG neurons expressing NMNAT1 or Wld s pro- 25 (expressedas %of wild type mRNAlevel) using qRT- tein, mutants of these proteins that lack NAD-syn- PCR in infected NIH3T3 cells; and thesis activity NMNAT1(W170A) and Wld s(W258A), right: immunoblot using antibodies to SIRT1 to show or EGFP wherein the bar chart shows the quantita- decreased expression of SIRT1 in the presence of tive analysis data of the number of remaining neur- SIRT1 siRNA which effectively blocked NAD de- ites at indicated time-point for each construct (per- 30 pendent axonal protection. centage of remaining neurites relative to pre- transection 6 S.D.) and the "*" indicates significant [0013] Figure 5 illustrates the mammalian NAD biosyn- difference (p<0.0001) with EGFP-infected neurons; thetic pathway in which predicted mammalian NAD bio- C) Protein expression in lentivirus-infected cells de- synthesis is illustrated based on the enzymatic expres- tected by immunoblot analysis using antibodies to 35 sion analysis and studies from yeast and lower eukary- the 6XHis tag; and otes (Abbreviation used; QPRT, quinolinate phosphori- D) DRG neuronal explant expressing eitherbosyltransferase; NaPRT, nicotinic acid phosphoribosyl- NMNAT1 or EGFP (control) cultured with 0.5 mM vin- transferase; NmPRT, nicotinamide phosphoribosyltrans- cristine wherein representative images of neurites ferase; Nrk, nicotinamide riboside kinase; NMNAT, nico- (phase-contrast; Bar=1mm) are shown at the indi- 40 tinamide mononucleotide adenylyltransferase; QNS, cated times after vincristine addition and quantifica- NAD synthetase) tion of the protective effect at the indicated time [0014] Figure 6 illustrates expression analysis of NAD points is plotted as the area covered by neurites rel- biosynthetic enzymes in mammal showing (A) NAD bio- ative to that covered by neurites prior to treatment. synthesis enzyme mRNA levels after 1, 3, 7, and 14 days 45 after nerve transection in rat DRG were determined by [0011] Figure 3 illustrates that axonal protection re- qRT-PCR in which the expression level was normalized quires pre-treatment of neurons with NAD prior to injury to glyceraldehydes-3-phosphate dehydrogenase ex- showing: pression in each sample and is indicated relative to the expression level in non-axotomized DRG; (B) neurite de- A) in vitro Wallerian degeneration using DRG ex- 50 generation introduced by incubation DRG in 1 or 0.1 mM plants cultured in the presence of various concen- rotenone for indicated time and NAD synthesis enzyme trations of NAD added 24 hr prior to axonal transec- mRNAlevels were determinedby qRT-PCR as described tion; and in the text. B) DRG explants preincubated with 1mM NAD for 4, [0015] Figure7 illustrates the subcellular localization of 8, 12, 24, or 48 h prior to transection wherein the bar 55 NMNAT enzymes and their ability to protect axon show- chart shows the number of remaining neurites in ing (A) in vitro Wallerian degeneration assay using len- each experiment (percentage of remaining neurites tivirus infected DRG neuronal explant cultures express- relative to pre-transection 6 S.D.) at each of the in- ing NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 in

4 5 EP 1 755 391 B1 6 which representative pictures taken at 12 and 72 hours mammal in need thereof, wherein the agent is NaMN, after transaction are shown; (B) Subcellular localization NMN, NmR, a nucleic acid encoding an NMNAT or res- of NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 in veratrol. It is believed that the increased NAD activity can HEK 293T cells using immunohistochemistry with anti- act to increase sirtuin activity which then produces a de- body against 6xHis tag to detect each proteins and stain- 5 crease in axonal degeneration of injured neuronal cells ing of the cells with the nuclear marker dye (bisbenzim- compared to axonal degeneration that occurs in injured ide) for comparison to determine the nuclear vs. cyto- neuronal cells not treated with the agent. Such decrease plasmic location of each protein (Scale bar = 25 mm); (C) in axonal degeneration can include a complete or partial enzymatic activity of wild type and mutant NMNAT1 and amelioration of the injury to the neuron. It is also believed NMNAT3in which 6xHis tagged each protein was purified 10 possible that the increase in NAD activity could act from lysate of HEK293T cells expressing NMNAT1, through other mechanisms not involving sirtuin mole- cytNMNAT1, NMNAT3, nucNMNAT3 in which the cules to produce or to contribute to the production of a amount of NAD generated after 1 hour at 37 deg was decrease in axonal degeneration. converted NADH, quantified and normalized to protein [0019] Seven known sirtuin molecules referenced as concentration; (D) protein expression of NMNAT1,15 SIRT’s make up the Sir2 family of histone/protein cytNMNAT1, NMNAT3, and nucNMNAT3 by lentivirus deacetylases in mammals and all such sirtuin molecules gene transfer confirmed by immunoblot analysis of are included within the scope of the present invention. HEK293T cells infected with each of the virus and (E) in The seven human sirtuins, SIRT1-SIRT7, are NAD-de- vitroWallerian degeneration assayusing lentivirus infect- pendent histone/protein deacetylases which are de- ed DRG neuronal explant cultures expressing NMNAT1, 20 scribed more fully in connection with NCBI LocusLink ID cytNMNAT1, NMNAT3, or nucNMNAT3 showing quan- Nos. 23411, 22933, 23410, 23409, 23408, 51548 and titative analysis data of remaining neurite numbers at 12, 51547, respectively (see http://www.nc- 24, 48, and 72 hours after axotomy. bi.nlm.hih.gov/LocusLink/). [0016] Figure 8 illustrates exogenous application of [0020] The compositions can increase activity of any NAD biosynthetic substrates and their ability to protect 25 one or more of the sirtuins and, in particular, various axon showing (A) in vitro Wallerian degeneration assay agents of the present invention increase activity of using DRG neuronal explant cultures after exogenous SIRT1. application of NAD, NmR with representative pictures [0021] By activity of a substance, reference is made to taken at 12, 24, 48, and 72 hours after transaction are either the concentration of the particular substance or shown; (B) in vitro Wallerian degeneration assay using 30 functional effectiveness of the substance. Concentration DRG neuronal explant cultures after exogenous applica- of a substance can be increased by numerous factors tion of Na, Nam, NaMN, NMN, NaAD, NAD, and NmR including, for example, increasing synthesis, decreasing showing quantitative analysis data of remaining neurite breakdown, increasing bioavailability of the substance or numbers at 12, 24, 48, and 72 hours after axotomy are diminishing binding of the substance or otherwise in- shown; (C) DRG neuronal explants infected with NaPRT 35 creasingthe available amountof free substance. Increas- expressing lentivirus and incubated with or without 1 mM ing functional effectiveness can result, for example, from of Na for 24 hours before axotomy, in in vitro Wallerian a change in molecular conformation, a change in the con- degeneration assay showing quantitative analysis data ditions under which the substance is acting, a change in of remaining neurite numbers at 12, 24, 48, and 72 hours sensitivity to the substance, and the like. Increasing ac- after axotomy. 40 tivity with respect to sirtuin molecules is intended to mean [0017] Figure 9 illustrates optic nerve transection after increasing concentration or enhancing functional effec- intravitreal injection of NAD biosynthetic substrates NAD, tiveness or increasing the availability of NAD or increas- NMN, NmR, or Nam was injected into intravitreal com- ing the flux through one or more biosynthetic pathways partment of left rat eye and allowed to incorporate retinal for NAD or any combination thereof. ganglion cells for 24 hours after which, left optic nerve 45 [0022] Axonal damage can be caused by traumatic in- was transected by eye enucleation and right and left optic jury or by non-mechanical injury due to diseases or con- nerves were collected at 4 days after transection and ditions and the result of such damage can be degener- analyzed by Western blotting in which optic nerves ation or dysfunction of the axon and loss of functional transected from mice without any treatment prior to ax- neuronal activity. Disease and conditions producing or otomy were used for negative control; showing in the50 associated with such axonal damage are among a large figure, the quantitative analysis data of percentage of re- number of neuropathic diseases and conditions. Such maining neurofilament immunoreactivity from transected neuropathies can include peripheral neuropathies, cen- optic nerve relative to non-transected 6 S.D. tral neuropathies, and combinations thereof. Further- more, peripheral neuropathic manifestations can be pro- DETAILED DESCRIPTION 55 duced by diseases focused primarily in the central nervous systems and central nervous system manifestations [0018] The present invention involves the use of an can be produced by essentially peripheral or systemic agent for treatment or prevention of an axonopathy in a diseases.

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[0023] Peripheral neuropathies involve damage to the neuropathies caused by adverse drug reactions and neu- peripheral nerves and such can be caused by diseases ropathies caused by vitamin deficiency. Ischemic optic of the nerves or as the result of systemic illnesses. Some neuropathies also include non-arteritic anterior ischemic such diseases can include diabetes, uremia, infectious optic neuropathy. diseases such as AIDs or leprosy, nutritional deficien- 5 [0026] Neurodegenerative diseases that are associat- cies, vascular or collagen disorders such as atheroscle- ed with axonopathy in the central nervous system include rosis, and autoimmune diseases such as systemic lupus a variety of diseases. Such diseases include those in- erythematosus, scleroderma, sarcoidosis, rheumatoid volving progressive dementia such as, for example, arthritis, and polyarteritis nodosa. Peripheral nerve de- Alzheimer’s disease, senile dementia, Pick’s disease, generation can also result from traumatic, i.e mechanical 10 and Huntington’s disease; central nervous system dis- damage to nerves as well as chemical or thermal damage eases affecting muscle function such as, for example, to nerves. Such conditions that injure peripheral nerves Parkinson’s disease, motor neuron diseases and pro- include compression or entrapment injuries such as glau- gressive ataxias such as amyotrophic lateral sclerosis; coma, carpal tunnel syndrome, direct trauma, penetrat- demyelinating diseases such as, for example multiple ing injuries, contusions, fracture or dislocated bones;15 sclerosis; viral encephalitides such as, for example, pressure involving superficial nerves (ulna, radial, or per- those caused by enteroviruses, arboviruses, and herpes oneal) which can result from prolonged use of crutches simplex virus; and prion diseases. Mechanical injuries or staying in one position for too long, or from a tumor; such as glaucoma or traumatic injuries to the head and intraneural hemorrhage; ischemia; exposure to cold or spine can also cause nerve injury and degeneration in radiation or certain medicines or toxic substances such 20 the brain and spinal cord. In addition, ischemia and stroke as herbacides or pesticides. In particular, the nerve dam- as well as conditions such as nutritional deficiency and age can result from chemical injury due to a cytotoxic chemical toxicity such as with chemotherapeutic agents anticancer agent such as, for example, a vinca alkaloid can cause central nervous system neuropathies. such as vincristine. Typical symptoms of such peripheral [0027] The term "treatment" as used herein is intended neuropathies include weakness, numbness, paresthesia 25 to include intervention either before or after the occur- (abnormal sensations such as burning, tickling, pricking renceof neuronal injury. As such, a treatment can prevent or tingling) and pain in the arms, hands, legs and/or feet. neuronal injury by administration before a primary insult The neuropathy can also be associated with mitochon- to the neurons occurs as well as ameliorate neuronal drial dysfunction. Such neuropathies can exhibit de- injury by administration after a primary insult to the neu- creased energy levels, i.e. decreased levels of NAD and 30 rons occurs. Such primary insult to the neurons can in- ATP. clude or result from any disease or condition associated [0024] The peripheral neuropathy can also be a met- with a neuropathy. "Treatment" also includes prevention abolic and endocrine neuropathy which includes a wide of progression of neuronal injury. "Treatment" as used spectrum of peripheral nerve disorders associated with hereincan include theadministration of drugsand/or syn- systemic diseases of metabolic origin. These diseases, 35 thetic substances, the administration of biological sub- some of which are mentioned earlier, include diabetes stances such as proteins, nucleic acids, viral vectors and mellitus, hypoglycemia, uremia, hypothyroidism, hepatic the like as well as the administration of substances such failure, polycythemia, amyloidosis, acromegaly, porphy- as neutraceuticals, food additives or functional foods. ria, disorders of lipid/glycolipid metabolism, nutritional/vi- [0028] The agents of the present invention are useful tamin deficiencies, and mitochondrial disorders, among 40 in treating mammals. Such mammals include humans as others. The common hallmark of these diseases is in- well as non-human mammals. Non-human mammals involvement of peripheral nerves by alteration of the struc- clude, for example, companion animals such as dogs ture or function of myelin and axons due to metabolic and cats, agricultural animals such live stock including pathway dysregulation. cows, horses and the like, and exotic animals, such as [0025] Neuropathies also include optic neuropathies 45 zoo animals. such as glaucoma; retinal ganglion degeneration such [0029] Substances that can increase sirtuin activity in as those associated with retinitis pigmentosa and outer mammals can include polyphenols some of which have retinal neuropathies; optic nerve neuritis and/or degen- been described earlier (see for example Howitz et al., eration including that associated with multiple sclerosis; Nature 425:191-196, 2003 and supplementary informa- traumatic injury to the optic nerve which can include, for 50 tion that accompanies the paper. example, injury during tumor removal; hereditary optic [0030] Such compounds can include stilbenes such as neuropathies such as Kjer’s disease and Leber’s hered- resveratrol, piceatannol, deoxyrhapontin, trans-stilbene itary optic neuropathy; ischemic optic neuropathies, such and rhapontin; chalcone such as butein, isoliquiritigen as those secondary to giant cell arteritis; metabolic optic and 3,4,2’,4’,6’-pentahydroxychalcone and chalcone; neuropathies such as neurodegenerative disesases in- 55 flavones such as fisetin, 5,7,3’,4’,5’-pentahydroxyfla- cluding Leber’s neuropathy mentioned earlier, nutritional vone, luteolin, 3,6,3’,4’-tetrahydroxyflavone, quercetin, deficiencies such as deficiencies in vitamins B12 or folic 7,3’,4’,5’-tetrahydroxyflavone, kaempferol, 6-hydroxya- acid, and toxicities such as due to ethambutol or cyanide; pigenin, apigenin, 3,6,2’,4’-tetrahydroxyflavone, 7,4’-di-

6 9 EP 1 755 391 B1 10 hydroxyflavone, 7,8,3’,4’-tetrahydroxyflavone, 3,6,2’,3’- for synthesizing NAD, an enzyme of the NAD salvage tetrahydroxyflavone, 4’-hydroxyflavone, 5,4’-dihydroxy- pathway or an enzyme of the nicotinamide riboside ki- flavone, 5,7-dihydroxyflavone, morin, flavone and 5-hy- nase pathway or a nucleic acid encoding an enzyme in droxyflavone; isoflavones such as daidzein and genis- the de novo pathway for synthesizing NAD, an enzyme tein; flavanones such as naringenin, 3,5,7,3’,4’-pentahy- 5 of the NAD salvage pathway or an enzyme of the nicoti- droxyflavanone, and flavanone or catechins such as namide riboside kinase pathway and, in particular, an (-)-epicatechin, (-)-catechin, (-)-gallocatechin, (+)-cate- enzyme of the NAD salvage pathway such as, for exam- chin and (+)-epicatechin. ple, a nicotinamide mononucleotide adenylyltransferase [0031] Additional polyphenols or other substance that (NMNAT) such as NMNAT1. Thus, in one non-limiting increase sirtuin deacetylase activity can be identified us- 10 example, administration of an NMNAT such as NMNAT1 ing assay systems described herein as well as in com- or NMNAT3 or a nucleic acid comprising a sequence mercially available assays such as fluorescent enzyme encoding an NMNAT such as NMNAT1 or NMNAT3 can assays (Biomol International L.P., Plymouth Meeting, diminish or prevent axonal degeneration in injured neu- Pennsylvania). Sinclair et al. also disclose substances rons. that can increase sirtuin activity (Sinclair et 15 al.,[0034] The human NMNAT1 enzyme (E.C.2.7.7.18) is WO2005/02672). represented according to the GenBank Assession num- [0032] Other substances can increase sirtuin activity bers for the human NMNAT1 gene and/or pro- indirectly by increasing NAD activity as a result of the tein:NP_073624; NM_022787; AAL76934; AF459819; particular sirtuin functioning through NAD-dependent and NP_073624; AF314163. A variant of this gene is histone/protein deacetylase activity. NAD activity can be 20 NMNAT-2 (KIAA0479), the human version of which can increased by administration of NAD or NADH as well as be found under GenBank Accession numbers by synthesizing NAD. NAD can be synthesised through NP_055854 and NM_015039. three majorpathways, the de novo pathway in which NAD [0035] As used herein, the term "percent identical" or is synthesized from tryptophan, the NAD salvage path- "percent identity" or "% identity" refers to sequence iden- way in which NAD is generated by recycling degraded 25 tity between two amino acid sequences or between two NAD products such as nicotinamide (Lin et al. Curent nucleotide sequences. Identity can each be determined Opin. Cell Biol. 15:241-246, 2003; Magni et al., Cell Mol. by comparing a position in each sequence which may be Life Sci. 61:19-34, 2004) and the nicotinamide riboside aligned for purposes of comparison. When an equivalent kinase pathway in which nicotinamide riboside is con- position in the compared sequences is occupied by the verted to nicotinamide mononucleotide by nicotinamide 30 same base or amino acid, then the molecules are iden- riboside kinase (Bieganowski et al., Cell 117:495-502, tical at that position; when the equivalent site occupied 2004). Thus, administering to injured neurons, a precur- by the same or a similar amino acid residue (e.g., similar sor of NAD in the de novo pathway such as, for example, in steric and/or electronic nature), then the molecules tryptophan or nicotinate and/or substances in the NAD can be referred to as homologous (similar) at that posi- salvage pathway such as, for example, nicotinamide, nic- 35 tion. Expression as a percentage of homology, similarity, otinic acid, nicotinic acid mononucleotide, or deamido- or identity refers to a function of the number of identical NAD and/or substances in the nicotinamide riboside ki- or similar amino acids at positions shared by the com- nase pathway such as, for example, nicotinamide ribo- pared sequences. Various alignment algorithms and/or side or nicotinamide mononucleotide, could potentially programs may be used, including FASTA, BLAST, or EN- increase NAD activity. As shown below, nicotinamide 40 TREZ. FASTA and BLAST are available as a part of the mononucleotide, nicotinic acid mononucleotide or nico- GCG sequence analysis package (University of Wiscon- tinamide riboside, in addition to NAD, protected against sin, Madison, Wis.), and can be used with, e.g., default axonal degeneration to a similar extent as did NAD, how- settings. ENTREZ is available through the National Cent- ever, nicotinic acid and nicotinamide did not. The in- erfor Biotechnology Information, NationalLibrary of Med- creased NAD activity can then increase sirtuin his-45 icine, National Institutes of Health, Bethesda, Md. The tone/protein deacetylase activity in the injured neurons percent identity of two sequences can be determined by and diminish or prevent axonal degeneration. In addition, the GCG program with a gap weight of 1, e.g., each amino it is believed that other substances can act by increasing acid gap is weighted as if it were a single amino acid or enzyme activity or by increasing levels of NAD, nicotina- nucleotide mismatch between the two sequences. Other mide mononucleotide, nicotinic acid mononucleotide, 50 techniques for alignment are described in Methods in En- nicotinamide riboside or sirtuin enzymes or by decreas- zymology, vol. 266: Computer Methods for Macromo- ing degredation of NAD, nicotinamide mononucleotide, lecular Sequence Analysis (1996), ed. Doolittle, Aca- nicotinic acid mononucleotide, nicotinamide riboside or demic Press, Inc., a division of Harcourt Brace & Co., sirtuin enzymes. San Diego, California, USA. Preferably, an alignment [0033] NAD can be increased in injured neurons by 55 program that permits gaps in the sequence is utilized to administering enzymes that synthesize NAD or nucleic align the sequences. The Smith-Waterman is one type acids comprising enzymes that synthesize NAD. Such of algorithm that permits gaps in sequence alignments. enzymes can include an enzyme in the de novo pathway See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP

7 11 EP 1 755 391 B1 12 program using the Needleman and Wunsch alignment identity with a human NMNAT1 or at least 70% identity method can be ut ilized to align s equences. An alternative with a human NMNAT3, at least 80% identity with a hu- search strategy uses MPSRCH software, which runs on man NMNAT1 or at least 80% identity with a human a MASPAR computer. MPSRCH uses a Smith-Water- NMNAT3, at least 90% identity with a human NMNAT1 man algorithm to score sequences on a massively par- 5 or at least 90% identity with a human NMNAT3, at least allel computer. This approach improves ability to pick up 95% identity with a human NMNAT1 or at least 95% iden- distantly related matches, and is especially tolerant of tity with a human NMNAT3. Moreover, the agent can be small gaps and nucleotide sequence errors. Nucleic acid- comprised by a human NMNAT1, a human NMNAT3 or encoded amino acid sequences can be used to search a conservatively substituted variants thereof. both protein and DNA databases. Databases with indi- 10 [0039] The agent can also be comprised by a polynu- vidual sequences are described in Methods in Enzymol- cleotide having at least 50% identity with a nucleic acid ogy, ed. Doolittle, supra. Databases include Genbank, encoding a human NMNAT1 or a polynucleotide having EMBL, and DNA Database of Japan (DDBJ). at least 50% identity with a nucleic acid encoding a hu- [0036] A "variant" of a polypeptide refers to a polypep- man NMNAT3, a polynucleotide having at least 60% tide having the amino acid sequence of the polypeptide 15 identity with a nucleic acid encoding a human NMNAT1 in which is altered in one or more amino acid residues. or a polynucleotide having at least 60% identity with a.nu- The variant may have "conservative" changes, wherein cleic acid encoding a human NMNAT3, a polynucleotide a substituted amino acid has similar structural or chem- having at least 70% identity with a nucleic acid encoding ical properties (e.g., replacement of leucine with isoleu- a human NMNAT1 or a polynucleotide having at least cine). A variant may have "nonconservative" changes 20 70% identity with a nucleic acid encoding a human (e.g., replacementof glycine with tryptophan). Analogous NMNAT3, a polynucleotide having at least 80% identity minor variations may also include amino acid deletions with a nucleic acid encoding a human NMNAT1 or a poly- or insertions, or both. Guidance in determining which nucleotide having at least 80% identity with a nucleic acid amino acid residues may be substituted, inserted, or de- encoding a human NMNAT3, a polynucleotide having at leted without abolishing biological or immunological ac- 25 least 90% identity with a nucleic acid encoding a human tivity may be found using computer programs well known NMNAT1 or a polynucleotide having at least 90% identity in the art, for example, LASERGENE software (DNAS- with a nucleic acid encoding a human NMNAT3, a poly- TAR). nucleotide having at least 95% identity with a nucleic acid [0037] The term "variant," when used in the context of encoding a human NMNAT1 or a polynucleotide having a polynucleotide sequence, may encompass a polynu- 30 at least 95% identity with a nucleic acid encoding a hu- cleotide sequence related to that of a particular gene or man NMNAT3. The agent can also be a polynucleotide the coding sequence thereof. This definition may also encoding a human NMNAT1, a human NMNAT3 or a include, for example, "allelic," "splice," "species," or "pol- variant thereof. ymorphic" variants. A splice variant may have significant [0040] Administration can be by any suitable route of identity to a reference molecule, but will generally have 35 administration including buccal, dental, endocervical, in- a greater or lesser number of polynucleotides due to al- tramuscular, inhalation, intracranial, intralymphatic, in- ternate splicing of exons during mRNA processing. The tramuscular, intraocular, intraperitoneal, intrapleural, in- corresponding polypeptide may possess additional func- trathecal, intratracheal, intrauterine, intravascular, intra- tional domains or an absence of domains. Species var- venous, intravesical, intranasal, ophthalmic, oral, otic, iants are polynucleotide sequences that vary from one 40 biliary perfusion, cardiac perfusion, priodontal, rectal, species to another. The resulting polypeptides generally spinal subcutaneous, sublingual, topical, intravaginal, will have significant amino acid identity relative to each transermal, ureteral, or urethral. Dosage forms can be other. A polymorphic variation is a variation in the poly- aerosol including metered aerosol, chewable bar, cap- nucleotide sequence of a particular gene between indi- sule, capsule containing coated pellets, capsule contain- viduals of a given species. Polymorphic variants also may 45 ing delayed release pellets, capsule containing extended encompass "single nucleotide polymorphisms" (SNPs) release pellets, concentrate, cream, augmented cream, in which thepolynucleotide sequence variesby one base. suppository cream, disc, dressing, elixer, emulsion, en- The presence of SNPs may be indicative of, for example, ema, extended release fiber, extended release film, gas, a certain population, a disease state, or a propensity for gel, metered gel, granule, delayed release granule, ef- a disease state. 50 fervescent granule, chewing gum, implant, inhalant, in- [0038] An agent that can be used in treating or pre- jectable, injectable lipid complex, injectable liposomes, venting a neuropathy in accordance with the present in- insert, extended release insert, intrauterine device, jelly, vention can be comprised by a polynucleotide encoding liquid, extended release liquid, lotion, augmented lotion, an NMNAT. In particular, the agent can be an enzyme shampoo lotion, oil, ointment, augmented ointment, having NMNAT activity and at least 50% identity with a 55 paste, pastille, pellet, powder, ext ended release powder, human NMNAT1 or at least 50% identity with a human metered powder, ring, shampoo, soap solution, solution NMNAT3, at least 60% identity with a human NMNAT1 for slush, solution/drops, concentrate solution, gel form- or at least 60% identity with a human NMNAT3, at least ing solution/drops, sponge, spray, metered spray, sup-

8 13 EP 1 755 391 B1 14 pository, suspension, suspension/drops, extended re- EXAMPLE 1 lease suspension, swab, syrup, tablet, chewable tablet, tablet containing coated particles, delayed release tablet, [0046] This example demonstrates that transected ax- dispersible tablet, effervescent tablet, extended release ons from neurons tranfected with a vector expressing tablet, orally disintegrating tablet, tampon, tape or tro- 5 Wlds protein show a delayed degeneration compared to che/lozenge. control neurons. [0041] Intraocular admistration can include adminis- [0047] In wlds mice, Wallerian degeneration in re- tration by injection including intravitreal injection, by eye- sponse to axonal injury has been shown to be delayed drops and by trans-scleral delivery. (Gillingwater, et al., J Physiol, 534:627-639, 2001). Ge- [0042] Administration can also be by inclusion in the 10 netic analysis has shown that the wlds mutation compris- diet of the mammal such as in a functional food for hu- es an 85 kb tandem triplication, which results in overex- mans or companion animals. pression of a chimeric nuclear molecule (Wlds protein). [0043] It is also contemplated that certain formulations This protein is composed of the N-terminal 70 AAs of Ufd containing the compositions that increase sirtuin activity (ubiquitin fusion degradation protein)2a, a ubiquitin chain are to be administered orally. Such formulations are pref- 15 assembly factor, fused to the complete sequence of nico- erably encapsulated and formulated with suitable carri- tinamide mononucleotide adenylyltransferasel ers in solid dosage forms. Some examples of suitable (NMNAT1), an enzyme in the NAD salvage pathway that carriers, excipients, and diluents include lactose, dex- generates NAD within the nucleus. The Wld s protein has trose, sucrose, sorbitol, mannitol, starches, gum acacia, NMNAT activity but lacks ubiquitin ligase function, sug- calcium phosphate, alginates, calcium silicate, microc- 20 gesting that axonal protection is derived from either in- rystalline cellulose, polyvinylpyrrolidone, cellulose, gela- creased NMNAT1 activity or a ’dominant negative’ inhi- tin, syrup, methyl cellulose, methyl- and propylhydroxy- bition of Ufd2a function. benzoates, talc, magnesium, stearate, water, mineral oil, [0048] To identify the mechanism of delayed axonal and the like. The formulations can additionally include degeneration mediated by the Wld sprotein, we employed lubricating agents, wetting agents, emulsifying and sus- 25 an in-vitro Wallerian degeneration model. Primary DRG pending agents, preserving agents, sweetening agents explant neurons were infected with lentivirus expressing or flavoring agents. The compositions may be formulated the appropriate proteins, and axons were injured by ei- so as to provide rapid, sustained, or delayed release of ther removal of the neuronal cell body (transection) or the active ingredients after administration to the patient growth in vincristine (toxic). by employing procedures well known in the art. The for- 30 [0049] Lentiviral expression constructs were kindly mulations can also contain substances that diminish pro- provided by D. Baltimore (Lois, et al., Science teolytic degradation and promote absorption such as, for 295:868-72, 2002). We modified the FUGW vector to example, surface active agents. generate a general expression shuttle FUIV (ubiquitin [0044] The specific dose can be calculated according promoter - gene of interest-IRES-enhanced YFP (Ve- to the approximate body weight or body surface area of 35 nus)) vector that enables enhanced YFP expression in the patient or the volume of body space to be occupied. cells that express the gene-of-interest. The following pro- The dose will also depend upon the particular route of teins, each with a hexahistidine tag at the C-terminus, administration selected. Further refinement of the calcu- were cloned into the FUIV vector: Wld s chimeric mutant lations necessary to determine the appropriate dosage protein; Ufd2a containing a point mutation (P1140A), for treatment is routinely made by those of ordinary skill 40 which has previously been shown to inhibit wild-type in the art. Such calculations can be made without undue Ufd2a functionas a "dominant-negative"(Ufd2a(P1140)). experimentation by one skilled in the art in light of the The following genes were cloned into FUGW vector: 1) activity in assay preparations such as has been de- The first 70 AAs of Ufd2a (the portion contained in Wld s scribed elsewhere for certain compounds (see for exam- protein) fused to the N-terminus of EGFP ple, Howitz et al., Nature 425:191-196, 2003 and supple- 45 (Ufd2a(1-70)-EGFP) or EGFP with nuclear localization mentary information that accompanies the paper). Exact signal at the C-terminal (Ufd2a(1-70)-nucEGFP). 2) The dosages can be determined in conjunction with standard NMNAT1 portion of Wld s protein fused to the C-terminus dose-response studies. It will be understood that the of EGFP (EGFP-NMNAT1). amount of the composition actually administered will be [0050] The murine cDNA for Ufd2a/Ube4b determined by a practitioner, in the light of the relevant 50 (mKIAA0684) was provided by Kazusa DNA Research circumstances including the condition or conditions to be Institute. Murine cDNAs for NMNAT1 (accession treated, the choice of composition to be administered, number: BC038133) were purchased from ATCC. PCR- the age, weight, and response of the individual patient, mediated mutagenesis was used to generate point mu- the severity of the patient’s symptoms, and the chosen tations in Ufd2a, NMNAT1 and Wlds. route of administration. 55 [0051] We generated siRNA constructs in the FSP-si [0045] The invention can be further understood by ref- vector generated from the FUGW backbone by replacing erence to the examples which follow. the ubiquitin promoter and GFP cDNA with the human U6 promoter and Pol I termination signal followed by the

9 15 EP 1 755 391 B1 16

SV40 promoter-puromycin-N-acetyl-transferase gene. shown in Figure 1B. Cloning of siRNA construct was performed as described [0057] We found that expression of EGFP-NMNAT1 previously, so that the siRNA is transcribed from the U6 delayed axonal degeneration comparable to Wld sprotein promoter (Castanotto, et al., RNA, 8:1454-60, 2002). Se- itself, whereas the N-terminal 70 AA of Ufd2a (fused to quences used for siRNA downregulation of protein ex- 5 EGFP), either targeted to the nucleus or cytoplasm, did pression were 1692~1710 of SIRT1, 1032~1050 of not affect axonal degeneration. Quantification of these SIRT2, 538∼556 of SIRT3, 1231∼1249 of SIRT4, 37∼55 effects was performed by counting the percentage of re- of SIRT5, 1390∼1408 of SIRT6, and 450∼468 of SIRT7. maining neurites at various times after removal of neu- The integrity of each lentiviral expression and siRNA con- ronal cell bodies. This analysis showed that EGFP- struct was confirmed by DNA sequencing. 10 NMNAT1, like Wlds protein itself, resulted in a >10-fold [0052] Mouse DRGexplants from E12.5embryos were increase in intact neurites 72 hr after injury. To further cultured in the presence of 1 nM nerve growth factor. exclude direct involvement of the UPS in Wlds protein- Non-neuronal cells were removed from the cultures by mediated axonal protection, we examined the effect of adding 5-fluorouracil to the culture medium. Transection Ufd2a inhibition using either a dominant-negative Ufd2a of neurites was performed at 10-20 DIV using an 18-15 mutant or an Ufd2a siRNA construct. However, neither gauge needle to remove the neuronal cell bodies. Incu- of these methods resulted in delayed axonal degradation bation with β-nicotinamide adenine dinucleotide (Sigma) in response to axotomy. Together, these experiments or Sirtinol (Calbiochem) was performed using conditions demonstrated that the NMNAT1 portion of the Wld s pro- indicated in the text or figures. tein is responsible for the delayed axonal degeneration [0053] Lentiviral expression vectors were generated 20 observed in wlds mice. using HEK293T cells as described above. For confirmation of lentivirus-derived protein expression, HEK293T EXAMPLE 2 cells were infected with lentivirus and cells were lysed 3 days after infection. These lysates were analyzed by im- [0058] This example shows that mutations in the full munoblot to using anti-His tag monoclonal antibody (Qia- 25 length NMNAT1 and in Wlds protein abolish the axonal gen) to detect expression of the respective hexahistidine- protective effects of the proteins. tagged proteins. Lentiviral infection of DRG neurons was [0059] NMNAT1 is an enzyme in the nuclear NAD sal- performed by incubating ∼106-107 pfu/ml virus with the vage pathway that catalyzes the conversion of nicotina- DRG explant for 24 h beginning 3-7 days prior to axonal mide mononucleotide (NMN) and nicotinate mononucle- transection. The infected neurons were examined under 30 otide (NaMN) to NAD and nicotinate adenine mononu- an inverted fluorescent microscope to insure detectable cleotide (NaAD), respectively. The axonal protection ob- lentivirus-mediated transgene expression in >95% of served in NMNAT1 overexpressing neurons could be neurons. mediated by its ability to synthesize NAD (i.e. its enzy- [0054] Quantitative analysis of axonal degeneration matic activity), or perhaps, by other unknown functions was performed as previously described (Zhai, et al., Neu- 35 of this protein. To address this question, we used the ron 39:217-25, 2003). Briefly, the cultures were exam- NMNAT1 crystal structure to identify several residues ined using phase contrast microscopy at the indicated predicted to participate in substrate binding. A mutation times. Axons with a fragmented, non-refractile appear- in one of these residues (W170A) was engineered into ance were designated as "degenerated." At each time full length NMNAT1 and Wld s protein. In vitro enzymatic point, at least 200 singly distinguishable axons were40 assays confirmed that both of these mutant proteins were blindly scored from several randomly taken images of severely limited in their ability to synthesize NAD (Fig. each culture. Each condition was tested in triplicate ex- 2A). Each of these mutants and their respective wild type plants in each experiment. Results were obtained from counterparts were introduced into neurons to assess 2-4 independent experiments for each condition. Statis- their ability to protect axons from degradation. We found tical analysis was performed by Student’s T test. For cal- 45 that neurons expressing these enzymatically inactive culations of neurite-covered area, digitally captured im- mutants had no axonal protective effects (Fig. 2A), indi- ages from quadruplicate samples of two independent ex- cating that NAD/NaAD-production is responsible for the periments were analyzed using analysis 3.1 software ability of NMNAT1 to prevent axonal degradation. (Soft Imaging System, Lakewood, CO). [0055] We found that transected axons from neurons 50 EXAMPLE 3 expressing the Wlds protein degenerated with the delayed kinetics characteristic of neurons derived from wlds [0060] This example illustrates that increased NMNAT (Buckmaster, et al., Eur J Neurosci 7:1596-602, 1995) activity in neurons injured with vincristine also show a mice as shown in Figure 1A. delayed axonal degradation. [0056] Next, we compared axonal degeneration after 55 [0061] In addition to mechanical transection, axonal transection in neurons that overexpress Wld sprotein with protection in wlds mice is also observed against other those that express the Ufd2a or NMNAT1 portions that damaging agents such as ischemia and toxins (Coleman, make up the Wlds protein linked to EGFP. Results are et al., Trends Neurosci 25:532-37, 2002; Gillingwater, et

10 17 EP 1 755 391 B1 18 al., J Cereb Blood Flow Metab 24:62-66, 2004). We lational modification within the axons themselves. sought to determine whether increased NMNAT activity [0067] The requirement for extended exposure to NAD would also delay axonal degradation in response to other of the intact neurons to prevent axonal degradation in types of axonal injury such as vincristine, a cancer chem- response to injury suggests that the protective process otherapeutic reagent with well-characterized axonal tox- 5 requires de novo transcriptional and/or translational icity. Neurons expressing either NMNAT1 or EGFP (con- events. Interestingly, both the Wld s protein and NMNAT1 trol) were grown in 0.5mM vincristine for up to 9 d. We arelocated within the nucleus(data not shown).Similarly, found that axons of neurons expressing NMNAT1 main- most enzymes that make up the NAD salvage pathway tained their original length and refractility, whereas axons in yeast are also compartmentalized in the nucleus. We emanating from neurons expressing EGFP gradually re- 10 compared NAD levels in wild type and NMNAT1 express- tracted and had mostly degenerated by day 9 (Fig. 2B). ing DRG neurons using sensitive microscale enzymatic These results indicate that NMNAT activity by itself can assays (Szabo, et al., Proc Natl Acad Sci USA, protect axons from a number of insults and mediate the 93:1753-58 ,1996), however no changes in overall cel- protective effects observed in wlds mice. lular NAD levels were found (data not shown). This is 15 similar to observations in yeast, in which activation of this EXAMPLE 4 nuclear pathway did not change overall levels of NAD (Anderson, et al., J Biol Chem, 277:18881-90,2002; Huh, [0062] This example shows that exogenously admin- et al., Nature, 425:686-91, 2003). Furthermore, levels of istered NAD can protect injured neurons from axonal de- tissue NAD in the brains of wild type and wlds mice are generation. 20 similar despite the increased levels of NMNAT activity in [0063] Previous experiments have shown that neuro- wlds mice (Mack, et al., Nat Neurosci, 4:1199-206, 2001). nal cells express membrane proteins that can bind and These data suggest that an NAD-dependent enzymatic transport extracellular NAD into the cell (Bruzzone, et al., activity in the nucleus, as opposed to cytoplasmic NAD- Faseb J 15:10-12, 2001). This encouraged us to inves- dependent processes, is likely to mediate the axonal pro- tigate whether exogenously administered NAD could25 tection observed in response to increased NMNAT ac- prevent axonal degeneration. We added various concen- tivity. trations of NAD to neuronal cultures prior to axonal transection and examined the extent of axonal degrada- EXAMPLE 6 tion. We found that 0.1-1 mM NAD added 24 hr prior to axotomy significantly delayed axonal degeneration, al- 30 [0068] This example shows that inhibition of Sir2 is in- though exogenously applied NAD was slightly less effec- volved in NAD-dependent axonal protection. tive in protecting axons than lentivirus mediated[0069] The Sir2 family of protein deacetylases and po- NMNAT1 expression (Fig. 3A). These results provide di- ly(ADP-ribose) polymerase (PARP) are the major NAD- rect support for the idea that increased NAD supply can dependent nuclear enzymatic activities. Sir2 is an NAD- prevent axonal degradation. 35 dependent deacetylase of histones and other proteins, and its activation is central to promoting increased lon- EXAMPLE 5 gevity in yeast and C. elegans (Bitterman, et al., Microbiol Mol Biol Rev, 67:376-99, 2003; Hekimi, et al., Science [0064] This example illustrates that NAD was required 299:1351-54, 2003). PARP is activated by DNA damage prior to the removal of the neuronal cell bodies to protect 40 and is involved in DNA repair (S.D. Skaper, Ann NYAcad the injured neurons from axonal degeneration. Sci, 993:217-28 and 287-88, 2003). These enzymes, in [0065] To gain insights into the mechanism of NAD- particular the Sir2 proteins, have generated great interest dependent axonal protection (NDAP), we examined in recent years as they provide a potential link between whether NAD was required prior to the removal of the caloric restriction and its effects on the ageing process. neuronal cell bodies, or whether direct exposure of the 45 The importance of these NAD-dependent enzymes in severed axons to high levels of NAD was sufficient to regulating gene activity, prompted us to investigate their provide protection (Fig. 3B). Neuronal cultures were pre- role in the self-destructive process of axonal degradation. pared and 1 mM NAD was added to the culture medium We therefore tested whether inhibitors of Sir2 (Sirtinol) at the time of axonal transection or at various times (4 to and PARP (3-aminobenzamide (3AB)) could affect NAD- 48 hr) prior to injury. 50 dependent axonal protection (NDAP) (Fig. 4A). Neurons [0066] We found that administering NAD at the time of were cultured in the presence of 1 mM NAD and either axonal transection or, for up to 8 hr prior to injury, had Sirtinol (100 mM) or 3AB (20 mM). Axonal transection no protective effects on axons. However, significant axon was performed by removal of the neuronal cell bodies sparingwas observed when neurons wereincubated with and the extent of axonal degradation was assessed 12 NAD for longer periods of time prior to injury, with the 55 to 72 hr later. We found that Sirtinol effectively blocked greatest effects occurring after at least 24 h of NAD pre- NDAP, indicating that Sir2 proteins are likely effectors of treatment, These results indicate that NAD dependent this process. In contrast, 3AB had no effect on NDAP, axonal protection is not mediated by a rapid post-trans- indicating that PARP does not play a role in axonal pro-

11 19 EP 1 755 391 B1 20 tection. To further examine the role of Sir2 proteins in EXAMPLES 8-11 NDAP, we tested the effects of resveratrol (10 ∼100mM), a polyphenol compound that enhances Sir2 activity [0073] The following Materials and Methods were used (Howitz, et al., Nature, 425:191-96, 2003). We found that in Examples 8-11. neurons treated with resveratrol prior to axotomy showed 5 [0074] Construction of expression plasmids and a decrease in axonal degradation that was comparable mutagenesis. Coding regions of the NAD biosynthetic to that obtained using NAD (Fig. 4A), providing further enzymes were PCR amplified from EST clones support for the idea that Sir2 proteins are effectors of the BC038133 for murine NMNAT1 and BC005737 for axonal protection mediated by increased NMNAT activ- murine nicotinamide mononucleotide adenylyltrans- ity. 10 ferase3 (NMNAT3), using Herculase (Stratagene). Hu- man NAD synthetase (QNS) hexahistidine-tagged cDNA EXAMPLE 7 was kindly provided by Dr. N. Hara (Shimane University, Shimane, Japan). Hexahistidine tag was added at the 3’- [0070] This example shows that SIRT1 is involved in end of each cDNA. NMNAT1 cytosolic mutant NAD-dependent axonal protection. 15 (cytNMNAT1) was generated by PCR-mediated site-di- [0071] In humans and rodents, seven molecules shar- rected mutagenesis. Nuclear form of NMNAT3 ing Sir2 conserved domain (sirtuin (SIRT)1 through 7) (nucNMNAT3) was generated by adding a nuclear local- have been identified, although some of these proteins do ization signal to the C-terminal end of NMNAT3. Each not appear to have histone/protein deacetylase activity PCR amplified NAD synthetic enzyme fragment was (Buck, et al., J Leukoc Biol, S0741-5400, 2004). SIRT1 20 cloned into FCIV lentiviral shuttle vector as previously is located in the nucleus and is involved in chromatin described. The integrity of all the constructs was se- remodeling and the regulation of transcription factors quenced using Taq DyeDeoxy Terminator cycle se- such as p53 (J. Smith, Trends Cell Biol, 12:404-406, quencing kits (Applied Biosystems) and an Applied Bio- 2002). The cellular location of other SIRT proteins is less systems 373 DNA sequencer. clear, but some have been found in the cytoplasm and 25 [0075] NAD biosynthetic substrates. All substrates in mitochondria. To determine which SIRT protein(s) is for NAD biosynthetic enzymes were purchased from Sig- involved in NAD-dependent axonal protection, we per- ma (Na, Nam, NMN, NaMN, nicotinine acid adenine di- formed knockdown experiments using siRNA constructs nucleotide (NaAD), and NAD). NmR was synthesized to specifically target each member of the SIRT family. from NMN. Conversion of NMN to NmR was confirmed Neurons were infected with lentiviruses expressing spe- 30 by HPLC (Waters) using reverse phase column LC-18T cific SIRT siRNA constructs that effectively suppressed (Supelco). NmR is eluted 260 6 10 seconds and NMN expression of their intended target (Fig. 4B). The infected is eluted 150 6 10 seconds under 1 ml/min flow rate of neurons were cultured in 1 mM NAD and axonal transec- buffer containing 50mM K 2HPO4 and 50mM KH 2PO4 (pH tion was performed by removing the cell bodies. We 7.0). Biological activity of NmR was accessed as previ- found that the SIRT1 siRNA construct was just as effec- 35 ously described by using yeast strains kindly provided tive at blocking the axonal protective effects of NAD as from Dr. Charles Brenner (Dartmouth Medical School, the Sirtinol inhibitor. In contrast, inhibition of the other New Hampshire, USA). SIRT proteins did not have significant effects on NDAP [0076] Real-time quantitative reverse transcrip- (Fig. 4B). These results indicate that SIRT1 is the major tion-PCRanalysis. All the surgical procedures were per- effector of the increased NAD supply that effectively pre- 40 formed according to National Institute of Health guide- vents axonal self destruction. Although, SIRT1 may lines for care and use of laboratory animals at Washing- deacetylate proteins directly involved in axonal stability, ton University. For the expression analysis following its predominantlynuclear location,along withthe require- nerve injury, the sciatic nerves of a C57BL/6 mouse was ment for NAD ~24 hr prior to injury for effective protection, transected and L4 to L5. DRGs were collected at indicat- suggest that SIRT1 regulates a genetic program that45 ed time points and pooled to extract RNA. Rat DRG ex- leads to axonal protection. plants from E14.5 embryo were cultured for 14 days ac- [0072] Axonal degeneration is an active, self-destruc- cording to the method desctribed and cultured with media tive phenomenon observed not only after injury and in containing 10 nM vincristin for indicated period and ex- response to chemotherapy, but also in association with tracted RNA. Total RNAs from pooled tissue sources or aging, metabolic diseases such as diabetic neuropathy, 50 DRG explant cultures were prepared. First-strand cDNA and neurodegenerative diseases. Our results indicate templates were prepared from 1 mg of each RNA using that the molecular mechanism of axonal protection in the standard methods. Two independent cDNA syntheses wlds mice is due to the increased supply of NAD resulting were performed for each RNA sample. Quantitative re- from enhanced activity of the NAD salvage pathway and verse transcription (RT)-PCR was performed by moni- consequent activation of the histone/protein deacetylase 55 toring in real-time the-increase in fluorescence of the SIRT1. SYBR-GREEN dye on a TaqMan 7700 Sequence De- tection System (Applied Biosystems). [0077] Cell culture, in vitro axotomy, and quantifi-

12 21 EP 1 755 391 B1 22 cation of axonal degeneration. Mouse DRG explants at 24 hours after intravitreal injection and optic nerve was from E12.5 embryos were cultured in the DMEM contain- recovered at indicated time. Optic nerve tissue was ho- ing 10% FCS and 1 nM nerve growth factor. Non-neuro- mogenized in 100 ml of a buffer containing 100mM tris- nal cells were removed from the cultures by adding 5- HCl (pH 6.8), 1 % SDS, and 1mM DTT. Fifty mg of protein fluorouracil to the culture media. Transection of neurites 5 for each sample was analyzed by the Western blotting was performed at 14-21 DIV using an 18-gauge needle using anti-neurofilament antibody 2H3 (Developmental to remove the neuronal cell bodies. Lentiviral expression Studies Hybridoma Center) and peroxidase-conjugated vectors were generated. Lentiviral infection was per- secondary antibody (Jackson ImmunoResearch). The formed 3-7 days prior to axonal transection for 24 hr. degeneration rate was calculated from the ratio of the Quantitative analysis of neurite degeneration was per- 10 neurofilament immunoreactivity of transected vs. contral- formed. ateral nerves. [0078] Determination of protein expression and localization. For confirmation of protein expression, EXAMPLE 8 HEK293T cells were infected with a virus that expresses each of NAD biosynthetic enzymes. Cells were lysed 5 15 [0081] This example illustrates the NAD biosynthetic days after infection to be analyzed by immunoblot to de- pathway and expression analysis of mammalian NAD tect expression of each protein with a hexa-histidine tag biosynthetic enzymes. by anti-6xHis tag monoclonal antibody (R&D Systems). [0082] NAD is synthesized via three major pathways Subcellular localization of each protein was analyzed us- in both prokaryotes and eukaryotes. In the de novo path- ing HEK293T cells transiently transfected with a viral20 way, NAD is synthesized from tryptophan (Fig.5). In the shuttle vector for each NAD biosynthetic enzymes. Cells salvage pathway, NAD is generated from vitamins includ- were fixed in 4% paraformaldehyde in PBS containing ing nicotinic acid and nicotinamide. A third route from 0.1% tween-20 (PBS-T) and incubated with PBS-T con- nicotinamide riboside called Preiss-Handler independent taining 5% BSA for 1 hour, and then covered with 1:1000 pathway has recently been discovered. The last enzy- diluted anti-6xHis tag antibody (R&D Systems) in PBS- 25 matic reaction of the de novo pathway involves the con- T containing 1% BSA and for 16 hours at 4°C. Cells were version of quinolinate to NaMN by QPRT (EC 2.4.2.19). washed with PBS-T and incubated with Alexa Fluor 594- At this point, the de novo pathway converges with the conjugated secondary antibody (Molecular Probes) in salvage pathway. NaPRT (EC 2.4.2.11) converts Na to TBS-T for 1 hour and examined by fluorescence micro- NaMN, which is then converted to NaAD by NMNAT (EC scopy (Nikon). 30 2.7.7.1). QNS1 (EC 6.3.5.1) converts NaAD to NAD. [0079] NMNAT protein overexpression, affinity pu- NmPRT (EC 2.4.2.12); also reported as visfatin) converts rification and enzymatic assay. HEK293T cells were Nam to NMN. NMN is also converted to NAD by NMNAT. transfected with an expression plasmid for each enzyme Nicotinamidase (PNC, EC 3.5.1.19), which converts by using calcium phosphate precipitation. Three days lat- Nam to Na in yeast and bacteria salvage pathway has er, cells were washed with PBS twice and then suspend- 35 not been identified in mammals. In the Preiss-Handler ed in the buffer containing 50 mM Sodium Phosphate independent pathway, Nrk (EC 2.7.1.22) converts NmR (pH8.0), and 300 mM NaCl (buffer A). Cells were then to NMN and converge to salvage pathway. Most of these homogenized by SONIFIRE 450 (BRANSON) and su- mammalian enzymes including QPRT, NmPRT, QNS1, pernatant was collected by centrifugation at 10,000 g for Nrk1/2 and NMNAT1/2/3 have previously cloned and 10 min. His-select Nickel Affinity Gel (Sigma) was40 characterized. A mammalian homologue of NaPRT was washed with buffer A and 0.1 ml of 50% gel suspension also identified as an EST annotated as a mammalian was added to 1 ml of supernatant and incubated for 10 homolog of a bacterial NaPRT. min at 4°C, then beads binding hexa-histidine -tagged [0083] To investigate the expression of mammalian protein was extensively washed with the buffer A. Pro- NAD biosynthetic enzymes in the nervous system, we teins were eluted by adding 100 ml of the solution con- 45 performed quantitative RT-PCR using RNA from mouse taining 50 mM Sodium Phosphate (pH 8.0), 300 mM Na- brain, retina, spinal code, and DRG at age of E14, P0, Cl, and 250 mM imidazole. Relative NMNAT enzymatic P7, P14 and P21. All enzymes are expressed ubiquitous- activity was measured by using affinity purified proteins ly in the nervous system throughout the development and as described before and subtracted the value obtained in adulthood, with an exception of Nrk2, whose expres- from mock transfected cells and normalized by the50 sion is very low in all examined tissues (data not shown). amount of recombinant protein determined by densitom- To identify inducibility of NAD-synthesizing enzymes in etry. response to neuronal insults, we compared the RNA ex- [0080] Administration of NAD biosynthetic sub- pression of each enzyme in DRGs at 1, 3, 7, and 14 days strates and optic Nerve transection. Nam, NMN, NmR, after sciatic nerve transection against non-injured DRG. or NAD was dissolved in PBS at the concentration of 100 55 As shown in Fig. 6A, most of the enzymes were up-reg- mM or 1 M. Each of 5m l solution was injected into left ulated 2 to 8-fold after injury. Among those, Nrk2 expres- intravitreal component under the anesthesia at a rate of sion is exceptionally highly induced (more than 20-fold) 0.5 ml ml per second. The left optic nerve was transected at 14 days after axotomy. We also analyzed expression

13 23 EP 1 755 391 B1 24 of NAD synthetic enzymes during the axonal degenera- showed same extent of delay in neurite degeneration as tion caused by neurotoxin in cultured rat DRG neuron. well as NMNAT1 (Fig. 7A, E). The lentivirus mediated DRG neurons were treated with 0.1 mM and 1 mM roten- expression of each enzyme was confirmed by Western one to cause axonal degeneration and collected RNA at blotting (Fig. 7D). These experiments confirmed that 24 hours after the addition of rotenone. The expression 5 NMNAT targeted to either the nucleus or cytosol protects of Nrk2 was increased more than 6 folds after rotenone neurite from degeneration. treatment (Fig. 6B). These results suggest that, while all enzymatic activities in NAD synthesis pathway is ubiqui- EXAMPLE 10 tously present, Nrk2 may be responsible for supplying NAD synthesizing substrate after neuronal insults. 10 [0087] This example illustrates that exogenous application of substrates for NAD biosynthetic enzymes pro- EXAMPLE 9 tects axon from degeneration. [0088] We have previously shown that exogenously [0084] This example illustrates that both nuclear and applied NAD in the culture medium shows axonal saving cytoplasmic Nmat enzymes save axons from degenera- 15 effectin vitro. Here weshowed that expression of NmPRT tion. also shows axonal protection suggesting that Nam is [0085] To determine whether nuclear localization of used as a substrate for NAD synthesis in neurons. To NMNAT1 is essential to provide the axonal protection, determine which substrate shown in Fig. 5 is used for we analyzed the effect of subcellular distribution of NAD synthesis in neurons and to identify whether any of NMNAT enzyme in the in vitro Wallerian degeneration 20 NAD precursors may be able to save axons similar to or assay and compared the extent of axonal protection be- possibly better than NAD, we applied Na, Nam, NmR, tween overexpression of cytoplasmic and nuclear NaMN, NMN, or NaAD in the culture media and per- NMNAT. NMNAT1 has putative nuclear localization sig- formed in vitro Wallerian degeneration assay. An appli- nal PGRKRKW in the 211-217 amino-acids of NMNAT1 cation of 1 mM NMN for 24 hours before neurite transec- protein. We generated a mutant NMNAT1 designated as 25 tion successfully saved neurites from degeneration. cytNMNAT1 in which this nuclear localization signal was Quantitative analysis revealed that NMN treatment re- altered as PGAAAAW and examined subcellular distri- sults in neurite protection to an extent similar to that bution. As shown in Fig. 7B, the majority of cytNMNAT1 achieved by exogenously applied NAD (Fig. 8B). These located in the cytosol as we expected. results further suggested the possibility that increased [0086] Next we confirmed enzymatic activity 30 of supply of other NAD biosynthetic substrates have an abil- cytNMNAT1, NMNAT1 and its mutant cytNMNAT1 were ity to save neurites from degeneration. We then exoge- purified from the cell lysate expressing either of proteins nously applied 1 mM of NAD biosynthetic substrates in- by using affinity gel. The enzymatic activity of affinity pu- cluding Na, Nam, NaMN, NaAD, and NmR to the DRG rified proteins was measured as described above and neurons for 24 hours and performed neurite transection. we found that cytNMNAT1 activity did not altered by its 35 As shown in Fig. 8A and B, NaMN or NmR treatment also mutation (Fig. 7C). After the overexpression ofsaved neurites as well as NAD. NaAD showed slight pro- cytNMNAT1 in DRG neurons, we observed strong neu- tection but Na failed to save neurites, while Na and Nam rite protection as well as nuclear wild NMNAT1 (Fig.7A, had no effect. Quantitative analysis revealed that exog- E). We further confirmed this result by using NMNAT1 enous application of 1mM NaMN, NMN, NmR, or NAD isoenzyme that lacks nuclear localization signal. Among 40 caused comparable increase in intact neurites at 48 two NMNAT isoenzymes, NMNAT3 is previously report- hours after transection (Fig. 8B). Because the protective ed to locate outside nucleus and mitochondria, and have effect of NaMN is equal to NMN, a step synthesize NAD comparable enzymatic activity to NMNAT1. We added from NaAD by QNS is active enough to save neurites nuclear localization signal KPKKIKTED of human topoi- under the increased supply of NaAD. Nevertheless, ex- somerase I to the C-terminal of NMNAT3 to generate 45 ogenous application of NaAD shows less increase in in- nuclear NMNAT3. We expressed hexa-histidine tagged tact neurites at 48 hours compared with NAD (Fig. 8B). NMNAT3 or nucNMNAT3 in HEK293T cells and ana- This indicates insufficient incorporation into the cell or lyzed subcellular localization and its enzymatic activity. instability of NaAD in our assay condition. These exper- NMNAT3 was distributed outside the nucleus including iments suggest that there are several different ways to bright punctuate staining as reported before 50 andsave neurites including exogenous application of NMN, nucNMNAT3 mainly localized in the nucleus with some NaMN, and NmR. All of these treatments seem to cause punctuate staining in the cytosol (Fig. 7B). The enzymatic increased supply of NAD and it is consistent to the pre- activity of NMNAT3 and nucNMNAT3 were measured vious experiments showing NAD application or NMNAT1 and both proteins have comparable enzymatic activity overexpression save neurites from degeneration. compared with NMNAT1 (Fig. 7C). Then, in vitro Walle- 55 rian degeneration assay was performed after overex- EXAMPLE 11 pression of these two NMNAT3 enzymes, and we found that overexpression of both NMNAT3 and nucNMNAT3 [0089] This example demonstrates that intraviteal ap-

14 25 EP 1 755 391 B1 26 plication of NAD biosynthetic substrates delays the ax- with neurodegenerative disease, motor neuron dis- onal degeneration of retinal ganglion cells. ease, neoplasia, endocrine disorder, metabolic dis- [0090] Transection of optic nerve is an in vivo model ease, nutritional deficiency, atherosclerosis, an au- which can be used to investigate mechanisms leading to toimmune disease, mechanical injury, chemical or Wallerian degeneration and following retinal ganglion cell 5 drug-induced injury, thermal injury, radiation injury, (RGC) death observed in human diseases such as glau- nerve compression, retinal or optic nerve disorder, coma. In the C57BL/Wlds mouse strain, optic nerve de- mitochondrial dysfunction, progressive dementia generation during Wallerian degeneration after axotomy demyelinating diseases ischemia and/or stroke in- is dramatically slowed. In addition, intravitreal injection fectious disease; or inflammatory disease. is used for screening of compounds that protect RGC 10 axon from degeneration in vivo and thus we can asses the axon protective effect of each NAD biosynthetic sub- Patentansprüche strates in vivo by intraocular injection of compounds including NAD, NMN, NmR, and Nam. From in vitro Walle- 1. Verwendung eines Mittels zur Behandlung oder Prä- rian degeneration assay, 1mM of NAD, NMN, and NmR 15 vention einer Axonopathie bei einem Säuger, der in the culture media is enough to protect axon from de- dessen bedarf, wobei das Mittel NaMN, NMN, NmR, generation. We initially injected 5 ml of 100 mM or 1 M eine Nukleinsäure, die für eine NMNAT kodiert, oder NAD solution into left intravitreal compartment. After 24 Resveratrol ist. hours incubation, left optic nerve was transected and control (right) and axotomized (left) optic nerve were col- 20 2. Verwendung nach Anspruch 1, wobei das Mittel eine lected at 3, 4, and 5 days after transection. Neurofilament Nukleinsäure ist, die für eine NMNAT kodiert. immunoreactivity from the axotomized optic nerve was measured and normalized against the value obtained 3. Verwendung nach Anspruch 1, wobei das Mittel eine from the right side of the optic nerve. We found that the Nukleinsäure ist, die für eine humane NMNAT1 oder immunoreactivity at 4days after transection 25 was eine humane NMNAT3 kodiert. 77627% and 78622% of non-axotomized optic nerve in 1 M and 100 mM NAD injected rats respectively, while 4. Verwendung nach einem der Ansprüche 1, 2 oder control animal showed only 7616 % (Fig. 9) 3, wobei die Axonopathie ererbt oder angeboren ist [0091] We then injected 5 ml of 100 mM NMN, NmR, oder mit neurodegenerativer Erkrankung, Motoneu- and Nam into left intravitreal compartment and collected 30 ronerkrankung, Neoplasie, endokriner Erkrankung, optic nerves at 4 days after left optic nerve transaction. Stoffwechselerkrankung, Ernährungsmängeln, The immunoreactivity obtained from NMN and NmR in- Atherosklerose, einer Autoimmunerkrankung, me- jected optic nerve was 60625 and 72619 % of non-ax- chanischer Schädigung, chemischer oder arzneimit- otomized nerve. Nam injected animals did not show any telinduzierter Schädigung, thermischer Schädigung, difference from the control animals. These results are 35 Strahlungsschädigung, Nervenkompression, Reti- consistent with the in vitro study that showed NAD, NMN, na- oder Sehnerverkrankung, mitochondrialer Dya- and NmR have axon saving activity but Nam does not. funktion, progressiver Demenz, demyelinisierenden Our in vivo study revealed that these small molecules Erkrankungen, Ischämie und/oder Schlaganfall, In- that are involved in the NAD biosynthetic pathway are fektionserkrankung oder entzündlicher Erkrankung useful tools to save axon from degeneration. 40 assoziiert ist.

Claims Revendications

1. The use of an agent for treatment or prevention of 45 1. Utilisation d’un agent pour le traitement ou la pré- an axonopathy in a mammal in need thereof, wherein vention d’une axonopathie chez un mammifère qui the agent is NaMN, NMN, NmR, a nucleic acid en- en a besoin, dans lequel l’agent est la NaMN, la coding an NMNAT or resveratrol. NMN, la NmR, un acide nucléique codant pour la NMNAT ou le resveratrol. 2. The use of claim 1 wherein the agent is a nucleic 50 acid encoding an NMNAT. 2. Utilisationde la revendication 1, dans laquelle l’agent est un acide nucléique codant pour une NMNAT. 3. The use of claim 1, wherein the agent is a nucleic acid encoding a human NMNAT1 or a human 3. Utilisationde la revendication 1, dans laquelle l’agent NMNAT3. 55 est un acide nucléique codant pour la NMNAT1 humaine ou la NMNAT3 humaine. 4. The use of any one of claims 1, 2 or 3, wherein the axonopathy is hereditary or congenital or associated 4. Utilisation selon l’une quelconque des revendica-

15 27 EP 1 755 391 B1 28 tions 1, 2 ou 3, dans laquelle l’axonopathie est hé- réditaire ou congénitale ou associée à une maladie neurodégénérative, une maladie du neurone mo- teur, d’une néoplasie, d’un trouble endocrinien, d’une maladie métabolique, d’une insuffisance nu- 5 tritionnelle, de l’athérosclérose, d’une maladie autoimmune, d’une blessure mécanique, d’une blessure chimique ou induite par un médicament, d’une blessure thermique, d’une blessure par rayonnement, d’une compression nerveuse, d’un trouble de la ré- 10 tine ou du nerf optique, d’un dysfonctionnement mitochondrial, de la démence progressive, des maladies démyélinisantes, l’ischémie et/ou une crise car- diaque, des maladies infectieuses ; ou des maladies inflammatoires. 15

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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