(19) TZZ¥ZZZZ_T

(11) EP 3 006 040 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 13.04.2016 Bulletin 2016/15 A61K 38/50 (2006.01) A61K 38/45 (2006.01) A61K 31/7084 (2006.01) C07H 19/20 (2006.01) (2006.01) (2006.01) (21) Application number: 15193068.2 A61K 48/00 C12N 9/12

(22) Date of filing: 03.06.2005

(84) Designated Contracting States: • ARAKI, Toshiyuki AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Kodaira, Tokyo 187-0031 (JP) HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR • SASAKI, Yo St. Louis, MO 63144 (US) (30) Priority: 04.06.2004 US 577233 P 04.01.2005 US 641330 P (74) Representative: Smaggasgale, Gillian Helen WP Thompson (62) Document number(s) of the earlier application(s) in 138 Fetter Lane accordance with Art. 76 EPC: London EC4A 1BT (GB) 05790283.5 / 1 755 391 Remarks: (71) Applicant: Washington University This applicationwas filed on 4.11.2015 as a divisional Saint Louis, MO 63130 (US) application to the application mentioned under INID code 62. (72) Inventors: • MILBRANDT, Jeffrey St. Louis, MO 63105 (US)

(54) METHODS AND COMPOSITIONS FOR TREATING NEUROPATHIES

(57) Methods of treating or preventing axonal degra- dation in neuropathic diseases in mammals are dis- closed. The methods can comprise administering to the mammal an effective amount of an agent that acts by increasing sirtuin activity in diseased and/or injured neu- rons. The methods can also comprise administering to the mammal an effective amount of an agent that acts by increasing NAD activity in diseased and/or injured neurons.Also disclosed aremethods of screening agents for treating a neuropathies and recombinant vectors for treating or preventing neuropathies. EP 3 006 040 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 006 040 A1 2

Description SIRT1. The activation of SIRT1 results in a decrease in severity of axonal degeneration or a prevention of axonal GOVERNMENT INTERESTS degeneration. It is also believed possible that the in- crease in NAD activity could act through other mecha- [0001] This work was supported at least in part with 5 nisms not involving sirtuin. Thus, increasing NAD activity, funds from the federal government under U.S.P.H.S. which may act through increasing SIRT1 activity or 5RO1 NS40745. The U.S. Government may have certain through one or more other mechanisms or both can di- rights in the invention. minish or prevent axonal degeneration in injured mam- malian axons. RELATED APPLICATION DATA 10 [0006] Thus, in various embodiments, the present in- vention is directed to a method of treating or preventing [0002] This application claims benefit under 35 U.S.C. a neuropathy in a mammal and, in particular, in a human § 119(e) to United States Provisional Application Serial in need thereof. The method can comprise administering No. 60/577,233, filed June 4, 2004 and United States an effective amount of an agent that acts to increase Provisional Application Serial No. 60/641,330, filed Jan- 15 sirtuin activity and, in particular, SIRT1 activity in dis- uary 4,2005. These applications are incorporated herein eased and/or injured neurons. in their entireties by reference. [0007] In various embodiments, the agent can in- crease SIRT1 activity through increasing NAD activity. It FIELD is believed that increasing NAD activity can increase sir- 20 tuin activity because NAD can act as a substrate of [0003] This invention relates generally to diseases and SIRT1. Such agents can include NAD or NADH, a pre- conditions involving neurons and, more particularly, to cursor of NAD, an intermediate in the NAD salvage path- methodsand compositions for treatingor preventingneu- way or a substance that generates NAD such as a nico- ropathies and other diseases and conditions involving tinamide mononucleotide adenylyltransferase (NMNAT) neurodegeneration. Also included are methods of iden- 25 or a nucleic acid encoding a nicotinamide mononucle- tifying agents for treating or preventing neuropathies. otide adenylyltransferase. The nicotinamide mononucle- otide adenylyltransferase can be an NMNAT1 protein. BACKGROUND [0008] In various embodiments, the agent can also act to directly increase SIRT1 activity and as such, the agent [0004] Axon degeneration occurs in a variety of neu- 30 can be a sirtuin polypeptide or a nucleic acid encoding a rodegenerative diseases such as Parkinson’s and Alzhe- sirtuin polypeptide or a substance such as a stilbene, a imer’s diseases as well as upon traumatic, toxic or chalcone, a flavone, an isoflavanone, a flavanone or a ischemicinjury to neurons. Such diseases and conditions catechin. Such compounds can include a stilbene select- are associated with axonopathies including axonal dys- ed from the group consisting of resveratrol, piceatannol, function. One example of axonopathy is Wallerian de- 35 deoxyrhapontin, trans-stilbene and rhapontin; a chal- generation (Waller, Philos Trans R. soc. Lond. cone selected from the group consisting of butein, isoli- 140:423-429,1850), whichoccurs when the distal portion quiritigen and 3,4,2’,4’,6’-pentahydroxychalcone; a fla- of the axon is severed from the cell body. The severed vone selected from the group consisting of fisetin, axon rapidly succumbs to degeneration. Axonopathy 5,7,3’,4’,5’-pentahydroxyflavone, luteolin, 3,6,3’,4’-tet- can, therefore, be a critical feature of neuropathic dis- 40 rahydroxyflavone, quercetin, 7,3’,4’,5’-tetrahydroxyfla- eases and conditions and axonal deficits can be an im- vone, kaempferol, 6-hydroxyapigenin, apigenin, portant component of the patient’s disability. 3,6,2’,4’-tetrahydroxyflavone, 7,4’-dihydroxyflavone, 7,8,3’,4’-tetrahydroxyflavone, 3,6,2’,3’-tetrahydroxyfla- SUMMARY vone, 4’-hydroxyflavone, 5,4’-dihydroxyflavone, 5,7-di- 45 hydroxyflavone, morin, flavone and 5-hydroxyflavone; an [0005] Accordingly, the present inventors have suc- isoflavone selected from the group consisting of daidzein ceeded in discovering that axonal degeneration can be and genistein; a flavanone selected from the group con- diminished or prevented by increasing NAD activity in sisting of naringenin, 3,5,7,3’,4’-pentahydroxyflavanone, diseased and/or injured neurons. It is believed that the and flavanone or a catechin selected from the group con- increased NAD activity can act to increase sirtuin activity 50 sisting of (-)-epicatechin, (-)-catechin, (-)-gallocatechin, which then produces a decrease in axonal degeneration (+)-catechin and (+)-epicatechin. of injured neuronal cells. Thus, one approach to prevent- [0009] In various embodiments, the invention can also ing axonal degeneration can be by activating sirtuin mol- involve methods of treating a neuropathy by administer- ecules, i.e. SIRT1 in injured mammalian axons. The ac- ing to a mammal and, in particular, a human, an effective tivation of SIRT1 can be through direct action on the55 amount of an agent that acts by increasing nuclear NAD SIRT1 molecule or by increasing the supply of nicotina- activity in diseased and/or injured neurons and/or sup- mide adenine dinucleotide (NAD) which acts as a sub- porting cells such as, for example, glia, muscle cells, fi- strate for the histone/protein deacetylase activity of broblasts, etc.

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[0010] Such agent can be NAD or NADH, nicotinamide activity can be an increase in nuclear NAD activity. mononucleotide, nicotinic acid mononucleotide or nico- [0015] Methods are also provided for screening agents tinamide riboside or derivatives thereof; or an enzyme that increase sirtuin activity in neurons as well as for that generates NAD such as a nicotinamide mononucle- screening agents that increase NAD biosynthetic activity otide adenylyltransferase or a nucleic acid encoding an 5 in neurons. The methods can comprise administering to enzyme that generates NAD such as a nucleic acid en- mammalian neuronal cells in vitro or in vivo a candidate coding a nicotinamide mononucleotide adenylyltrans- agent, producing an axonal injury to the neuronal cells ferase or an agent that increases expression of a nucleic and detecting a decrease in axonal degeneration of the acid encoding an enzyme in a pathway that generates injured neuronal cells. Such methods can in some em- NAD or an agent that increases activity and/or stability 10 bodiments be primary screening methods in which sec- of an enzyme in a pathway that generates NAD or an ondary assays further delineate activity as associated agent that increases NAD activity. The nicotinamide with sirtuin activity or with NAD and enzymes or compo- mononucleotide adenylyltransferase can be an NMNAT1 nents of NAD biosynthetic or salvage pathways. protein. [0016] In various embodiments of the screening meth- [0011] In various embodiments, the invention can also 15 ods of the present invention, axonal injury can be pro- involve methods of treating or preventing an optic neu- duced by a number of methods including chemically in- ropathy in a mammal in need thereof. The methods can juring the neuronal cells, thermally injuring the neuronal comprise administering to the mammal an effective cells, oxygen-depriving the neuronal cells, and physically amount of an agent that acts by increasing NAD activity injuring the neuronal cells. in diseased and/or injured neurons. Administering to the 20 [0017] A recombinant vector is also provided in various mammal can comprise administering to the eye, in par- embodiments. The vector can comprise a promoter op- ticular by administering the agent with a sustained re- eratively linked to a sequence encoding a mammalian lease delivery system or by administering a sustain re- NMNAT1 protein or NMNAT3 protein. In various aspects lease pellet comprising the agent to the eye. of such embodiments, the recombinant vector can be a [0012] The agent can be NAD or NADH, nicotinamide 25 lentivirus or an adeno-associated virus. mononucleotide, nicotinic acid mononucleotide or nico- [0018] Also provided in various embodiments, is a re- tinamide riboside; or an enzyme that generates NAD combinant vector comprising a promoter operatively such as a nicotinamide mononucleotide adenylyltrans- linked toa sequence encoding a SIRT1 protein. Invarious ferase; or a nucleic acid encoding an enzyme that gen- aspects of such embodiments, the recombinant vector erates NAD such as a nucleic acid encoding a nicotina- 30 can be a lentivirus or an adeno-associated virus. mide mononucleotide adenylyltransferase or an agent that increases NAD activity. The nicotinamide mononu- BRIEF DESCRIPTION OF THE DRAWINGS cleotide adenylyltransferase can be an NMNAT1 protein or an NMNAT3 protein. [0019] Figure 1 illustrates that NMNAT1 activity of the [0013] In various embodiments of the methods of the 35 Wlds fusion protein produces a delayed degeneration of present invention, the neuropathy associated with axonal injured axons showing: degradationcan be any of anumber ofneuropathies such as, for example, those that are hereditary or congenital A) in vitro Wallerian degeneration in lentivirus-infect- or associated with Parkinson’s disease, Alzheimer’s dis- ed dorsal root ganglia (DRG) neuronal explant cul- ease, Herpes infection, diabetes, amyotrophic lateral40 tures expressing Wlds protein or EGFP wherein tu- sclerosis, a demyelinating disease, ischemia or stroke, bulin βIII-immunoreactive neurites are shown before chemical injury, thermal injury, AIDS and the like. In ad- transection and 12,24,48, and 72 hr after transection dition,neurodegenerative diseases not mentioned above (Scale Bar=1mm and the "*" denotes the location of as well as a subset of the above mentioned diseases can the cell bodies prior to removal; and also be treated with the methods of the present invention. 45 B) in vitro Wallerian degeneration in lentivirus-infect- Such subsets of diseases can include Parkinson’s dis- ed DRG neurons expressing EGFP only, Wlds pro- ease or non-Parkinson’s diseases, Alzheimer’s disease tein, Ufd2a portion (70 residues) of Wlds protein or non-Alzheimer’s diseases and so forth. fused to EGFP (Ufd2a(1-70)-EGFP), [0014] In various embodiments, the present invention Ufd2a(1-70)-EGFP with C-terminal nuclear localiza- is also directed to methods of screening agents for treat- 50 tion signal, NMNAT1 portion of Wlds protein fused ing a neuropathy in a mammal. The methods can com- to EGFP, dominant-negative Ufd2a prise administering to neuronal cells in vitro or in vivo, a (Ufd2a(P1140A)), or Ufd2a siRNA construct in which candidate agent, producing an axonal injury to the neu- representative images of neurites and quantitative ronal cells and detecting a decrease in axonal degener- analysis data of remaining neurite numbers (per- ation of the injured neuronal cells. In various embodi- 55 centage of remaining neurites relative to pre- ments, the method can comprise detecting an increase transection 6 S.D.) at the indicated time-point with in NAD activity produced by a candidate agent, in a cell each construct (bottom left) are shown and the "*" and, in particular, in a neuronal cell. The increase in NAD indicates significant difference (p<0.0001) with

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EGFP-infected neurons; also showing EGFP signal [0022] Figure 4 illustrates that NAD-dependent Axonal before transection confirming transgene expression 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 5 culturespreincubated with 1 mM NADalone (control) protein (bottom right panels). or in the presence of either 100 mM Sirtinol (a Sir2 inhibitor) or 20 mM 3-aminobenzimide (3AB, a PARP [0020] Figure 2 illustrates that increased NAD supply inhibitor); protects axons from degeneration after injury showing: B) invitro Wallerian degeneration using DRG explant 10 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- 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 the15 (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- (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 20 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- (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- 25 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- pendent axonal protection. centage of remaining neurites relative to pre- transection 6 S.D.) and the "*" indicates significant [0023] Figure 5 illustrates the mammalian NAD biosyn- difference (p<0.0001) with EGFP-infected neurons; 30 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 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- 35 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- tinamide mononucleotide adenylyltransferase; QNS, cated times after vincristine addition and quantifica- NAD synthetase) tion of the protective effect at the indicated time [0024] Figure 6 illustrates expression analysis of NAD points is plotted as the area covered by neurites rel- 40 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 after nerve transection in rat DRG were determined by [0021] 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: 45 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- 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 50 in the text. B) DRG explants preincubated with 1mM NAD for 4, [0025] Figure7 illustrates the subcellular localization of 8, 12, 24, or 48 h prior to transection wherein the bar 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- 55 ing NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 in dicated time points and the "*" indicates significant which representative pictures taken at 12 and 72 hours axonal protection compared to control (p<0.0001). after transaction are shown; (B) Subcellular localization of NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 in

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HEK 293T cells using immunohistochemistry with anti- NAD activity can act to increase sirtuin activity which then body against 6xHis tag to detect each proteins and stain- produces a decrease in axonal degeneration of injured ing of the cells with the nuclear marker dye (bisbenzim- neuronal cells compared to axonal degeneration that oc- ide) for comparison to determine the nuclear vs. cyto- curs in injured neuronal cells not treated with the agent. plasmic location of each protein (Scale bar = 25 mm); (C) 5 Such decrease in axonal degeneration can include a enzymatic activity of wild type and mutant NMNAT1 and complete or partial amelioration of the injury to the neu- NMNAT3in which 6xHis tagged each protein was purified ron. It is also believed possible that the increase in NAD from lysate of HEK293T cells expressing NMNAT1, activity could act through other mechanisms not involving cytNMNAT1, NMNAT3, nucNMNAT3 in which the sirtuin molecules to produce or to contribute to the pro- amount of NAD generated after1 hour at 37 deg was10 duction of a decrease in axonal degeneration. converted NADH, quantified and normalized to protein [0029] Seven known sirtuin molecules referenced as concentration; (D) protein expression of NMNAT1, 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 15 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, 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. 20 bi.nlm.hih.gov/LocusLink/). Said NCBI LocusLink refer- [0026] Figure 8 illustrates exogenous application of ence sites are hereby incorporated by reference. In var- NAD biosynthetic substrates and their ability to protect ious embodiments, the methods and compositions of the axon showing (A) in vitro Wallerian degeneration assay present invention can increase activity of any one or more using DRG neuronal explant cultures after exogenous of the sirtuins and, in particular, various methods of the application of NAD, NmR with representative pictures 25 present invention increase activity of SIRT1. taken at 12, 24, 48, and 72 hours after transaction are [0030] By activity of a substance, reference is made to shown; (B) in vitro Wallerian degeneration assay using either the concentration of the particular substance or DRG neuronal explant cultures after exogenous applica- functional effectiveness of the substance. Concentration tion of Na, Nam, NaMN, NMN, NaAD, NAD, and NmR of a substance can be increased by numerous factors showing quantitative analysis data of remaining neurite 30 including, for example, increasing synthesis, decreasing numbers at 12, 24, 48, and 72 hours after axotomy are breakdown, increasing bioavailability of the substance or shown; (C) DRG neuronal explants infected with NaPRT diminishing binding of the substance or otherwise in- expressing lentivirus and incubated with or without 1 mM creasingthe available amountof free substance. Increas- of Na for 24 hours before axotomy, in in vitro Wallerian ing functional effectiveness can result, for example, from degeneration assay showing quantitative analysis data 35 a change in molecular conformation, a change in the con- of remaining neurite numbers at 12, 24, 48, and 72 hours ditions under which the substance is acting, a change in after axotomy. sensitivity to the substance, and the like. Increasing ac- [0027] Figure 9 illustrates optic nerve transection after tivity with respect to sirtuin molecules is intended to mean intravitreal injection of NAD biosynthetic substrates NAD, increasing concentration or enhancing functional effec- NMN, NmR, or Nam was injected into intravitreal com- 40 tiveness or increasing the availability of NAD or increas- partment of left rat eye and allowed to incorporate retinal ing the flux through one or more biosynthetic pathways ganglion cells for 24 hours after which, left optic nerve for NAD or any combination thereof. was transected by eye enucleation and right and left optic [0031] Neuropathies can include any disease or con- nerves were collected at 4 days after transection and dition involving neurons and/or supporting cells, such as analyzed by Western blotting in which optic nerves45 for example, glia, muscle cells, fibroblasts, etc., and, in transected from mice without any treatment prior to ax- particular, those diseases or conditions involving axonal otomy were used for negative control; showing in the damage. Axonal damage can be caused by traumatic figure, the quantitative analysis data of percentage of re- injury or by non-mechanical injury due to diseases or maining neurofilament immunoreactivity from transected conditions and the result of such damage can be degen- optic nerve relative to non-transected 6 S.D. 50 eration or dysfunction of the axon and loss of functional neuronal activity. Disease and conditions producing or DETAILED DESCRIPTION associated with such axonal damage are among a large number of neuropathic diseases and conditions. Such [0028] The present invention involves methods and neuropathies can include peripheral neuropathies, cen- compositions for treating neuropathies. The methods can 55 tral neuropathies, and combinations thereof. Further- comprise administering to a mammal an effective amount more, peripheral neuropathic manifestations can be pro- of a substance that increases NAD activity in diseased duced by diseases focused primarily in the central nerv- and/or injured neurons. It is believed that the increased ous systems and central nervous system manifestations

5 9 EP 3 006 040 A1 10 can be produced by essentially peripheral or systemic deficiencies such as deficiencies in vitamins B12 or folic diseases. acid, and toxicities such as due to ethambutol or cyanide; [0032] 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 5 neuropathies also include non-arteritic anterior ischemic such diseases can include diabetes, uremia, infectious optic neuropathy. diseases such as AIDs or leprosy, nutritional deficien- [0035] Neurodegenerative diseases that are associat- cies, vascular or collagen disorders such as atheroscle- ed with neuropathy or axonopathy in the central nervous rosis, and autoimmune diseases such as systemic lupus system include a variety of diseases. Such diseases in- erythematosus, scleroderma, sarcoidosis, rheumatoid 10 clude those involving progressive dementia such as, for arthritis, and polyarteritis nodosa. Peripheral nerve de- example, Alzheimer’s disease, senile dementia, Pick’s generation can also result from traumatic, i.e mechanical disease, and Huntington’s disease; central nervous sys- damage to nerves as well as chemical or thermal damage tem diseases affecting muscle function such as, for ex- to nerves. Such conditions that injure peripheral nerves ample, Parkinson’s disease, motor neuron diseases and include compression or entrapment injuries such as glau- 15 progressive ataxias such as amyotrophic lateral sclero- coma, carpal tunnel syndrome, direct trauma, penetrat- sis;demyelinating diseases such as, for example multiple ing injuries, contusions, fracture or dislocated bones; 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; 20 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 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 25 can cause central nervous system neuropathies. such as vincristine. Typical symptoms of such peripheral [0036] The term "treatment" as used herein is intended neuropathies include weakness, numbness, paresthesia 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- 30 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 rons occurs. Such primary insult to the neurons can in- ATP. clude or result from any disease or condition associated [0033] The peripheral neuropathy can also be a met- with a neuropathy. "Treatment" also includes prevention abolic and endocrine neuropathy which includes a wide 35 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, 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- 40 as neutraceuticals, food additives or functional foods. ria, disorders of lipid/glycolipid metabolism, nutritional/vi- [0037] The methods and compositions of the present tamin deficiencies, and mitochondrial disorders, among invention areuseful in treating mammals. Such mammals others. The common hallmark of these diseases is in- include humans as well as non-human mammals. Non- volvement of peripheral nerves by alteration of the struc- human mammals include, for example, companion ani- ture or function of myelin and axons due to metabolic 45 mals such as dogs and cats, agricultural animals such pathway dysregulation. live stock including cows, horses and the like, and exotic [0034] Neuropathies also include optic neuropathies animals, such as zoo animals. such as glaucoma; retinal ganglion degeneration such [0038] 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- 50 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 tion that accompanies the paper all of which is incorpo- example, injury during tumor removal; hereditary optic rated herein by reference). Such compounds can include neuropathies such as Kjer’s disease and Leber’s hered- stilbenes such as resveratrol, piceatannol, deoxyrhapon- itary optic neuropathy; ischemic optic neuropathies, such 55 tin, trans-stilbene and rhapontin; chalcone such as as those secondary to giant cell arteritis; metabolic optic butein, isoliquiritigen and 3,4,2’,4’,6’-pentahydroxychal- neuropathies such as neurodegenerative disesases in- cone and chalcone; flavones such as fisetin, 5,7,3’,4’,5’- cluding Leber’s neuropathy mentioned earlier, nutritional pentahydroxyflavone, luteolin, 3,6,3’,4’-tetrahydroxyfla-

6 11 EP 3 006 040 A1 12 vone, quercetin, 7,3’,4’,5’-tetrahydroxyflavone, kaemp- [0041] In various embodiments, NAD can be increased ferol, 6-hydroxyapigenin, apigenin, 3,6,2’,4’-tetrahydrox- in injured neurons by administering enzymes that syn- yflavone, 7,4’-dihydroxyflavone, 7,8,3’,4’-tetrahydroxy- thesize NAD or nucleic acids comprising enzymes that flavone, 3,6,2’,3’-tetrahydroxyflavone, 4’-hydroxyfla- synthesize NAD. Such enzymes can include an enzyme vone, 5,4’-dihydroxyflavone, 5,7-dihydroxyflavone,5 in the de novo pathway for synthesizing NAD, an enzyme morin, flavone and 5-hydroxyflavone; isoflavones such of the NAD salvage pathway or an enzyme of the nicoti- as daidzein and genistein; flavanones such as naringen- namide riboside kinase pathway or a nucleic acid encod- in, 3,5,7,3’,4’-pentahydroxyflavanone, and flavanone or ing an enzyme in the de novo pathway for synthesizing catechins such as (-)-epicatechin, (-)-catechin, (-)-gallo- NAD, an enzyme of the NAD salvage pathway or an en- catechin, (+)-catechin and (+)-epicatechin. 10 zyme of the nicotinamide riboside kinase pathway and, [0039] Additional polyphenols or other substance that in particular, an enzyme of the NAD salvage pathway increase sirtuin deacetylase activity can be identified us- such as, for example, a nicotinamide mononucleotide ing assay systems described herein as well as in com- adenylyltransferase (NMNAT) such as NMNAT1. Thus, mercially available assays such as fluorescent enzyme in one non-limiting example, administration of an NMNAT assays (Biomol International L.P., Plymouth Meeting, 15 such as NMNAT1 or NMNAT3 or a nucleic acid compris- Pennsylvania). Sinclair et al. also disclose substances ing a sequence encoding an NMNAT such as NMNAT1 that can increase sirtuin activity (Sinclair et al.,or NMNAT3 can diminish or prevent axonal degeneration WO2005/02672 which is incorporated in its entirety by in injured neurons. reference). [0042] The human NMNAT1 enzyme (E.C.2.7.7.18) is [0040] In various embodiments, other substances can 20 represented according to the GenBank Assession num- increase sirtuin activity indirectly by increasing NAD ac- bers for the human NMNAT1 gene and/or pro- tivity as a result of the particular sirtuin functioning tein:NP_073624; NM_022787; AAL76934; AF459819; through NAD-dependent histone/protein deacetylase ac- and NP_073624; AF314163. A variant of this gene is tivity. NAD activity can be increased by administration of NMNAT-2 (KIAA0479), the human version of which can NAD or NADH as well as by synthesizing NAD. NAD can 25 be found under GenBank Accession numbers be synthesised through three major pathways, thede NP_055854 and NM_015039. novo pathway in which NAD is synthesized from tryp- [0043] As used herein, the term "percent identical" or tophan, the NAD salvage pathway in which NAD is gen- "percent identity" or "% identity" refers to sequence iden- erated by recycling degraded NAD products such as tity between two amino acid sequences or between two nicotinamide (Lin et al. Curent Opin. Cell 30 Biol.nucleotide sequences. Identity can each be determined 15:241-246, 2003; Magni et al., Cell Mol. Life Sci. by comparing a position in each sequence which may be 61:19-34, 2004) and the nicotinamide riboside kinase aligned for purposes of comparison. When an equivalent pathway in which nicotinamide riboside is converted to position in the compared sequences is occupied by the nicotinamide mononucleotide by nicotinamide riboside same base or amino acid, then the molecules are iden- kinase (Bieganowski et al., Cell 117:495-502, 2004).35 tical at that position; when the equivalent site occupied Thus, administering to injured neurons, a precursor of by the same or a similar amino acid residue (e.g., similar NAD in the de novo pathway such as, for example, tryp- in steric and/or electronic nature), then the molecules tophan or nicotinate and/or substances in the NAD sal- can be referred to as homologous (similar) at that posi- vage pathway such as, for example, nicotinamide, nico- tion. Expression as a percentage of homology, similarity, tinic acid, nicotinic acid mononucleotide, or deamido- 40 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 TREZ. FASTA and BLAST are available as a part of the mononucleotide, nicotinic acid mononucleotide or nico- 45 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- icine, National Institutes of Health, Bethesda, Md. In one tone/protein deacetylase activity in the injured neurons 50 embodiment, the percent identity of two sequences can and diminish or prevent axonal degeneration. In addition, be determined by the GCG program with a gap weight it is believed that other substances can act by increasing of 1, e.g., each amino acid gap is weighted as if it were enzyme activity or by increasing levels of NAD, nicotina- a single amino acid or nucleotide mismatch between the mide mononucleotide, nicotinic acid mononucleotide, two sequences. Other techniques for alignment are de- nicotinamide riboside or sirtuin enzymes or by decreas- 55 scribed in Methods in Enzymology, vol. 266: Computer ing degredation of NAD, nicotinamide mononucleotide, Methods for Macromolecular Sequence Analysis (1996), nicotinic acid mononucleotide, nicotinamide riboside or ed. Doolittle, Academic Press, Inc., a division of Harcourt sirtuin enzymes. Brace & Co., San Diego, California, USA. Preferably, an

7 13 EP 3 006 040 A1 14 alignment program that permits gaps in the sequence is prised by a nicotinamide mononucleotide adenylyltrans- utilized to align the sequences. The Smith-Waterman is ferase (NMNAT) or a polynucleotide encoding an onetype of algorithm thatpermits gaps in sequence align- NMNAT. In particular, the agent can be an enzyme hav- ments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, ing NMNAT activity and at least 50% identity with a hu- the GAP program using the Needleman and Wunsch 5 man NMNAT1 or at least 50% identity with a human alignment method can be utilized to align sequences. An NMNAT3, at least 60% identity with a human NMNAT1 alternative search strategy uses MPSRCH software, or at least 60% identity with a human NMNAT3, at least which runs on a MASPAR computer. MPSRCH uses a identity with a human NMNAT1 or at least 70% identity Smith-Waterman algorithm to score sequences on a with a human NMNAT3, at least 80% identity with a hu- massively parallel computer. This approach improves 10 man NMNAT1 or at least 80% identity with a human ability to pick up distantly related matches, and is espe- NMNAT3, at least 90% identity with a human NMNAT1 cially tolerant of small gaps and nucleotide sequence er- or at least 90% identity with a human NMNAT3, at least rors. Nucleic acid-encoded amino acid sequences can 95% identity with a human NMNAT1 or at least 95% iden- be used to search both protein and DNA databases. Da- tity with a human NMNAT3. Moreover, the agent can be tabases with individual sequences are described in Meth- 15 comprised by a human NMNAT1, a human NMNAT3 or ods in Enzymology, ed. Doolittle, supra. Databases in- a conservatively substituted variants thereof. clude Genbank, EMBL, and DNA Database of Japan [0047] The agent can also be comprised by a polynu- (DDBJ). cleotide having at least 50% identity with a nucleic acid [0044] A "variant" of a polypeptide refers to a polypep- encoding a human NMNAT1 or a polynucleotide having tide having the amino acid sequence of the polypeptide 20 at least 50% identity with a nucleic acid encoding a hu- in which is altered in one or more amino acid residues. man NMNAT3, a polynucleotide having at least 60% The variant may have "conservative" changes, wherein identity with a nucleic acid encoding a human NMNAT1 a substituted amino acid has similar structural or chem- or a polynucleotide having at least 60% identity with a.nu- ical properties (e.g., replacement of leucine with isoleu- cleic acid encoding a human NMNAT3, a polynucleotide cine). A variant may have "nonconservative" changes 25 having at least 70% identity with a nucleic acid encoding (e.g., replacementof glycine with tryptophan). Analogous a human NMNAT1 or a polynucleotide having at least minor variations may also include amino acid deletions 70% identity with a nucleic acid encoding a human or insertions, or both. Guidance in determining which NMNAT3, a polynucleotide having at least 80% identity amino acid residues may be substituted, inserted, or de- with a nucleic acid encoding a human NMNAT1 or a poly- leted without abolishing biological or immunological ac- 30 nucleotide having at least 80% identity with a nucleic acid tivity may be found using computer programs well known encoding a human NMNAT3, a polynucleotide having at in the art, for example, LASERGENE software (DNAS- least 90%.identity with a nucleic acid encoding a human TAR). NMNAT1 or a polynucleotide having at least 90% identity [0045] The term "variant," when used in the context of with a nucleic acid encoding a human NMNAT3, a poly- a polynucleotide sequence, may encompass a polynu- 35 nucleotide having at least 95% identity with a nucleic acid cleotide sequence related to that of a particular gene or encoding a human NMNAT1 or a polynucleotide having the coding sequence thereof. This definition may also at least 95% identity with a nucleic acid encoding a hu- include, for example, "allelic," "splice," "species," or "pol- man NMNAT3. The agent can also be a polynucleotide ymorphic" variants. A splice variant may have significant encoding a human NMNAT1, a human NMNAT3 or a identity to a reference molecule, but will generally have 40 variant thereof. a greater or lesser number of polynucleotides due to al- [0048] The agent can also be comprise by a sirtuin ternate splicing of exons during mRNA processing. The polypeptide or a nucleic acid encoding a sirtuin polypep- corresponding polypeptide may possess additional func- tide. In particular, the agent can comprise an enzyme tional domains or an absence of domains. Species var- having SIRT activity and at least 50% identity with a hu- iants are polynucleotide sequences that vary from one 45 man SIRT1, at least 60% identity with a human SIRT1,at species to another. The resulting polypeptides generally least 70% identity with a human SIRT1,at least 80% iden- will have significant amino acid identity relative to each tity with a human SIRTl,at least 90% identity with a human other. A polymorphic variation is a variation in the poly- SIRT1, or at least 95% identity with a human SIRT1. nucleotide sequence of a particular gene between indi- Moreover, the agent can be comprised by a human viduals of a given species. Polymorphic variants also may 50 SIRT1 or a conservatively substituted variants thereof. encompass "single nucleotide polymorphisms" (SNPs) The agent can also be comprised by a polynucleotide in which thepolynucleotide sequence variesby one base. having at least 50% identity with a nucleic acid encoding The presence of SNPs may be indicative of, for example, a human SIRT1, a polynucleotide having at least 60% a certain population, a disease state, or a propensity for identity with a nucleic acid encoding a human SIRT1, a a disease state. 55 polynucleotide having at least 70% identity with a nucleic [0046] An agent that can be used in treating or pre- acid encoding a human SIRT1, a polynucleotide having venting a neuropathy in accordance with the methods at least 80% identity with a nucleic acid encoding a hu- and compositions of the present invention can be com- man SIRT1, a polynucleotide having at least 90% identity

8 15 EP 3 006 040 A1 16 with a nucleic acid encoding a human SIRT1 or a poly- the active ingredients after administration to the patient nucleotide having at least 95% identity with a nucleic acid by employing procedures well known in the art. The for- encoding a human SIRT1. Moreover, the agent can com- mulations can also contain substances that diminish pro- prise a polynucleotide encoding a human SIRT1 or a var- teolytic degradation and promote absorption such as, for iant thereof. 5 example, surface active agents. [0049] Administration can be by any suitable route of [0053] The specific dose can be calculated according administration including buccal, dental, endocervical, in- to the approximate body weight or body surface area of tramuscular, inhalation, intracranial, intralymphatic, in- the patient or the volume of body space to be occupied. tramuscular, intraocular, intraperitoneal, intrapleural, in- The dose will also depend upon the particular route of trathecal, intratracheal, intrauterine, intravascular, intra- 10 administration selected. Further refinement of the calcu- venous, intravesical, intranasal, ophthalmic, oral, otic, lations necessary to determine the appropriate dosage biliary perfusion, cardiac perfusion, priodontal, rectal, for treatment is routinely made by those of ordinary skill spinal subcutaneous, sublingual, topical, intravaginal, in the art. Such calculations can be made without undue transermal, ureteral, or urethral. Dosage forms can be experimentation by one skilled in the art in light of the aerosol including metered aerosol, chewable bar, cap- 15 activity in assay preparations such as has been de- sule, capsule containing coated pellets, capsule contain- scribed elsewhere for certain compounds (see for exam- ing delayed release pellets, capsule containing extended ple, Howitz et al., Nature 425:191-196, 2003 and supple- release pellets, concentrate, cream, augmented cream, mentary information that accompanies the paper). Exact suppository cream, disc, dressing, elixer, emulsion, en- dosages can be determined in conjunction with standard ema, extended release fiber, extended release film, gas, 20 dose-response studies. It will be understood that the gel, metered gel, granule, delayed release granule, ef- amount of the composition actually administered will be fervescent granule, chewing gum, implant, inhalant, in- determined by a practitioner, in the light of the relevant jectable, injectable lipid complex, injectable liposomes, circumstances including the condition or conditions to be insert, extended release insert, intrauterine device, jelly, treated, the choice of composition to be administered, liquid, extended release liquid, lotion, augmented lotion, 25 the age, weight, and response of the individual patient, shampoo lotion, oil, ointment, augmented ointment, the severity of the patient’s symptoms, and the chosen paste, pastille, pellet, powder, extended release powder, route of administration. metered powder, ring, shampoo, soap solution, solution [0054] In various embodiments, the present invention for slush, solution/drops, concentrate solution, gel form- also provides methods of screening candidate agents. ing solution/drops, sponge, spray, metered spray, sup- 30 In one such assay method, agents are tested for effec- pository, suspension, suspension/drops, extended re- tiveness in decreasing or preventing axonal degenera- lease suspension, swab, syrup, tablet, chewable tablet, tion of injured neuronal cells. Candidate agents are thus tablet containing coated particles, delayed release tablet, administered to neuronal cells subjected to injury and a dispersible tablet, effervescent tablet, extended release decrease in axonal degeneration of the injured neuronal tablet, orally disintegrating tablet, tampon, tape or tro- 35 cells is detected. Typically, the agent is added prior to che/lozenge. producing the injury, however, in some instances, the [0050] Intraocular admistration can include adminis- injury can be produced before addition of the candidate tration by injection including intravitreal injection, by eye- compound. The method can be performed in vitro or in drops and by trans-scleral delivery. vivo. The in vitro tests can be performed using any of a [0051] Administration can also be by inclusion in the 40 number of mammalian neuronal cells under a variety of diet of the mammal such as in a functional food for hu- experimental conditions in which injury is elicited. An ex- mans or companion animals. ample of mammalian neuronal cell-types that can be [0052] It is also contemplated that certain formulations used are primary dorsal root ganglion cells injured by containing the compositions that increase sirtuin activity either transection and removal of the neuronal cell body are to be administered orally. Such formulations are pref- 45 or growth in media containing vincristine as described erably encapsulated and formulated with suitable carri- below. The in vivo tests can be performed in intact ani- ers in solid dosage forms. Some examples of suitable mals such as, for example, a mouse model of peripheral carriers, excipients, and diluents include lactose, dex- nerve regeneration (Pan et al., J. Neurosci. trose, sucrose, sorbitol, mannitol, starches, gum acacia, 23:11479-11488, 2003) or mouse model of progressive calcium phosphate, alginates, calcium silicate, microc- 50 motor neuronopathy (Schmalbruch et al., J. Neuropathol. rystalline cellulose, polyvinylpyrrolidone, cellulose, gela- Exp. Neurol. 50:192-204, 1991; Ferri et al., Current Biol. tin, syrup, methyl cellulose, methyl- and propylhydroxy- 13:669-673, 2003). benzoates, talc, magnesium, stearate, water, mineral oil, [0055] Because, the mechanism of decreasing or pre- and the like. The formulations can additionally include venting neuronal injury results from an increase in NAD- lubricating agents, wetting agents, emulsifying and sus- 55 dependent histone/protein deacetylase activity of sirtuin pending agents, preserving agents, sweetening agents molecules, the assay method can also be used as a pri- or flavoring agents. The compositions may be formulated mary screen for substances that either increase sirtuin so as to provide rapid, sustained, or delayed release of activity directly or through increasing NAD activity. Thus

9 17 EP 3 006 040 A1 18 the methods above can be used to screen for agents that 1140)). The following genes were cloned into FUGW vec- inrease NAD biosynthetic activity or agents that increase tor: 1) The first 70 AAs of Ufd2a (the portion contained sirtuin activity in neurons. in Wlds protein) fused to the N-terminus of EGFP [0056] Recombinant vectors that serve as carriers for (Ufd2a(1-70)-EGFP) or EGFP with nuclear localization a nucleic acid encoding a sirtuin molecule or an enzyme 5 signal at the C-terminal (Ufd2a(1-70)-nucEGFP). 2) The for biosynthesis of NAD are also within the scope of the NMNAT1 portion of Wld s protein fused to the C-terminus present invention. Such recombinant vectors can com- of EGFP (EGFP-NMNAT1). prise a promoter operatively linked to a sequence encod- [0062] The murine cDNA for Ufd2a/Ube4b ing a mammalian NMNAT1 protein or a mammalian sir- (mKIAA0684) was provided by Kazusa DNA Research tuin protein such as a SIRT1 protein. Such recombinant 10 Institute. Murine cDNAs for NMNAT1 (accession vectors can be any suitable vector such as, for example number: BC038133) were purchased from ATCC. PCR- a lentivirus or an adeno-associated virus. Any suitable mediated mutagenesis was used to generate point mu- promoter can be also used such as, for example a ubiq- tations in Ufd2a, NMNAT1 and Wlds. uitin promoter, a CMV promoter or a β-actin promoter. [0063] We generated siRNA constructs in the FSP-si [0057] The invention can be further understood by ref- 15 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 EXAMPLE 1 SV40 promoter-puromycin-N-acetyl-transferase gene. Cloning of siRNA construct was performed as described [0058] This example demonstrates that transected ax- 20 previously, so that the siRNA is transcribed from the U6 ons from neurons tranfected with a vector expressing promoter (Castanotto, et al., RNA, 8:1454-60, 2002). Se- Wlds protein show a delayed degeneration compared to quences used for siRNA downregulation of protein ex- control neurons. pression were 1692-1710 of SIRT1, 1032-1050 of SIRT2, [0059] In wlds mice, Wallerian degeneration in re- 538~556 of SIRT3, 1231~1249 of SIRT4, 37~55 of sponse to axonal injury has been shown to be delayed 25 SIRT5, 1390~1408 of SIRT6, and 450-468 of SIRT7. The (Gillingwater, et al., JPhysiol, 534:627-639, 2001). Ge- integrity of each lentiviral expression and siRNA con- netic analysis has shown that the wlds mutation compris- struct was confirmed by DNA sequencing. es an 85 kb tandem triplication, which results in overex- [0064] MouseDRG explants fromE12.5 embryos were pression of a chimeric nuclear molecule (Wlds protein). cultured in the presence of 1 nM nerve growth factor. This protein is composed of the N-terminal 70 AAs of Ufd 30 Non-neuronal cells were removed from the cultures by (ubiquitin fusion degradation protein)2a, a ubiquitin chain adding 5-fluorouracil to the culture medium. Transection assembly factor, fused to the complete sequence of nico- of neurites was performed at 10-20 DIV using an 18- tinamide mononucleotide adenylyltransferase1gauge needle to remove the neuronal cell bodies. Incu- (NMNAT1), an enzyme in the NAD salvage pathway that bation with β-nicotinamide adenine dinucleotide (Sigma) generates NAD within the nucleus. The Wld s protein has 35 or Sirtinol (Calbiochem) was performed using conditions NMNAT activity but lacks ubiquitin ligase function, sug- indicated in the text or figures. gesting that axonal protection is derived from either in- [0065] Lentiviral expression vectors were generated creased NMNAT1 activity or a ’dominant negative’ inhi- using HEK293T cells as described above. For confirma- bition of Ufd2a function. tion of lentivirus-derived protein expression, HEK293T [0060] To identify the mechanism of delayed axonal 40 cells were infected with lentivirus and cells were lysed 3 degenerationmediated by the Wld s protein,we employed days after infection. These lysates were analyzed by im- an in-vitro Wallerian degeneration model. Primary DRG munoblot to using anti-His tag monoclonal antibody (Qia- explant neurons were infected with lentivirus expressing gen) to detect expression of the respective hexahistidine- the appropriate proteins, and axons were injured by ei- tagged proteins. Lentiviral infection of DRG neurons was ther removal of the neuronal cell body (transection) or 45 performed by incubating ~106-107 pfu/ml virus with the growth in vincristine (toxic). DRG explant for 24 h beginning 3-7 days prior to axonal [0061] Lentiviral expression constructs were kindly transection. The infected neurons were examined under provided by D. Baltimore (Lois, et al., Sciencean inverted fluorescent microscope to insure detectable 295:868-72, 2002). We modified the FUGW vector to lentivirus-mediated transgene expression in >95% of generate a general expression shuttle FUIV (ubiquitin 50 neurons. promoter - gene of interest-IRES-enhanced YFP (Ve- [0066] Quantitative analysis of axonal degeneration nus)) vector that enables enhanced YFP expression in was performed as previously described (Zhai, et al., Neu- cells that express the gene-of-interest. The following pro- ron 39:217-25, 2003). Briefly, the cultures were exam- teins, each with a hexahistidine tag at the C-terminus, ined using phase contrast microscopy at the indicated were cloned into the FUIV vector: Wld s chimeric mutant 55 times. Axons with a fragmented, non-refractile appear- protein; Ufd2a containing a point mutation (P1140A), ance were designated as "degenerated." At each time which has previously been shown to inhibit wild-type point, at least 200 singly distinguishable axons were Ufd2a function as a "dominant-negative"(Ufd2a(P blindly scored from several randomly taken images of

10 19 EP 3 006 040 A1 20 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- that neurons expressing these enzymatically inactive culations of neurite-covered area, digitally captured im- 5 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). [0067] We found that transected axons from neurons EXAMPLE 3 expressing the Wlds protein degenerated with the de- 10 layed kinetics characteristic of neurons derived from wlds [0072] 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. [0068] Next, we compared axonal degeneration after [0073] In addition to mechanical transection, axonal transection in neurons that overexpress Wld sprotein with 15 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 shown in Figure 1B. al., J Cereb Blood Flow Metab 24:62-66, 2004). We [0069] We found that expression of EGFP-NMNAT1 sought to determine whether increased NMNAT activity delayed axonal degeneration comparable to Wld s protein 20 would also delay axonal degradation in response to other itself, whereas the N-terminal 70 AA of Ufd2a (fused to types of axonal injury such as vincristine, a cancer chem- EGFP), either targeted to the nucleus or cytoplasm, did otherapeutic reagent with well-characterized axonal tox- not affect axonal degeneration. Quantification of these icity. Neurons expressing either NMNAT1 or EGFP (con- effects was performed by counting the percentage of re- trol) were grown in 0.5mM vincristine for up to 9 d. We maining neurites at various times after removal of neu- 25 found that axons of neurons expressing NMNAT1 main- ronal cell bodies. This analysis showed that EGFP- tained their original length and refractility, whereas axons NMNAT1, like Wlds protein itself, resulted in a >10-fold emanating from neurons expressing EGFP gradually re- increase in intact neurites 72 hr after injury. To further tracted and had mostly degenerated by day 9 (Fig. 2B). exclude direct involvement of the UPS in Wlds protein- These results indicate that NMNAT activity by itself can mediated axonal protection, we examined the effect of 30 protect axons from a number of insults and mediate the Ufd2a inhibition using either a dominant-negative Ufd2a protective effects observed in wlds mice. mutant or an Ufd2a siRNA construct. However, neither of these methods resulted in delayed axonal degradation EXAMPLE 4 in response to axotomy. Together, these experiments demonstrated that the NMNAT1 portion of the Wld s pro- 35 [0074] This example shows that exogenously admin- tein is responsible for the delayed axonal degeneration istered NAD can protect injured neurons from axonal de- observed in wlds mice. generation. [0075] Previous experiments have shown that neuro- EXAMPLE 2 nal cells express membrane proteins that can bind and 40 transport extracellular NAD into the cell (Bruzzone, et al., [0070] This example shows that mutations in the full Faseb J 15:10-12, 2001). This encouraged us to inves- length NMNAT1 and in Wlds protein abolish the axonal tigate whether exogenously administered NAD could protective effects of the proteins. prevent axonal degeneration. We added various concen- [0071] NMNAT1 is an enzyme in the nuclear NAD sal- trations of NAD to neuronal cultures prior to axonal vage pathway that catalyzes the conversion of nicotina- 45 transection and examined the extent of axonal degrada- mide mononucleotide (NMN) and nicotinate mononucle- tion. We found that 0.1-1 mM NAD added 24 hr prior to otide (NaMN) to NAD and nicotinate adenine mononu- axotomy significantly delayed axonal degeneration, al- cleotide (NaAD), respectively. The axonal protection ob- though exogenously applied NAD was slightly less effec- served in NMNAT1 overexpressing neurons could be tive in protecting axons than lentivirus mediated mediated by its ability to synthesize NAD (i.e. its enzy- 50 NMNAT1 expression (Fig. 3A). These results provide di- matic activity), or perhaps, by other unknown functions rect support for the idea that increased NAD supply can of this protein. To address this question, we used the prevent axonal degradation. NMNAT1 crystal structure to identify several residues predicted to participate in substrate binding. A mutation EXAMPLE 5 in one of these residues (W170A) was engineered into 55 full length NMNAT1 and Wld s protein. In vitro enzymatic [0076] This example illustrates that NAD was required assays confirmed that both of these mutant proteins were prior to the removal of the neuronal cell bodies to protect severely limited in their ability to synthesize NAD (Fig. the injured neurons from axonal degeneration.

11 21 EP 3 006 040 A1 22

[0077] 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 The importance of these NAD-dependent enzymes in severed axons to high levels of NAD was sufficient to 5 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. dependent axonal protection (NDAP) (Fig. 4A). Neurons [0078] We found that administering NAD at the time of 10 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 to 72 hr later. We found that Sirtinol effectively blocked greatest effects occurring after at least 24 h of NAD pre- 15 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- lational modification within the axons themselves. tection. To further examine the role of Sir2 proteins in [0079] The requirement for extended exposure to NAD NDAP, we tested the effects of resveratrol (10~100 mM), of the intact neurons to prevent axonal degradation in 20 a polyphenol compound that enhances Sir2 activity response to injury suggests that the protective process (Howitz, et al., Nature, 425:191-96, 2003). We found that requires de novo transcriptional and/or translational neurons treated with resveratrol prior to axotomy showed events. Interestingly, both the Wld s protein and NMNAT1 a decrease in axonal degradation that was comparable are located within the nucleus (data not shown). Similarly, to that obtained using NAD (Fig. 4A), providing further most enzymes that make up the NAD salvage pathway 25 support for the idea that Sir2 proteins are effectors of the in yeast are also compartmentalized in the nucleus. We axonal protection mediated by increased NMNAT activ- compared NAD levels in wild type and NMNAT1 express- ity. ing DRG neurons using sensitive microscale enzymatic assays (Szabo, et al., Proc Natl Acad Sci USA,EXAMPLE 7 93:1753-58 ,1996), however no changes in overall cel- 30 lular NAD levels were found (data not shown). This is [0082] This example shows that SIRT1 is involved in similar to observations in yeast, in which activation of this NAD-dependent axonal protection. nuclear pathway did not change overall levels of NAD [0083] In humans and rodents, seven molecules shar- (Anderson, et al., J Biol Chem, 277:18881-90,2002; Huh, ing Sir2 conserved domain (sirtuin (SIRT)1 through 7) et al., Nature, 425:686-91, 2003). Furthermore, levels of 35 have been identified, although some of these proteins do tissue NAD in the brains of wild type and wlds mice are not appear to have histone/protein deacetylase activity similar despite the increased levels of NMNAT activity in (Buck, et al., J Leukoc Biol, S0741-5400, 2004). SIRT1 wlds mice (Mack, et al., Nat Neurosci, 4:1199-206, 2001). is located in the nucleus and is involved in chromatin These data suggest that an NAD-dependent enzymatic remodeling and the regulation of transcription factors activity in the nucleus, as opposed to cytoplasmic NAD- 40 such as p53 (J. Smith, Trends Cell Biol, 12:404-406, dependent processes, is likely to mediate the axonal pro- 2002). The cellular location of other SIRT proteins is less tection observed in response to increased NMNAT ac- clear, but some have been found in the cytoplasm and tivity. in mitochondria. To determine which SIRT protein(s) is involved in NAD-dependent axonal protection, we per- EXAMPLE 6 45 formed knockdown experiments using siRNA constructs to specifically target each member of the SIRT family. [0080] This example shows that inhibition of Sir2 is in- Neurons were infected with lentiviruses expressing spe- volved in NAD-dependent axonal protection. cific SIRT siRNA constructs that effectively suppressed [0081] The Sir2 family of protein deacetylases and po- expression of their intended target (Fig. 4B). The infected ly(ADP-ribose) polymerase (PARP) are the major NAD- 50 neurons were cultured in 1 mM NAD and axonal transec- dependent nuclear enzymatic activities. Sir2 is an NAD- tion was performed by removing the cell bodies. We dependent deacetylase of histones and other proteins, found that the SIRT1 siRNA construct was just as effec- and its activation is central to promoting increased lon- tive at blocking the axonal protective effects of NAD as gevity in yeast and C. elegans (Bitterman, et al., Microbiol the Sirtinol inhibitor. In contrast, inhibition of the other Mol Biol Rev, 67:376-99, 2003; Hekimi, et al., Science 55 SIRT proteins did not have significant effects on NDAP 299:1351-54, 2003). PARP is activated by DNA damage (Fig. 4B). These results indicate that SIRT1 is the major and is involved in DNA repair (S.D. Skaper, Ann NYAcad effector of the increased NAD supply that effectively pre- Sci, 993:217-28 and 287-88, 2003). These enzymes, in vents axonal self destruction. Although, SIRT1 may

12 23 EP 3 006 040 A1 24 deacetylate proteins directly involved in axonal stability, nerve injury, the sciatic nerves of a C57BL/6 mouse was its predominantlynuclear location,along withthe require- transected and L4 to L5. DRGs were collected at indicat- ment for NAD -24 hr prior to injury for effective protection, ed time points and pooled to extract RNA. Rat DRG ex- suggest that SIRT1 regulates a genetic program that plants from E14.5 embryo were cultured for 14 days ac- leads to axonal protection. 5 cording to the method desctribed and cultured with media [0084] Axonal degeneration is an active, self-destruc- containing 10 nM vincristin for indicated period and ex- tive phenomenon observed not only after injury and in tracted RNA. Total RNAs from pooled tissue sources or response to chemotherapy, but also in association with DRG explant cultures were prepared. First-strand cDNA aging, metabolic diseases such as diabetic neuropathy, templates were prepared from 1 mg of each RNA using and neurodegenerative diseases. Our results indicate 10 standard methods. Two independent cDNA syntheses that the molecular mechanism of axonal protection in the were performed for each RNA sample. Quantitative re- wlds mice is due to the increased supply of NAD resulting verse transcription (RT)-PCR was performed by moni- from enhanced activity of the NAD salvage pathway and toring in real-time the increase in fluorescence of the consequent activation of the histone/protein deacetylase SYBR-GREEN dye on a TaqMan 7700 Sequence De- SIRT1. 15 tection System (Applied Biosystems). [0089] Cell culture, in vitro axotomy, and quantifi- EXAMPLES 8-11 cation of axonal degeneration. Mouse DRG explants from E12.5 embryos were cultured in the DMEM contain- [0085] The following Materials and Methods were used ing 10% FCS and 1 nM nerve growth factor. Non-neuro- in Examples 8-11. 20 nal cells were removed from the cultures by adding 5- [0086] Construction of expression plasmids and fluorouracil to the culture media. Transection of neurites mutagenesis. Coding regions of the NAD biosynthetic was performed at 14-21 DIV using an 18-gauge needle enzymes were PCR amplified from EST clonesto remove the neuronal cell bodies. Lentiviral expression BC038133 for murine NMNAT1 and BC005737 for vectors were generated. Lentiviral infection was per- murine nicotinamide mononucleotide25 formed 3-7 days prior to axonal transection for 24 hr. adenylyltransferase3 (NMNAT3), using Herculase Quantitative analysis of neurite degeneration was per- (Stratagene). Human NAD synthetase (QNS) hexahisti- formed. dine-tagged cDNA was kindly provided by Dr. N. Hara [0090] Determination of protein expression and lo- (Shimane University, Shimane, Japan). Hexahistidine calization. For confirmation of protein expression, tag was added at the 3’-end of each cDNA. NMNAT1 30 HEK293T cells were infected with a virus that expresses cytosolic mutant (cytNMNAT1) was generated by PCR- each of NAD biosynthetic enzymes. Cells were lysed 5 mediated site-directed mutagenesis. Nuclear form of days after infection to be analyzed by immunoblot to de- NMNAT3 (nucNMNAT3) was generated by adding a nu- tect expression of each protein with a hexa-histidine tag clear localization signal to the C-terminal end ofby anti-6xHis tag monoclonal antibody (R&D Systems). NMNAT3. Each PCR amplified NAD synthetic enzyme 35 Subcellular localization of each protein was analyzed us- fragment was cloned into FCIV lentiviral shuttle vector ing HEK293T cells transiently transfected with a viral as previously described. The integrity of all the constructs shuttle vector for each NAD biosynthetic enzymes. Cells was sequenced using Taq DyeDeoxy Terminator cycle were fixed in 4% paraformaldehyde in PBS containing sequencing kits (Applied Biosystems) and an Applied Bi- 0.1% tween-20 (PBS-T) and incubated with PBS-T con- osystems 373 DNA sequencer. 40 taining 5% BSA for 1 hour, and then covered with 1:1000 [0087] NAD biosynthetic substrates. All substrates diluted anti-6xHis tag antibody (R&D Systems) in PBS- for NAD biosynthetic enzymes were purchased from Sig- T containing 1% BSA and for 16 hours at 4°C. Cells were ma (Na, Nam, NMN, NaMN, nicotininc acid adenine di- washed with PBS-T and incubated with Alexa Fluor 594- nucleotide (NaAD), and NAD). NmR was synthesized conjugated secondary antibody (Molecular Probes) in from NMN. Conversion of NMN to NmR was confirmed 45 TBS-T for 1 hour and examined by fluorescence micro- by HPLC (Waters) using reverse phase column LC-18T scopy (Nikon). (Supelco). NmR is eluted 260 6 10 seconds and NMN [0091] NMNAT protein overexpression, affinity pu- is eluted 150 6 10 seconds under 1 ml/min flow rate of rification and enzymatic assay. HEK293T cells were buffer containing 50mM K 2HPO4 and 50mM KH 2PO4 (pH transfected with an expression plasmid for each enzyme 7.0). Biological activity of NmR was accessed as previ- 50 by using calcium phosphate precipitation. Three days lat- ously described by using yeast strains kindly provided er, cells were washed with PBS twice and then suspend- from Dr. Charles Brenner (Dartmouth Medical School, ed in the buffer containing 50 mM Sodium Phosphate New Hampshire, USA). (pH8.0), and 300 mM NaCl (buffer A). Cells were then [0088] Real-time quantitative reverse transcrip- homogenized by SONIFIRE 450 (BRANSON) and su- tion-PCR analysis. All the surgical procedures were per- 55 pernatant was collected by centrifugation at 10,000 g for formed according to National Institute of Health guide- 10 min. His-select Nickel Affinity Gel (Sigma) was lines for care and use of laboratory animals at Washing- washed with buffer A and 0.1 ml of 50% gel suspension ton University. For the expression analysis following was added to 1 ml of supernatant and incubated for 10

13 25 EP 3 006 040 A1 26 min at 4°C, then beads binding hexa-histidine -tagged [0095] 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- 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 5 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 the 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. 10 response to neuronal insults, we compared the RNA ex- [0092] 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 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 15 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 at 24 hours after intravitreal injection and optic nerve was of NAD synthetic enzymes during the axonal degenera- recovered at indicated time. Optic nerve tissue was ho- tion caused by neurotoxin in cultured rat DRG neuron. mogenized in 100 ml of a buffer containing 100mM tris- DRG neurons were treated with 0.1 mM and 1 mM roten- HCl (pH 6.8), 1 % SDS, and 1mM DTT. Fifty mg of protein 20 one to cause axonal degeneration and collected RNA at for each sample was analyzed by the Western blotting 24 hours after the addition of rotenone. The expression using anti-neurofilament antibody 2H3 (Developmental of Nrk2 was increased more than 6 folds after rotenone Studies Hybridoma Center) and peroxidase-conjugated treatment (Fig. 6B). These results suggest that, while all secondary antibody (Jackson ImmunoResearch). The enzymatic activities in NAD synthesis pathway is ubiqui- degeneration rate was calculated from the ratio of the 25 tously present, Nrk2 may be responsible for supplying neurofilament immunoreactivity of transected vs. contral- NAD synthesizing substrate after neuronal insults. ateral nerves. EXAMPLE 9 EXAMPLE 8 30 [0096] This example illustrates that both nuclear and [0093] This example illustrates the NAD biosynthetic cytoplasmic Nmat enzymes save axons from degenera- pathway and expression analysis of mammalian NAD tion. biosynthetic enzymes. [0097] To determine whether nuclear localization of [0094] NAD is synthesized via three major pathways NMNAT1 is essential to provide the axonal protection, in both prokaryotes and eukaryotes. In the de novo path- 35 we analyzed the effect of subcellular distribution of way, NAD is synthesized from tryptophan (Fig.5). In the NMNAT enzyme in the in vitro Wallerian degeneration salvage pathway,NAD isgenerated fromvitamins includ- assay and compared the extent of axonal protection be- ing nicotinic acid and nicotinamide. A third route from tween overexpression of cytoplasmic and nuclear nicotinamide riboside called Preiss-Handler independent NMNAT. NMNAT1 has putative nuclear localization sig- pathway has recently been discovered. The last enzy- 40 nal PGRKRKW in the 211-217 amino-acids of NMNAT1 matic reaction of the de novo pathway involves the con- protein. We generated a mutant NMNAT1 designated as version of quinolinate to NaMN by QPRT (EC 2.4.2.19). cytNMNAT1 in which this nuclear localization signal was At this point, the de novo pathway converges with the altered as PGAAAAW and examined subcellular distri- salvage pathway. NaPRT (EC 2.4.2.11) converts Na to bution. As shown in Fig. 7B, the majority of cytNMNAT1 NaMN, which is then converted to NaAD by NMNAT (EC 45 located in the cytosol as we expected. 2.7.7.1). QNS1 (EC 6.3.5.1) converts NaAD to NAD. [0098] Next we confirmed enzymatic activity of NmPRT (EC 2.4.2.12); also reported as visfatin) converts cytNMNAT1, NMNAT1 and its mutant cytNMNAT1 were Nam to NMN. NMN is also converted to NAD by NMNAT. purified from the cell lysate expressing either of proteins Nicotinamidase (PNC, EC 3.5.1.19), which converts by using affinity gel. The enzymatic activity of affinity pu- Nam to Na in yeast and bacteria salvage pathway has 50 rified proteins was measured as described above and not been identified in mammals. In the Preiss-Handler we found that cytNMNAT1 activity did not altered by its independent pathway, Nrk (EC 2.7.1.22) converts NmR mutation (Fig. 7C). After the overexpression of to NMN and converge to salvage pathway. Most of these cytNMNAT1 in DRG neurons, we observed strong neu- mammalian enzymes including QPRT, NmPRT, QNS1, rite protection as well as nuclear wild NMNAT1 (Fig.7A, Nrk1/2 and NMNAT1/2/3 have previously cloned and 55 E). We further confirmed this result by using NMNAT1 characterized. A mammalian homologue of NaPRT was isoenzyme that lacks nuclear localization signal. Among also identified as an EST annotated as a mammalian two NMNAT isoenzymes, NMNAT3 is previously report- homolog of a bacterial NaPRT. ed to locate outside nucleus and mitochondria, and have

14 27 EP 3 006 040 A1 28 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 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- 5 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 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 10 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- rian degeneration assay was performed after overex- EXAMPLE 11 pression of these two NMNAT3 enzymes, and we found 15 that overexpression of both NMNAT3 and nucNMNAT3 [0101] This example demonstrates that intraviteal ap- showed same extent of delay in neurite degeneration as plication of NAD biosynthetic substrates delays the ax- well as NMNAT1 (Fig. 7A, E). The lentivirus mediated onal degeneration of retinal ganglion cells. expression of each enzyme was confirmed by Western [0102] Transection of optic nerve is an in vivo model blotting (Fig. 7D). These experiments confirmed that20 which can be used to investigate mechanisms leading to NMNAT targeted to either the nucleus or cytosol protects Walleriandegeneration and following retinal ganglion cell neurite from degeneration. (RGC) death observed in human diseases such as glau- coma. In the C57BL/Wlds mouse strain, optic nerve de- EXAMPLE 10 generation during Wallerian degeneration after axotomy 25 is dramatically slowed. In addition, intravitreal injection [0099] This example illustrates that exogenous appli- is used for screening of compounds that protect RGC cation of substrates for NAD biosynthetic enzymes pro- axon from degeneration in vivo and thus we can asses tects axon from degeneration. the axon protective effect of each NAD biosynthetic sub- [0100] We have previously shown that exogenously strates in vivo by intraocular injection of compounds in- applied NAD in the culture medium shows axonal saving 30 cluding NAD, NMN, NmR, and Nam. From in vitro Walle- effectin vitro. Herewe showed thatexpression ofNmPRT rian degeneration assay, 1mM of NAD, NMN, and NmR also shows axonal protection suggesting that Nam is in the culture media is enough to protect axon from de- used as a substrate for NAD synthesis in neurons. To generation. We initially injected 5 ml of 100 mM or 1 M determine which substrate shown in Fig. 5 is used for NAD solution into left intravitreal compartment. After 24 NAD synthesis in neurons and to identify whether any of 35 hours incubation, left optic nerve was transected and NAD precursors may be able to save axons similar to or control (right) and axotomized (left) optic nerve were col- possibly better than NAD, we applied Na, Nam, NmR, lected at 3, 4, and 5 days after transection. Neurofilament NaMN, NMN, or NaAD in the culture media and per- immunoreactivity from the axotomized optic nerve was formed in vitro Wallerian degeneration assay. An appli- measured and normalized against the value obtained cation of 1 mM NMN for 24 hours before neurite transec- 40 from the right side of the optic nerve. We found that the tion successfully saved neurites from degeneration. immunoreactivity at 4days after transection was Quantitative analysis revealed that NMN treatment re- 77627% and 78622% of non-axotomized optic nerve in sults in neurite protection to an extent similar to that 1 M and 100 mM NAD injected rats respectively, while achieved by exogenously applied NAD (Fig. 8B). These control animal showed only 7616 % (Fig. 9) results further suggested the possibility that increased 45 [0103] We then injected 5 ml of 100 mM NMN, NmR, supply of other NAD biosynthetic substrates have an abil- and Nam into left intravitreal compartment and collected ity to save neurites from degeneration. We then exoge- optic nerves at 4 days after left optic nerve transaction. nously applied 1 mM of NAD biosynthetic substrates in- The immunoreactivity obtained from NMN and NmR in- cluding Na, Nam, NaMN, NaAD, and NmR to the DRG jected optic nerve was 60625 and 72619 % of non-ax- neurons for 24 hours and performed neurite transection. 50 otomized nerve. Nam injected animals did not show any As shown in Fig. 8A and B, NaMN or NmR treatment also difference from the control animals. These results are saved neurites as well as NAD. NaAD showed slight pro- consistent with the in vitro study that showed NAD, NMN, tection but Na failed to save neurites, while Na and Nam and NmR have axon saving activity but Nam does not. had no effect. Quantitative analysis revealed that exog- Our in vivo study revealed that these small molecules enous application of 1mM NaMN, NMN, NmR, or NAD 55 that are involved in the NAD biosynthetic pathway are caused comparable increase in intact neurites at 48 useful tools to save axon from degeneration. hours after transection (Fig. 8B). Because the protective [0104] All references cited in this specification are effect of NaMN is equal to NMN, a step synthesize NAD hereby incorporated by reference. Any discussion of ref-

15 29 EP 3 006 040 A1 30 erences cited herein is intended merely to summarize 8. A method according to clause 7, wherein the agent the assertions made by their authors and no admission has at least 70% identity with a human NMNAT1 or is made that any reference or portion thereof constitutes at least 70% identity with a human NMNAT3. relevant prior art. Applicants reserve the right to chal- lenge the accuracy and pertinency of the cited referenc- 5 9. A method according to clause 6, wherein the agent es. is selected from the group consisting of a human NMNAT1, a human NMNAT3 and conservatively CLAUSES: substituted variants thereof.

[0105] 10 10. A method according to clause 6, wherein the agent comprises a nucleic acid having at least 50% 1. A method of treating or preventing a neuropathy identitywith a nucleic acid encodinga human NMNA- or axonopathy in a mammal in need thereof, the Tor a nucleic acid having at least 50% identity with method comprising administering to the mammal an a nucleic acid encoding a human NMNAT3. effective amount of an agent that acts by increasing 15 sirtuin activity in diseased and/or injured neurons 11. A method according to clause 10, wherein the and supporting cells. agent comprises a nucleic acid having at least 70% identity with a nucleic acid encoding a human 2. A method according to clause 1, wherein the agent NMNAT1 or a nucleic acid having at least 70% iden- acts by increasing SIRT1 activity. 20 tity with a nucleic acid encoding a human NMNAT3.

3. A method according to clause 1, wherein the agent 12. A method according to clause 11 wherein the isNAD, NADH, an intermediateof a denovo pathway agent comprises a nucleic acid encoding a human for synthesizing NAD, an intermediate of a NAD sal- NMNATor a human NMNAT3 or a nucleic acid var- vage pathway, an intermediate of a nicotinamide ri- 25 iant thereof. boside kinase pathway or a combination thereof. 13. A method according to clause 1, wherein the 4. A method according to clause 1, wherein the agent agent comprises a sirtuin polypeptide or a nucleic is NAD, nicotinamide mononucleotide, nicotinic acid acid encoding a sirtuin polypeptide. mononucleotide or nicotinamide riboside. 30 14. A method according to clause 6, wherein the 5. A method according to clause 1, wherein the agent agent comprises an enzyme having SIRT activity comprises an enzyme of a de novo pathway for syn- and at least 50% identity with a human SIRT1. thesizing NAD, an enzyme of a NAD salvage path- way or an enzyme of a nicotinamide riboside kinase 35 15. A method according to clause 14, wherein the pathway; a nucleic acid encoding an enzyme of a de agent has at least 70% identity with a human SIRT1. novo pathway for synthesizing NAD, an enzyme of a NAD salvage pathway or an enzyme of a nicotina- 16. A method according to clause 15, wherein the mide riboside kinase pathway; an agent that increas- agent is selected from the group consisting of a hu- es expression of an enzyme of a de novo pathway 40 man SIRT1 and conservatively substituted variants for synthesizing NAD, an enzyme of a NAD salvage thereof. pathway or an enzyme of a nicotinamide riboside kinase pathway; or an agent that increases catalytic 17. A method according to clause 6, wherein the activity and/or stability of an enzyme of ade novo agent comprises a nucleic acid having at least 50% pathway for synthesizing NAD, an enzyme of a NAD 45 identity with a nucleic acid encoding a human SIRT1. salvage pathway or an enzyme of a nicotinamide riboside kinase pathway. 18. A method according to clause 17, wherein the agent comprises a nucleic acid having at least 70% 6. A method according to clause 5, wherein the agent identity with a nucleic acid encoding a human SIRT1. comprises a nicotinamide mononucleotide adenylyl- 50 transferase (NMNAT) or a nucleic acid encoding an 19. A method according to clause 18 wherein the NMNAT. agent comprises a nucleic acid encoding a human SIRT1 or a nucleic acid variant thereof. 7. A method according to clause 6, wherein the agent comprises an enzyme having NMNAT activity and 55 20. A method according to clause 1, wherein the at least 50% identity with a human NMNAT1 or at agent is a stilbene, a chalcone, a flavone, an isofla- least 50% identity with a human NMNAT3. vanone, a flavanone or a catechin.

16 31 EP 3 006 040 A1 32

21. A method according to clause 20, wherein the 27. A method of treating or preventing a neuropathy agent is a stilbene selected from the group consisting or axonopathy in a mammal in need thereof, the of resveratrol, piceatannol, deoxyrhapontin, method comprising administering to the mammal an trans-stilbene and rhapontin; a chalcone selected effective amount of an agent that acts by increasing from the group consisting of butein, isoliquiritigen 5 NAD activity in diseased and/or injured neurons and 3,4,2’,4’,6’-pentahydroxychalcone; a flavone and/or supporting cells. selected from the group consisting of fisetin, 5,7,3’,4’,5’-pentahydroxyflavone, luteolin, 3,6,3’,4’- 28. A method according to clause 27, wherein the tetrahydroxyflavone, quercetin, 7,3’,4’,5’-tetrahy- agent is NAD, NADH, an intermediate of a de novo droxyflavone, kaempferol, 6-hydroxyapigenin, api- 10 pathway for synthesizing NAD, an intermediate of a genin, 3,6,2’,4’-tetrahydroxyflavone, 7,4’-dihydroxy- NAD salvage pathway, an intermediate of a nicoti- flavone, 7,8,3’,4’-tetrahydroxyflavone, 3,6,2’,3’-tet- namide riboside kinase pathway or a combination rahydroxyflavone, 4’-hydroxyflavone, 5,4’-dihydrox- thereof. yflavone, 5,7-dihydroxyflavone, morin, flavone and 5-hydroxyflavone; an isoflavone selected from the 15 29. A method according to clause 28, wherein the group consisting of daidzein and genistein; a fla- agent is NAD, nicotinamide mononucleotide, nico- vanone selected from the group consisting of narin- tinic acid mononucleotide or nicotinamide riboside. genin, 3,5,7,3’,4’-pentahydroxyflavanone, and fla- vanone or a catechin selected from the group con- 30. A method according to clause 27, wherein the sisting of (-)-epicatechin, (-)-catechin, (-)-gallocate- 20 agent comprises an enzyme of a de novo pathway chin, (+)-catechin and (+)-epicatechin. for synthesizing NAD, an enzyme of a NAD salvage pathway or an enzyme of a nicotinamide riboside 22. A method according to clause 1, wherein the neu- kinase pathway; a nucleic acid encoding an enzyme ropathy or axonopathy is hereditary or congenital or of a de novo pathway for synthesizing NAD, an en- associated with neurodegenerative disease, motor 25 zyme of a NAD salvage pathway or an enzyme of a neuron disease, neoplasia, endocrine disorder, met- nicotinamide riboside kinase pathway; an agent that abolic disease, nutritional deficiency, atherosclero- increases expression of an enzyme of ade novo sis, an autoimmune disease, mechanical injury, pathway for synthesizing NAD, an enzyme of a NAD chemical or drug-induced injury, thermal injury, ra- salvage pathway or an enzyme of a nicotinamide diation injury, nerve compression, retinal or optic30 riboside kinase pathway; or an agent that increases nerve disorder, mitochondrial dysfunction, progres- catalytic activity and/or stability of an enzyme of a sive dementia demyelinating diseases ischemia de novo pathway for synthesizing NAD, an enzyme and/or stroke infectious disease; or inflammatory of a NAD salvage pathway or an enzyme of a nico- disease. tinamide riboside kinase pathway. 35 23. A method according to clause 22, wherein the 31. A method according to clause 30, wherein the neuropathy or axonopathy is induced by a cytotoxic agent comprises a nicotinamide mononucleotide anticancer agent. adenylyltransferase (NMNAT) or a nucleic acid en- coding an NMNAT. 24. A method according to clause 22, wherein the 40 optic neuropathy is glaucoma, retinal ganglion de- 32. A method according to clause 31, wherein the generation, optic neuritis and/or degeneration, mac- agent comprises an enzyme having NMNAT activity ular degeneration, ischemic optic neuropathy, trau- and at least 50% identity with a human NMNAT1 or matic injury to the optic nerve, hereditary optic neu- at least 50% identity with a human NMNAT3. ropathy, metabolic optic neuropathy, neuropathy45 due to a toxic agent or that caused by adverse drug 33. A method according to clause 32, wherein the reactions or vitamin deficiency. agent has at least 70% identity with a human NMNAT1 or at least 70% identity with a human 25. A method according to clause 22, wherein the NMNAT3. neuropathy associated with mitochondrial dysfunc- 50 tion results from oxidative damage, from mutations 34. A method according to clause 31, wherein the in mitochondrial proteins encoded either in the mito- agent is selected from the group consisting of a hu- chondrial genome or nuclear genome, from expo- man NMNAT1, a human NMNAT3 and conserva- sure to toxins, or from the process of aging. tively substituted variants thereof. 55 26. A method according to clause 1, wherein the 35. A method according to clause 31, wherein the mammal is a human. agent comprises a nucleic acid having at least 50% identity with a nucleic acid encoding a human

17 33 EP 3 006 040 A1 34

NMNAT1 or a nucleic acid having at least 50% iden- ducing an axonal injury to the neuronal cells com- tity with a nucleic acid encoding a human NMNAT3. prises chemically injuring the neuronal cells, ther- mally injuring the neuronal cells, oxygen-depriving 36. A method according to clause 35, wherein the the neuronal cells, physically injuring the neuronal agent comprises a nucleic acid having at least 70% 5 cells, inhibiting energy metabolism or a combination identity with a nucleic acid encoding a human thereof. NMNAT1 or a nucleic acid having at least 70% iden- tity with a nucleic acid encoding a human NMNAT3. 45. A method of screening agents for treating a neu- ropathy in a mammal, the method comprising detect- 37. A method according to clause 36 wherein the 10 ing an increase in NAD activity produced by a can- agent comprises a nucleic acid encoding a human didate agent, in a cell. NMNAT1 or a human NMNAT3 or a nucleic acid var- iant thereof. 46. A method of screening for agents that increase sirtuin activity in neurons, the method comprising ad- 38. A method according to clause 27, wherein the 15 ministering to mammalian neuronal cells in vitro or neuropathy oraxonopathy ishereditary or congenital invivo, acandidate agent; producingan axonal injury or associated with neurodegenerative disease, mo- to the neuronal cells; and detecting a decrease in tor neuron disease, neoplasia, endocrine disorder, axonal degeneration of the injured neuronal cells. metabolic disease, nutritional deficiency, athero- sclerosis, an autoimmune disease, mechanical inju- 20 47. A method of screening for agents that increase ry, chemical or drug-induced injury, thermal injury, NAD activity in neurons, the method comprising ad- radiation injury, nerve compression, retinal or optic ministering to mammalian neuronal cells in vitro or nerve disorder, mitochondrial dysfunction, progres- invivo , acandidate agent; producingan axonal injury sive dementia demyelinating diseases ischemia to the neuronal cells; and detecting a decrease in and/or stroke infectious disease; or inflammatory 25 axonal degeneration of the injured neuronal cells. disease. 48. A recombinant vector comprising a promoter op- 39. A method according to clause 38, wherein the eratively linked to a polynucleotide having at least neuropathy or axonopathy is induced by a cytotoxic 50% identity with a nucleic acid encoding a human anticancer agent. 30 NMNAT1 or a polynucleotide having at least 50% identity with a nucleic acid encoding a human 40. A method according to clause 22, wherein the NMNAT3. optic neuropathy is glaucoma, retinal ganglion de- generation, optic neuritis and/or degeneration, mac- 49. A recombinant vector according to clause 48 ular degeneration, ischemic optic neuropathy, trau- 35 comprising a polynucleotide having at least 70% matic injury to the optic nerve, hereditary optic neu- identity with a nucleic acid encoding a human ropathy, metabolic optic neuropathy, neuropathy NMNAT1 or a polynucleotide having at least 70% due to a toxic agent or that caused by adverse drug identity with a nucleic acid encoding a human reactions or vitamin deficiency. NMNAT3. 40 41. A method according to clause 38, wherein the 50. A recombinant vector according to clause 48 neuropathy associated with mitochondrial dysfunc- comprising a polynucleotide encoding a human tion results from oxidative damage, from mutations NMNAT1 or a human NMNAT3 or a polynucleotide in mitochondrial proteins encoded either in the mito- variant thereof. chondrial genome or nuclear genome, from expo- 45 sure to toxins, or from the process of aging. 51. A recombinant vector according to clause 48 comprising a lentivirus or an adeno-associated virus. 42. A method according to clause 27, wherein the mammal is a human. 52. A recombinant vector comprising a promoter op- 50 eratively linked to a poloynucleotide having at least 43. A method of screening agents for treating a neu- 50% identity with a nucleic acid encoding a human ropathy in a mammal, the method comprising admin- SIRT1. istering to mammalian neuronal cellsin vitro or in vivo, a candidate agent; producing an axonal injury 53. A recombinant vector according to clause 52 to the neuronal cells; and detecting a decrease in 55 comprising a polynucleotide having at least 70% axonal degeneration of the injured neuronal cells. identity with a nucleic acid encoding a human SIRT1.

44. A method according to clause 43, wherein pro- 54. A recombinant vector according to clause 54

18 35 EP 3 006 040 A1 36 comprising a polynucleotide acid encoding a human 64. A method according to clause 62, wherein the SIRT1 or a polynucleotide variant thereof. agent comprises a nucleic acid having at least 50% identity with a nucleic acid encoding a human 55. A recombinant vector according to clause 52 NMNAT1 or a nucleic acid having at least 50% iden- comprising a lentivirus or an adeno-associated virus. 5 tity with a nucleic acid encoding a human NMNAT3.

56. A method of treating or preventing an optic neu- 65. A method according to clause 64, wherein the ropathy in a mammal in need thereof, the method agent comprises a nucleic acid having at least 70% comprising administering to the mammal an effective identity with a nucleic acid encoding a human amount of an agent that acts by increasing NAD ac- 10 NMNAT1 or a nucleic acid having at least 70% iden- tivity in diseased and/or injured neurons. tity with a nucleic acid encoding a human NMNAT3.

57. A method according to clause 56, wherein ad- 66. A method according to clause 65 wherein the ministering to the mammal comprises intraocular ad- agent comprises a nucleic acid encoding a human ministering. 15 NMNAT1 or a human NMNAT3 or a nucleic acid var- iant thereof. 58. A method according to clause 57, wherein in- traocular administering comprises intraocular ad- 67. A method according to clause 22, wherein the ministering of a sustained release delivery system. optic neuropathy is glaucoma, retinal ganglion de- 20 generation, optic neuritis and/or degeneration, mac- 59. A method according to clause 57, wherein in- ular degeneration, ischemic optic neuropathy, trau- traocular administering comprises intravitrial injec- matic injury to the optic nerve, hereditary optic neu- tion, administration by eyedrops or administration by ropathy, metabolic optic neuropathy, neuropathy trans-scleral delivery. due to a toxic agent or that caused by adverse drug 25 reactions or vitamin deficiency. 60. A method according to clause 56, wherein the agent is NAD, NADH, an intermediate of a de novo 68. A method according to clause 56, wherein the pathway for synthesizing NAD, an intermediate of a mammal is a human. NAD salvage pathway, an intermediate of a nicoti- namide riboside kinase pathway or a combination 30 thereof. Claims

61. A method according to clause 60, wherein the 1. An agent for use in the treatment or prevention of an agent is NAD, nicotinamide mononucleotide, nico- axonopathy in a mammal in need thereof, wherein tinic acid mononucleotide or nicotinamide riboside. 35 the agent is NAD, NADH, an intermediate of a de novo pathway for synthesizing NAD, an intermediate 62. A method according to clause 56, wherein the of a NAD salvage pathway or an intermediate of the agent comprises an enzyme of a de novo pathway Preiss-Handler independent pathway. for synthesizing NAD, an enzyme of a NAD salvage pathway or an enzyme of a nicotinamide riboside 40 2. An agent in accordance with claim 1, wherein the kinase pathway; a nucleic acid encoding an enzyme intermediate of a de novo pathway for synthesizing of a de novo pathway for synthesizing NAD, an en- NAD is quinolinic acid or nicotinate adenine dinucle- zyme of a NAD salvage pathway or an enzyme of a otide (NaAD). nicotinamide riboside kinase pathway; an agent that increases expression of an enzyme of a de novo 45 3. An agent in accordance with claim 1, wherein the pathway for synthesizing NAD, an enzyme of a NAD intermediate of a NAD salvage pathway is nicotinic salvage pathway or an enzyme of a nicotinamide acid. riboside kinase pathway; or an agent that increases catalytic activity and/or stability of an enzyme of a 4. An agent in accordance with claim 1, wherein the de novo pathway for synthesizing NAD, an enzyme 50 intermediate of a NAD salvage pathway is nicotina- of a NAD salvage pathway or an enzyme of a nico- mide. tinamide riboside kinase pathway. 5. An agent in accordance with claim 1, wherein the 63. A method according to clause 62, wherein the agent is nicotinic acid mononucleotide (NMN). agent comprises a nicotinamide mononucleotide55 adenylyltransferase (NMNAT) or a nucleic acid en- 6. An agent in accordance with claim 1, wherein the coding an NMNAT. agent is nicotinate mononucleotide (NaMN).

19 37 EP 3 006 040 A1 38

7. An agent in accordance with claim 1, wherein the agent is nicotinamide riboside (NmR)

8. An agent in accordance with claim 1, wherein the mammal is a companion animal, an agricultural an- 5 imal, or an exotic animal.

9. An agent in accordance with claim 8, wherein the mammal is a dog, a cat, a cow or a horse. 10 10. An agent in accordance with claim 1, wherein the mammal is mouse.

11. An agent in accordance with claim 1, wherein the mammal is a human. 15

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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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