WO2005080573A1

WO2005080573A1 - Recombinant viral vectors to promote neuronal cell survival and uses thereof

Info

Publication number: WO2005080573A1
Application number: PCT/CA2005/000225
Authority: WO
Inventors: Adriana Dipolo; Vincent Pernet; William W. Hauswirth
Original assignee: Universite de Montreal; University of Florida; University of Florida Research Foundation Inc
Current assignee: Universite de Montreal; University of Florida; University of Florida Research Foundation Inc
Priority date: 2004-02-20
Filing date: 2005-02-21
Publication date: 2005-09-01
Anticipated expiration: 2006-08-20

Abstract

The present invention relates to apoptosis. More specifically, the present invention is concerned with recombinant viral vectors to promote neural survival in glaucoma and other retinal diseases. A vector which expresses MEK nucleic acid sequence encoding a recombinant MEK product which is more active than a wild type MEK product, wherein said recombinant MEK is expressed to a level which is sufficient to enable an increase in activation of at least one of Erk1 and Erk2, thereby enabling neuronal survival of cells undergoing cell death is taught herein.

Description

TITLE OF THE INVENTION

[0001] RECOMBINANT VIRAL VECTORS TO PROMOTE NEURONAL

CELL SURVIVAL AND USES THEREOF

FIELD OF THE INVENTION

[0002] The present invention broadly relates to neurαprotection. The invention also relates to apoptosis and more specifically to delaying apoptosis in fully differentiated neurons. The present invention relates to survival, and/or maintenance or increase in growth and/or neural function of neuronal cells. More specifically, the present invention is concerned with recombinant viral vectors to promote neuronal cell survival. The present invention mote specifically relates to the promotion or maintenance of growth of fully differentiated neuronal cells of the central nervous system (CNS). In one particular embodiment, the invention relates to recombinant viral vectors to promote the survival of fully differentiated neurσtrσphic factor-responsive neurons. In one embodiment, the invention relates to recombinant viral vectors to promote the survival in glaucoma and other retinal diseases and Uses thereof.

BACKGROUND OF THE INVENTION

[0003] Glaucoma is a leading cause of blindness worldwide [1]. The incidence of glaucoma increases dramatically with age. More than 2.2 million people in North America age 40 and older have glaucoma and every hour, someone goes blind from this sight-threatening disease (www.preventblindness.grg). As the elderly population continues to grow rapidly, glaucoma has become an imminent social as well as medical problem. The characteristic visual field changes and loss of vision in glaucoma are caused by the selective degeneration of retinal ganglion cells (RGCs). RGC apoptosis has been detected in experimental glaucoma in rats, monkeys and humans (Nickells. 1999). Elevated intraocular pressure (IOP) is a key risk factor for RGC loss in glaucoma [2], however, this condition worsens in a group of patients despite the Use of IOP lowering medication [3-5]. Thus, there is great need for the development of alternative strategies that slow RGC death and the progression of glaucomatous optic neuropathy. At present, there are no effective neuroprotective strategies for the treatment of this disease-

[0004] The potent effect of neurotrophins on the survival of adult central nervous system (CNS) neurons [6] has led to interest in using them to develop therapeutic strategies applicable to glaucoma. Among neurotrophins, brain-derived neurotrophic factor (BDNF) is the most potent survival factor for injured RGCs [7- 12]. Consistent with this, RGCs express TrkB, the high-affinity receptor for BDNF [13, 14]. An important limitation of applying exogenous BDNF as neuroprotective therapy for RGCs is that this factor will affect all other retinal cells that express the receptor TrkB, including amacrine cells, Mϋller glia and cone photσreceptors tl3, 15, 16]. In addition, it is known that exogenous neurotrophic factors may produce adverse side effects. For example, BDNF may limit its neuroprotective action on axotomized RGCs by upregulating nitric oxide synthase activity [17] or by suppressing the expression of the heat shock protein 27 [18]. Systemic administration of ciliary neurotrophic factor (CNTF). which protects several classes of neurons and glia [ 9], has been shown to produce rapid weight loss resulting in death [20]. In addition, recent studies demonstrated that CNTF has deleterious effects on visual function as assessed by electroretinography [21. 22]

[0005] An alternative strategy is to target the intracellular events that lead to

RGC survival, bypassing the use of exogenous peptide factors, which may have a broad base of targets and are thus less specific. Upon binding to Trk receptors. BDNF stimulates multiple signaling pathways, including the extracellular signal- regulated kiπase 1/2 (Erk1/2) and the phosphatidylinositol-3 (PI-3) kinase pathways [23]. Although both Erk 1/2 and Pl-3 kinase are stimulated in RGCs following TrkB activation in vivo, we recently demonstrated that Erk1/2 is a key signaling component mediating adult RGC survival [24]. Thus, we tested the hypothesis that selective stimulation of the Erk 1/2 pathway would promote RGC survival in a rat model of ocular hypertension. Recombinant adeπo-associated virus (AAV) was used for in vivo gene delivery of constitutively active or wild-type MEKI into RGCs. This vector system was selected based on our finding that RGCs are the primary cellular target for AAV serotype 2 transduction upon intravitreal virus administration [24-26]. In addition, AAV evokes minimal immune response in the host t27, 28] and mediates long-term traπsgene expression that can persist in the retina for at least one year after vector administration [29, 30].

[0006] The present invention seeks to meet these needs and other needs.

[0007] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0008] The present invention relates to vectors and methods which aim to overcome the defects of the prior art. In particular the present invention relates to recombinant adeno-assσciated (rAAV)-based genetic constructs that selectively increase the level of phosphorylated Erk1/2, which is the active state of this kinase. so as to treat or ameliorate a disease or disorder associated with a degeneration or apoptosis of fully differentiated, post-mitotic neurons

[0009] More specifically, the present invention relates to vectors and methods, which can be used in neuroprotective strategies and to mediate the survival of fully differentiated neuronal cells of the CNS. [0010] In one particular embodiment the neuroprotective strategies of the present invention not only delay apoptosis of the fully differentiated neuronal cells of the CNS. but also maintain the structural integrity of axσnal processes, which is an essential requisite for proper neural function.

[0011] More particularly, the present invention relates to an increase in

Erk1/2 activation to protect fully differentiated neuronal ceils of the CNS. In one embodiment the invention relates to the effect of MEK, an upstream activator of Erk1/2, in a gene transfer protocol enabling a neuroprotective strategy in fully differentiated neuronal cells. In one particular embodiment, the neuroprotective strategy of the present invention is exemplified with several models of optic nerve injury.

[0012] In one embodiment the present invention relates to a recombinant adeno-associated virus (rAAV) genetic construct containing a nucleic acid sequence (e.g. gene) that encodes a constitutively active (CA) MEK (MEK-CA) or active portion thereof, to test its effect on the survival of fully differentiated neuronal cells of the CNS, such as RGCs.

[001 ] The present invention provides an alternative strategy to thdse of the prior art, to promote survival of fully differentiated neuronal cells of the CNS . This strategy does not depend on diffusible and short-lived factors such as neurotrophins (e.g. brain-derived neurotrophic factor: BDNF). Using RGCs as a model system, the rAAV-MEK-ca (for constitutively active) virus permitted a direct modulation of intracellular MAPK activity in retinal ganglion neurons.

[0014] The present invention also relates to the demonstration that MEK activation stimulates cell survival after traumatic injury and importantly, in a clinically relevant model of glaucoma. [0015] In addition, and of importance, the present invention shows that the constitutively active MEK not only enables neuroprotection of RGCs but also of the axons in the optic nerve, further demonstrating the power and applicability of the present invention to fully differentiated neurons of the CNS.

[0016] The present invention further relates to a method to neuroprotect fully differentiated neuronal cells of the CNS by providing an increase in constitutively active MEK (MEK-CA) at the protein level.

[0017] As alluded to above, glaucoma is the second leading cause of vision loss in the world (Quigley, -1996, supra; also see Quigley, 1995, Aust. NZ J Ophthalmol. 1995 May, 23(2).85-91). With the significant increase in the incidence of diabetis, glaucoma could become an even more critical health issue. It has been reported that the leading causes of visual impairment and blindness are diabetic retinopathy and age-related eye diseases (e.g.. cataracts, macular degeneration, and glaucoma, in Morb Mortal Wkly Rep. (MMWR) 2004 Nov 19;53(45). 069-71). Indeed, a substantially higher prevalence of visual impairment and eye disease is encountered amongst those with diabetes as compared to those without diabetes (MMWR, 2004, supra). Furthermore, angle closure glaucoma is emerging as a leading cause of blindness in the densely populated countries of Asia (Foster, 2002, Semin. Ophthalmol. Jun;17(2):50-8).

[0018] While the present invention is exemplified using glaucoma as a model system for a disease leading to retinal ganglion cell (RGC), and optic nerve degeneration, the present invention should not be so limited. Indeed, having demonstrated that RGC and axon survival is increased, the instant invention can be applied to other diseases or conditions in which RGCs or optic nerve degeneration occurs. Non-limiting examples of such diseases or conditions also include: optic neuritis and multiple sclerosis (Frohman et al., Lancet NeUrσl. 2005 Feb; 4(2):111-21; and Foroσzan et al.. Curr. Opin. Ophthalmol. 2002 Dec; 3(6):375-80); optic neuropathies (Johns et al., Semin. Ophthalmol. 2002 Mar; 17(1):33-8; Barboni et al.. Ophthalmology. 2005 Jan;112(1):120-6); orbital trauma (Levin. Ophthalmol. Clin. North Am. 2004 Sep.- 17(3):455-B4, vii; Girkin et al., Am. J. Ophthalmol. 2005 Jan; 139(1): 100-5; Chang et al Curr. Opin. Ophthalmol. 2004 Oct;15(5).4 1-5); optic disk and nerve cancer (Zografos et al., Am. J. Ophthalmol. 2004 Dec; 38(6):964-9; Carrasco et al., Curr. Opin. Ophthalmol. 2004 Oct: 15(5):406-10; Schick et al., J. Neurosurg. 2004 Dec;.101(6):951-9).

[0019] Also, while the present invention is exemplified by showing that the increase in the presence of MEK-CA protein in RGCs and axons enable neuroprotection and neuronal cell survival, the present invention is not so limited. Indeed, RGCs and their axons are but one example of fully differentiated neυtrophic factαr-(NF)respσns.ve cells. Non-limiting examples of such fully differentiated neutrophic factor-responsive cells are knowb in the art (Salehi et al., J. Neural Transm. 2004 1 (3);323-345; Tuszynski et al.. Prog. Brain Res. 2004; 2004;146:441-9; and Lad et al.. Curr. Drug Targets CNS Neurol. Disord. 2003 Oct; 2(5):315-34); Dopaminergic neurons of substantia nigra projecting to striatum {Parkinson's Disease) (Behrstock, et al., Ann. NY Acad. Sci. 2004 Jun; 1019:5-14; Fernandez-Espejo; Mol. Neurobiol. 2004 Feb; 29(1 ):15-30; Kirik et al., Nat. Neurosci. 2004 Feb; 7(2): 105- 0. Epub 2004 Jan 27); striatal neurons and cortical neurons (Huntingtσn's Disease) (Alberch et al.. Prog. Brain Res. 2004; 146:195- 229; Kordower et al., Prog Brain Res. 2000; 127:414-30); Motoheurons (Amyotrophic lateral sclerosis) (Bohn, Exp. Neurol. 2004 Dec; 190(2):263-75; Hurko et al., J. Neurol. Sci. 2000 Nov 1; 180(1~2):21-S).

[0020] It shall also be recognized that the neuroprotection shown herein for the cell body (RGC) and for the axon of the fully differentiated neurotrophic factor (NF)-respohsive neurons, is a significant demonstration. It will be recognized that neuroprotection of only the cell body and not the axon (or the converse), does have value and utility, but clearly, the neuroprotection of all the neuronal parts is what is to be achieved, in view of providing the most efficient, and Useful neuroprotection and recovery of vision. In other words, delaying apoptosis of the neural cell does, not ensure that neuronal cell function will be maintained. Nickells (Brain Res. Bull. 2004 Feb 15; 62(6):439-46), for example teaches that "several independent laboratories have demonstrated that blocking cell death does not necessarily create a situation where the cell resumes normal function". Nickells mentions a study, with dopaminergic cells, which showed that while they were prevented from undergoing apoptosis, they lost their axonal terminals, and hence their function (von Coelln et al., J. Neurσchem. 2001; 77:263-267). Reference to Bcl-2 gene therapy, to keep neuron alive, was also shown not to enabte a preservation of neuron function, since the protected neurons no longer synthetized neural transmitters (Fink et al., Gene Ther. 2000 7:115-119). It is thus clear that the means for enhancing neuron cell survival or delay apoptosis thereof should also aim. when ever possible, at preserving neuronal function (Nickells supra).

[0021] It will also be recognized by the person of skill in the art that numerous types of vectors exist and are well-known in the art. In one embodiment of the present invention, through a transfer of genetic material an increase in the level of MEK-ca protein is effected (using rAAV). It shall be clear that the present invention is not limited to vectors comprising constitutively expressed promoters. Indeed, controllable expression sequences, tissue-specific expression sequences and regulated-expression sequences (e.g. promoters) are encompassed by the present invention. In one particular embodiment, the vector is a recombinant Adeno-Associated-Virus (rAAV). Of course other vectors, well known in the art could be used (e.g. adenoviral or lentiviral vectors) (retroviral vectors can't be used because they only infect actively dividing cells and post-mitotic neurons are not dividing any more). In addition virus-free delivery can also be used. Furthermore the delivery of protein directly, encapsulated or otherwise is also considered as being covered by the present invention. Non-limiting examples of particular rAAV vectors that can be used or adapted and used in accordance with the present invention include: Sustained tetracycline-regulated transgene expression in vivo using a single type 2 adeno-associated viral vector (Follϊσt, et al., J. Gene Med. 2003 Jυn; 5(6):493-5θ1 ); Tetracycline-inducible transgene expression mediated by a single AAV vector (Chtarto et al.. Gene Ther. 2003 Jan;10(1):84-94); Tightly regulated expression from a single tetracycline-off rAAV vector (Jiang et al.. Gene Ther. 2004 Jul; (13): 1057-67).

[0022] In a general sense therefore, the present invention provides compositions and methods for treating or ameliorating a disease or condition associated with a decrease in the number or function of fully differentiated NF- responsive neurons in a mammal, and particularly for treating or reducing the severity and extent of such disease or condition in a human. As alluded to above, such diseases or conditions include without being limited thereto, glaucoma, visual impairment, blindness, retinitis pigmentosa or age-related macular degeneration, optic nerve degeneration, optic neuritis and multiple sclerosis, optic neuropathies, orbital trauma, optic disk and nerve cancer, Parkinson's Disease, Huntington's Disease, and Amyotrophic lateral sclerosis.

[0023] In a general sense, in one embodiment the invention involves an administration of a genetic construct which enables an increase in the level of activated (i.e., phosphorylated) Erk1/2 in a pharmaceutically-acceptable vehicle to the mammal, in the amount and for a period of time which are sufficient to treat or ameliorate the disease or condition in the mammal suffering therefrom or at risk of developing same. In one particular embodiment, the genetic construct is a rAAV construct comprising at least an activating portion of MEK-ca. As will be shown herein, the activation of at least one of Erk1 and Erk2. to a level of the present invention is necessary for delaying apoptosis, neuroprotection and the like. Thus, the activation of only one of Erk1 and Erk2, is also within the scope of the present invention, provided that a sufficient level of activated Erk 1 or Erk 2 is reached. [0024] It is believed that prior to the present invention, a neuroprotective action of MEK-CA in fully differentiated NF-responsive neurons had not been demonstrated. The present invention demonstrates that the neuroprotective action of is effected through Erk1 and Erk2, which are direct substrates for MEK. In accordance with the present invention the phosphorylation of Erk1 and/or Erk2 needs to be increased as compared to wild-type levels, in order to have a neuroprotective effect. While at about 1.5 fold as compared to wild-type, a neuroprotective effect could be observed, an increase Of at least about 2 fold in the phosphorylation of Erk1 and/or Erk2 needs to occur as compared to wild-type levels, in order to have the neuroprotective effect of the present invention. In one particular embodiment, increases from about 2 fold to about 10 fold (over basal levels found in control or normal cells) are required to promote neuronal cell survival. Of course, at least any one of about 3, 4. 5, 6, 7, 8, or 9 fold to about 10 fold, are also within the scope of the present invention. While levels higher than about 10 folds are also contemplated, care must be taken to insure that no nonspecific effects of Erk1 and/or Erk2 phosphorylation is observed. It also follows that the level of overexpression of MEK needs to be monitored, to insure that nonspecific effects are not detrimental to the use of the present invention. The person skilled in the art to which the present invention pertain will adapt the levels of Erk1 and/or Erk2 phosphorylation, the level of overexpression of MEK-CA or the level of active MEK-CA protein, so as to avoid non-desirable or detrimental side-effects to the animal (e.g. human) to which the present invention pertains.

[0025] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings-

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In the appended drawings: [0027] Figure 1 shows an outline of the experimental protocol used to test the effect of AAV.MEK-CA on RGC survival in experimental glaucoma. Following intraocular injection of viral vectors, RGCs were back labeled with the fluorescent tracer Dil. Episcleral vein injection was performed one week after Dil application to assure that all RGCs were labeled prior to intraocular pressure increase. Retinas were examined histologically at 5 and 7 weeks following ocular hypertension surgery to determine the density of surviving RGCs;

[0028] Figure 2 shows that AAV mediates MEK-CA gene product expression in adult RGCs. AAV-mediafed MEK-CA (Panels A-C) or MEK-WT (Panels D-F) were visualized using an antibody against the HA tag present only in MEK1 transgenes. Robust HA staining was observed in a large number of cell bodies in the ganglion cell layer (GCL) and dendrites in the inner plexiform layer (IPL) (Panels A and D). RGCs were visualized using the retrograde tracer FluoroGold (FG) applied to the superior colliculus, the main target for these neurons in the rat brain (Panels B and E). Superimposition of the HA and FG staining demonstrated that the vast majority of RGCs expressed MEK-CA (Panel C) or MEK-WT (Panel F) gene product. HA immunσreactivity was not detected in retinal sections after intraocular injection of AAV.GFP (data not shown). High- power magnification demonstrated HA labeling on RGC so a and dendritic processes after infection with AAV.MEK-CA (Panel G) or AAV.MEK-WT (Panel J). Retrograde labeling with FG (Panels H and K) confirmed that RGCs expressed HA-tagged MEK proteins (Panels I and L). In vivo activation of Erk1/2 kinases was detected in retinal homogenates at 4 weeks after injection of AAV.MEK-CA compared to control eyes treated with AAV.MEK-WT (Panel M). Western blots of total retinal extracts were probed with an antibody that selectively recognizes both Erk1 and Erk2 phosphorylated on Thr202/Tyr204 residues. The bottom panel shows the same blot reprαbed with an antibody to visualize total Erk1/2 protein. Scale bars: A-F = 100 μm, G-L = 25 gm. PS: photoreceptors segments, ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer, shows the examination of retinas and optic nerves at four weeks following administration.

[0020] Figure 3 shows AAV.MEK-CA protects RGCs from hypertension- induced death. Quantitative analysis of RGC survival following injection of AAV.MEK-CA (solid bars), AAV.MEK-wt (hatched bars) or AAV.GFP (white bars) is shown for whole retina (Panel A) or superior hemisphere (also termed superior quadrant) only, where AAV vectors were injected (Panel B) (n *= 8-13 rats per group). The density of RGCs in intact, unoperated rat retinas is shown as reference (gray bar). MEK-CA gene transfer markedly increased the number of RGCs that survived at 5 or 7 weeks ocular hypertension surgery (ANOVA: ^*: P<0.001 ; **: P<0.0001). Data are expressed as the mean ± S.E.M.

[0030] Figure 4 shows AAV.MEK-CA protects RGC soma from hypertension-damage. Fluorescence photomicrographs of flat-mounted retinas showing DiMabeled RGCs in intact (Panel A) or glaucomatous retinas treated with AAV.MEK-CA (Panel B) or AAV.MEK-GFP (Panel C) at 5 weeks after ocular hypertension surgery. Images were taken from the superior, central retina. AAV.MEK-CA treatment led to higher neuronal densities and better preservation of cellular integrity. Scale bar: 100 μ .

[0031] Figure 5 shows Erk1/2 activation protects intraretinal RGC axons in glaucoma. Confocal microscopy images of intraretinal RGC axons visualized on flat-mounted retinas stained with RT-97, an antibody that recognizes the phosphorylated 200-kDa neurσfilament H subunit. Immunoreactive axons coursed in organized bundles toward the optic nerve head in normal retinas (Panel A). Treatment with AAV.MEK-CA remarkably preserved the overall structure of RGC axon bundles (Panel B), while retinas treated with the control vector AAV.GFP suffered significant fiber loss at 5 weeks after hypertension surgery (Panel C). Many remaining fibers had a beaded appearance confirming the progressive axonal degeneration after glaucomatous injury. Scale bars: 20 μm;

[0032] Figure 6 shows that AAV.MEK-CA treatment reduces optic nerve damage in glaucoma. Cross-sections of optic nerve segments from intact (Panel A) and glaucomatous eyes treated with AAV.MEK-CA (Panel B) or AAV.MEK-WT (Panel C) at 5 weeks after ocular hypertension surgery. AAV.MEK-CA-treated eyes displayed a larger number of axonal fibers with normal morphology compared to AAV.MEK-WT-treated control eyes, which showed extensive axon degeneration including disarray of fascicuiar organization and degradation of myelin sheaths. Panel D shows the quantitative analysis of RGC axons in the optic nerve following injection of AAV.MEK-CA (solid bars), AAV.MEK-WT (hatched bars) or AAV.GFP (white bars) (n = 4-7 rats per group). The number of axons in the intact, uninjured optic nerve is shown as reference (gray bar). MEK-CA gene transfer markedly protected RGC axons at 5 weeks ocular hypertension surgery (ANOVA: *: P<0.05). Data are expressed as the mean ± S.E.M.

[0033] Figure 7 shows a schematic diagram of the point mutations introduced in the kinase domain of the MEK gene to render it constitutively active. The mutations S218E and S222D in the kinase domain (SEQ ID NOs: 9 vs 11) as well as an 8-amino acid deletion in the regulatory domain (SEQ ID NOs: 8 vs 10) were introduced in wild type MEK to render it constitutively active (G1C variant). Another constitutively active MEK was produced by site-directed mutagenesis of S218D and S222D in the kinase domain as well as an 12-amino acid deletion in the regulatory domain (residues 44-55). Both cDNAs were used to generate the plasmid DNA rAAV.MEK-CA used to produce recombinant virus rAAV.MEK-CA used herein. The results herein presented used the former MEK-CA. Of note, a further MEK-ca was used, termed R4F having the same serine mutations and the same substitutions as G1C, but in addition it has a complete deletion of amino acids 32 to 51. A schematic representation of a vector used in accordance with the present invention, to produce the recombinant AAV is shown in Figure 9B (see below for details of construction).

[0034] Figure 8 shows a schematic diagram of the plasmid DNA rAAV.MEK-CA used to produce recombinant virus rAAV.MEK-CA. TR: 145-bp AAV terminal repeat sequence; CBA: chicken beta-actin promoter sequence; MEK-CA: MAP-ERK kinase-constitutively active cDNA; HA : hemaglutinin tag; SD/SA: simian virus 40 late viral protein 16S/19S splice donor and acceptor signal; pA1 and pA2: polyadenylation signals.

[0035] Figure 9A shows the nucleic acid sequence of pMCL MAPKK1 human (MEK-wt) MEK-wt is comprised between nucleotide positions 9,991 and 11 ,442 of Figure 9A (SEQ ID NO:1) see also Figure 11 B showing the alignment (SEQ ID NOs: 6 and 7); Figure 9B shows a schematic representation of the pXXUF12 vector used to create the rAAV; Figure 9 B; Figure 9C (SEQ ID NO:2) shows the nucleic acid of the vector domain. Figure 9D shows the nucleic acid sequence of the pXXUF12 vector harboring the GFP gene (SEQ ID NO:3); and Figure 9E shows features of the vector sequence of Figure 9D.

[0036] Figure 10 shows that AAV.MEK-CA leads to specific stimulation of the Erk1/2 pathway in vivo, and not to other signaling pathways which are stimulated by diffusible neurotrophic factors. Fig. 10A shows the level of Erk1/2 as well as that of phosphorylated (activated) Erk1/2a, as described above; Figure 10B shows the phosphorylation status of Akt, a critical downstream target of PI3K; and Figure 10C shows the phosphorylation status of Erk5, a critical factor having been shown to be involved in neuronal survival.

[0037] Figure 11 shows an alignment between human MEK-wt (SEQ ID

NO: 4) and mouse MEK-wt (SEQ ID NO: 5) at the protein level (showing only 4 differences (3 of which are conserved substitutions); Figure 11 B shows the same alignment but at the nucleic acid level (SEQ ID NOs: 6 and 7, respectively).

[0038] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0039] We previously demonstrated that brain-derived neurotrophic factor and its receptor, TrkB, promote the survival of retinal ganglion cells (RGC) via the mitogen-activated protein kinase (MAPK) pathway, one goal of the present study was to determine the effect of activation of MAPK on RGC survival and axon regeneration after acute optic nerve lesion using a direct system which was not dependent on a combination of gene therapy and the addition of a diffusible factor. Another goal was to assess whether an active form of MEK, an upstream activator of MAPK, protected RGCs in an experimental glaucoma animal model, and was therefore physiologically relevant.

[0040] The extracellular signal-regulated kinase (Erk) 1/2 pathway is an evolutionarily conserved mechanism used by several peptide factors to promote cell survival. It is demonstrated for the first time that selective activation of Erk1/2 protected RGCs in a rat model of experimental glaucoma. Recombinant adeno- associated virus (AAV) was used to selectively transduce RGCs with genes encoding constitutively active or wild-type MEK1 , the upstream activator of Erk1/2. MEK1 gene transfer into RGCs markedly increased neuronal survival: ~77% of the total number of RGCs remained alive in the dorsal retina at five weeks after ocular hypertension surgery, a time when >60% of these neurons were lost in control eyes. Hence, the Erk1/2 pathway plays a key role in the protection of RGCs from hypertension damage. The instant invention identifies a novel gene therapy strategy in which selective activation of the Erk1/2 signaling pathway effectively slows cell death in a fully differentiated neuron in a disease associated with apoptosis thereof (e.g. glaucoma).

[0041] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0042] Adeπovirus-associated virus-2 (AAV) is a human parvovirus which is routinely used in gene therapy strategies. AAV is a safe vector whose rescue mechanism is simple. In addition AAV is not pathogenic and not associated with a disease. The removal of coding sequences of AAV, enables insertion of nucleic acid sequences of the present invention. For more information on the biology and uses of AAV as a vector, the reader is referred to the following documents, the contents of which are herein incorporated by reference in their entirety: WO01/94605A2 of Zolotukhin et al., of University of Florida; US 2003/0 29 64A1 of Flannery et at., and US2004/0022766A1 of Acland et al.

[0043] It will be understood that the diseases and conditions for which the present invention finds utility have in common the fact that they are associated with loss of function and/or apoptosis of fully differentiated NF-responsive neurons. It will also be recognized that a number of such diseases or conditions are progressive, degenerative diseases that are often age-related and have a late- onset manifestation. Finally, it will be recognized that because of the progressive manifestation of the symptoms of a number of such diseases or conditions, that diagnosis thereof is often carrled-out after significant degenerecence has already occurred. [0044] Unless defined otherwise, the scientific and technical terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Commonly understood definitions of molecular biology terms can be found for example in Dictionary of Microbiology and Molecular Biology. 2nd ed. (Singleton et al.. 1994, John Wiley & Sons, New York, NY), the Harper Collins Dictionary of Biology. Hale S. Marham, 1991 , Harper Perennial. New York. NY); Rieger et al., Glossary of genetics: Classical and molecular, 5^lh edition. Springer-Verlag, New-York, 1991; Alberts et al., Molecular Biology of the Cell, 4^lh edition. Garland science, New-York, 2002; and. Lewin, Genes VII, Oxford University Press, New-York, 2000. Generally, the procedures of molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (2000, Molecular Cloning - A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).

[0045] In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

General terms

[0046] When referring to nucleic acid molecules, proteins or polypeptides, the term native refers to a naturally-occurring nucleic acid or polypeptide. A homolog is a gene sequence encoding a polypeptide isolated from an organism other than a human. As known in the art MEK and downstream effectors thereof (as well as upstream effectors) are very highly conserved throughout evolution. Similarly, a homolog of a native polypeptide is an expression product of a gene homolog. Figures 2A and 2B, show the extreme conservation of MEK between us mυscυlus and homo sapiens, demonstrating the importance of MEK, and its conservation throughout evolution. MEK has been described previously [31] and its sequence is well known in the art (human MEK is listed in GenBank as NM_002755. while the sequence of the mouse homolog is found as BC054754.

[0047] Purified. As used herein, the term "purified" refers to a molecule or molecules having been separated from a component of the composition in which it was originally contained. Thus, for example, a "purified protein" or a "purified nucleic" acid has been purified to a level not found in nature. A "substantially pure" molecule is a molecule that is lacking in most other components (e.g., 30, 40. 50. 60, 70. 75, 80. 85, 90, 95. 96, 97. 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. For the sake of brevity, the units (e.g. 66. 67...81 , 82....91 , 92%....) have not been specifically recited but are considered nevertheless within the scope of the present invention.

[0048] Expression. By the term "expression" is meant the process by which a gene or otherwise nucleic acid sequence produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s). When referring to a RNA nucleic acid, the term expression relates to its translation into a polypeptide(s). In accordance with the present invention one such polypeptide is MEK-CA.

[0049] As used herein, the designation "functional derivative" denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid as chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylϊcity and the like. The term "functional derivatives" is intended to include "functional fragments", "functional segments", "functional variants", "functional analogs" or "functional chemical derivatives" of the subject matter of the present invention. "Fragments" of the nucleic acid molecules according to the present invention refer to such molecules having at least 12 nt, more particularly at least 8 nt, and even more preferably at least 24 nt which have utility as diagnostic probes and/or primers. It Will become apparent to the person of ordinary skill that larger fragments of 100 nt, 1000 nt, 2000 nt and more also find utility in accordance with the present invention.

[0050] The term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention, to maintain at least one of its biological activity. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition, or secondary, tertiary or quaternary structure of one molecule is not identical to that found in the other, or if the amino acid sequence or nucleotide sequence is not identical. Particularly, in the instant case the functional activity is preferably the ability of the MEK-CA, or part thereof to phσsphσrylate Erk1/2.

[0051] The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, all these methods are well known in the art. Since numerous MEK and Erk1/2 have been characterized by function and sequence, the residues which can be mutated, can only tolerate a conservative change or cannot be changed (the same principle applies for deletion or insertion in an essential, important or non-important region), are known in the art.

[0052] The term "subject" or "patient" as used herein refers to an animal, preferably a mammal, most preferably a human who is the object of treatment, observation or experiment.

[0053] As used herein, the term "physiologically relevant" is meant to describe a function which is relevant to a function of the protein or gene in its natural setting in vivo.

[0054] As will be understood by the person of ordinary skill, macromόlecules having non-naturally occurring modifications are also within the scope of the term "molecule" or "variant". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above. Similarly, in a one embodiment in which a delivery of a polypeptide is carried-out, the polypeptides of the present invention (e.g. MEK-ca, Erk1/2) are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the physiologically relevant domains (e.g. in the case of MEK, the kϊnasing domain and domains responsible for interaction with Erk1/2). Having identified MEK and Erk1/2 as interacting partners whose interaction delay apoptosis in fully differentiated neurotrophic factor-responsive neurons, now validates these proteins and the nucleic acid encoding same as targets to improve the neuroprotection compositions and methods of the present invention. For example, screening assays to identify "molecules", "agents" or "compounds" which could enhance the activity of MEK or MEK-ca on Erk1/2, or on at least one of Erk1 and Erk2 sufficiently to neurσprotect the selected neuron which is targeted, fall within the scope of the present invention. Molecules identified in accordance with such an embodiment of the present invention have a therapeutic value in diseases or conditions in which the targeted neuron has function which is compromised or in which it is degenerating. Having identified by such assays an agonist of MEK function on Erk1 and/or Erk2, or on Erk1 and/or Erk2 directly, such an agonist could be used alone or together with one of the compositions of the present invention.

[0055] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0056] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device o method being employed to determine the value.

t0057] The use of the term "or" i the claims is Used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or".

[0058] As used in this specification and claim(s). the words "comprising"

(and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, υn-recited elements or method steps. [0059] As used herein the terminology "MEK-CA" refers to a constitutively active MEK, which enables an increase in Erk1/2 activation which is sufficient to neuroprotect. it should be understood, that the subject matter of the present invention should not be limited to the exact sequence of the MEK-CA which ϊs used and exemplified herein- Based on the knowledge of the sequence and structure-function relationship of MEK, variations to the MEK-CA of the present invention could be effected and used in accordance with the present invention, provided that the variant retains its ability to activate at least one of Erk1 and Erk2, enabling the sought after neuroprotection. One MEK-Ca in accordance with the present invention comprises a 12 amino acid deletion (aa 44-55 in Figure 12A; SEQ ID NO:*) and 2 mutations from Ser (S) to aspartic acid (D). The two mutated serine residues are in the kinasing domain. As well known in the art, mutating a serine to aspartate artificially introduces a negative charge which mimicks the phosphorylation that occurs naturally at the serine. Glutamic acid could also be used to introduce a negative charge. As well, other residues could be used, as known in the art. Other variants of MEK-CA that can be used in accordance with the present invention have one or more of the 4 non-identical residues, between mouse and human changed. The skilled artisan routinely knows and can test whether the variation retains the required function. In addition, the region of the deletion could be modified, without affecting the function of MEK-CA on Erk1/2. It will be noted that the two serine residues are present in human and mouse MEK, and that the region which was deleted is also 100% conserved between the two homologs.

[0060] As used herein the terminology "Erk1/2" refers to Erk1 and/or Erk2. or "at least one of Erk1 and Erk2". Thus while the activation of both Erk1 and Erk2 is shown herein, the invention should not be limited to the activation of both.

[0061] The terms "treatment", "treating", "treat" and the like refer herein generally to obtaining a desired pharmacologic and/or physiologic effect- The effect may be prophylactic when it completely or partially prevents a disease or symptom, aπdtor may be therapeutic when it partially or completely stabilizes or cures the disease, condition or the adverse effect attributable to same. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having It; (b) inhibiting or delaying the disease symptom, or (c) relieving the disease symptom (i.e., regression of the disease or symptom)

100621 As used herein "NF-responsive neurons" refers to any neuron of the central or peripheral nervous system that is capable of responding to a neurotrophic factor (NF) and that undergoes a substantial biological process (survival, axon elongation, proliferation, differentiation, synaptσgenesis or synaptic modification, etc) upon contact with such neurotrophic factor.

Molecular biology DNA

[0063] The present description refers to a number of routinely used recombinant DNA

(rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.

[0064] Nucleotide sequences are presented herein by single strand, in the 5', 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the lU AG-IUB Biochemical Nomenclature Commission.

[0065] As used herein, "nucleic acid motecule" or "polynucleotides". refers to a polymer of nucleotides and includes but should not be limited to DNA or RNA. Non-limiting examples thereof include, DNA (e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). The term MEK-wt nucleic add or MEK-wt polynucleotide refers to a native MEK. as opposed to a constitutively active MEK, designated MEK-CA nucleic acid sequence (shown in Figures 10 and 12, with an amino acid deletion and a mutation of serine to aspartate of glutamate of amino acids 118 and 222, at the amino acid level thereof) that encodes one embodiment of a number of MEK-CA proteins of the present invention. As used herein a "nucleic acid", a "nucleic acid molecule" or a "polynucleotide" means a chain of two or more nucleotides such as RNA (ribσnucleotide) and DNA (deoxyribonucleofide). A purified nucleic acid is one that is substantially separated from other nucleic acid sequences in a cell or in an organism in which the nucleic acid naturally occur (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95. 96, 97, 98, 99, 100% free of contaminants). The term includes, e.g., a recombinant nucleic acid incorporated into a vector, a plasmid, a virus (e.g. rAAV), or a genome of a prokaryotfe or eukaryote. Examples of purified nucleic acids include cDNAs, fragment of genomic nucleic acids, nucleic acid produced by amplification methods (PCR, NASBA, TMA, LCR etc), nucleic acid formed by restriction enzyme treatment of genomic nucleic acid and chemically synthesized nucleic acid molecules.

[0065] As used herein, "nucleic acid molecule" or "polynucleotides", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimeras thereof- The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the term "nucleic acid" and polynucleotides as are analogs thereof. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et a/., PCT Int'l Pub. No. WO 95/32305). phosphorothioate linkages, methylphσsphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribσse or deoxyribose, or similar compounds having known substitutions, e.g.. 2' methσxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2^* halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U). known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^th ed-, 1992). or known derivatives of purine or pyrimidine bases (see. Cook. PCT int'l Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methαxy backbone, or a nucleic acid including conventional bases and one or more base analogs).

[0066] The term "recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering. The same is true for "recombinant nucleic acid".

[0067] The term "DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.

[0068] Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA ("mRNA")

[0069] As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribσnucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to welt-known methods. While they are usually In a single-stranded form, they can be in a double- stranded form and even contain a "regulatory region". They can contain natural rare or synthetic nucleotides. They can be designed to enhance a chosen criterium like stability for example.

[0070] Gene. A DNA sequence related to a polypeptide chain or protein, and as used herein can include the 5' and 3' untranslated ends. The polypeptide can be encoded by a full-length sequence or any portion thereof, as long as the physiologically relevant functional activity of the protein is retained (e.g. interaction with and phosphorylation of Erk1/2).

[0071] Structural Gene. A DNA sequence that is transcribed into RNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide(s).

[0072] The term "aHele" defines an alternative form of a gene, which occupies a given locus on a chromosome.

[0073] As commonly known, a "mutation" is a detectable change in the genetic material, which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.

[0074] Vector. A plasmid or phage DNA or other DNA sequence into which DNA can be inserted to be cloned. The vector can replicate autonomously in a host cell, and can be further characterized by one or a small number of endoπuclease recognition sites at which such DNA sequences can be cut in a detenninable fashion and into which DNA can be inserted. The vector can further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, are tetracycline resistance or ampicillin resistance. The words "cloning vehicle" are sometimes used for "vector."

[0075] Expression Vector. A vector or vehicle similar to a cloning vector but which is capable of expressing a gene, which has been cloned into it. after transformation into a host. The cloned gene (or nucleic acid sequence) is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.

[0076] Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene (or nucleic acid sequence) in a prokaryotic or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.The DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.

[0077] "Promoter" refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of the present invention, the promoter is preferably bound at its 3' terminus by the transcription initiation site and extends upstream (5^* direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain -10 and -35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences, which serve as ribosome binding sequences during translation initiation.

[0078] Promoters and enhancers are well known and numerous in the art.

They can be constitutive (always expressed) or inducible (e.g. having an "on/off switch. Promoters can comprise enhancer elements (chosen from genes, viruses, retroviruses...). The inducers of inducible promoters (or regulatory sequences) are numerous. Of course, they are chosen so as to function together (e.g.MMTV and glucocorticoid; Mil and TFA or heavy metals; SV40 and TPA...). Promoters, enhancers, inducers, and the like are very well known in the art. They are listed in previously described references as well as in Table 1 of WO01/904605A2.

[0079] "Gene delivery vector" or the like is used herein to designate a construct that is adapted to deliver, to facilitate activation, to provide expression to or of one or more gene(s) or sequence(s) of interest in a host cell. Representative examples of such vectors include viral vectors, nucleic acid expression vectors, certain eukaryotic cells (e.g., producer cells) and naked DNA.

[0080] "Recombinant adeno-associated virus vector" or "rAAV vector" is one particular example of such gene delivery vectors based on an adeno- associated virus. rAAV vectors, generally contain 5' and 3' adeno-associated virus inverted terminal repeats (ITRs). and a transgene or gene of interest (e.g. MEK- CA) operatively linked to sequences which regulate its expression in a target cell. In accordance with certain embodiments, the transgene may be operably linked to a heterologous promoter (constitutive [CMV, CBA promoters], or inducible [tet promoter]. In addition, the rAAV vector may have a polyadenylatiσn sequence.

[0081] rAAV vectors are well known in the art. US2003/0129164A1 and US2004/0022766A1 , which both teache rAAVs and their uses to treat or prevent ocular diseases or conditions are herein incorporated by reference in their entirety. Generally, rAAV vectors comprise AAV ITRs at each end of the transgene or gene of interest. This allows replication, packaging, and efficient integration of the sequence into the chromosomes. It is usually preferred to provide a transgenic sequence between about 2 to 5 kb in length (or to add a "stuffer" or "filler" sequence to bring the total size of the recombinant transgene sequences between the two ITRs to about 2 to 5 kb). The transgene may be composed of a reiteration of the same heterologous sequence (e.g.. the same sequences separated by a riboso e readthrough, or alternatively, by an Internal Ribosome Entry Site or "IRES", or "ribosme landing pad").

[0082] Recombinant AAV vectors of the present invention may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through 6. IN one embodiment, the rAAV vector also contains additional adenoviral sequences, which assist for example in packaging the rAAV vector into virus particles.

[0083] Packaging cell lines suitable for producing adeno-associated viral vectors may be readily accomplished given readily available techniques (see e.g., U.S. Pat. No. 5,872,005). Methods for constructing and packaging rAAV vectors are described in. for example, WO 00/54813, WO01/904605A2, US2003/D129164A1 and US2004/0022766A .

Hybridization

[0084] Nucleic Acid Hybridization. Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single- stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern or Northern hybridization procedures, the latter situation occurs. The DNA/RNA of the individual to be tested may be digested with a restriction endonuclease, prior to its fractionation by agarose gel electrophoresis. conversion to the single-stranded form, and transfer to nitrocellulose paper, making it available for re-annealing to the hybridization probe. Non-limiting examples of hybridization conditions can be found in Ausubel, F.M. βt al., Current protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1994). A nitrocellulose filter is incubated overnight at 68°C with labeled probe in a solution, high salt (either 6x SSC[20X: 3M NaCI/0.3M trisodium citrate] or 6X SSPE [20X: 3.6M NaCl/0.2M NaH₂PO₄t0.02M EDTA. pH 7.7]), 5X Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. This is followed by several washes in 0.2X SSC/0.1% SDS at a temperature selected based on the desired stringency: room temperature (low stringency), 42^σC (moderate stringency) or 68°C (high stringency). The salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency. The temperature and salt concentration selected is determined based on the melting temperature (Tm) of the DNA hybrid. Other protocols or commercially available hybridization kits Using different annealing and washing solutions can also be used as well known in the art.

[0085] "Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodyna nically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 2000, supra and Ausubel et al., 1994, supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter (or other such support like nylon), as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing high salt (6 x SSC or 5 x SSPE). 5 x Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured carrier DNA (e.g. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0. % SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or βδ'C (high stringency). The salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency. The selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000. supra). Other protocols or commercially available hybridization kits (e.g., ExpressHyb™ from BD Biosciences Clonetech) using different annealing and washing solutions can also be used as well known in the art.

[0086] By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C. A:T or A:U pairing) or may contain one or more residues (including abasic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions. Contiguous bases of an oligomer are preferably at least about 80% (81, 82, 83, 84, 85, 86, 87. 88, 89, 90. 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be detennined empirically by using routine testing (see Sambrook et al., Molecular Cloning, A Laboratory Manual, 3^ ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY, 2000) at §§ 1.90-1.91 , 7.37-7.57, 9.47-9.51 and 1.47-1 .57, particularly at §§ 9-50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0087] Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Patent Nos. 5.503.980 (Cantor), 5,202,231 (Drmanac et al.), 5,149,625 (Church et al.), 5.112,736 (Caldwell et al.), 5,068,176 (Vijg et al.), and 5.002.867 (Macevicz)). Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ one nucleotide (see U.S. Patent Nos. 5,837,832 and 5,861.242 (Chee et al.)).

[0088] A detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to a probe oligonucleotide. One specific example of a detection step uses a homogeneous detection method such as described in detail previously in Arnold et al. Clinical Chemistry 35:1588- 1594 (1989), and U.S. Patent Nos. 5,658.737 (Nelson et al.), 5.118,801 , and 5,312,728 (Lizardi et al.). The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g. protein detection by far Western technology; Guichet et al.. 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001 , EMBO 20(3): 510-519). Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to Use a detection method amenable to automation. A πon-ϋmifing example thereof includes a chip or other support comprising one or more (e.g. an array) of different probes. Primers and Probes.

[0089] As used herein, a "primer" defines an oligonucleotide capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for nucleic acid synthesis under suitable conditions. Primers can be, for example, designed to be specific for certain alletes so as to be used in an allele-specific amplification system. For example, a primer can be designed so as to be complementary to a nucleic acid sequence of the present invention (MEK, Erk , or Erk2). The primer's 5' region may be non-complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a "promoter primer"). Those skilled in the art will appreciate that any oligomer that can function as a primer can be modified to include a 5' promoter sequence and thus function as a promoter primer. Similarly, any promoter primer can serve as a primer, independent of its functional promoter sequence. Of course the design of a primer from a known nucleic acid sequence is well known in the art. As for the oligos, it can comprise a number of types of different nucleotides.

[0090] Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 30 nucleotides, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with Its targeted sequence (see below and in Sambrook et al., 2000, Molecular Cloning - A Laboratory Manual. 3rd Edition. CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.). [0091] To enable hybridization to occur under the assay conditions of the present invention, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 75% (75%. 76%. 77%, 78%, 79%, 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%, 88%. 89%) and more preferably at least 90% (90%, 91%, 92%. 93%, 94%, 95%. 96%. 97%, 98%, 99%, 100%) identity to a portion of a polynucleotide encoding one of the proteins of the present invention. Probes and primers of the present invention are those that hybridizes to a nucleic acid (e.g. cDNA or mRNA) sequence under stringent hybridization conditions and those that hybridizes to gene homologs under at least moderately stringent conditions. Preferred probes and primers of the present invention have complete sequence identity to one of the three genetic sequences of the present invention (e.g., cDNA or mRNA). However, probes and primers differing from the native MEK, Erk1 or Erk2 gene sequences but keeping the ability to hybridize to these native gene sequences under stringent conditions may be used in the present invention. It should be understood that probes and primers can be easily designed and used in the present invention based on the nucleic acid sequence disclosed herein, which are well known in the art, using methods of computer alignment and sequence analysis known in the art (see in Sambrook et al., 2000, Molecular Cloning - A Laboratory Manual, 3rd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).

[0092] In a preferred embodiment, the oligonucleotide primers of the present invention comprises at least 10 contiguous nucleotides (preferably, 10, 11 , 12, 13. 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24. 25, 26, 27. 28, 29, 30, 31 , 32) of a nucleic acid molecule encoding one of the proteins of the present invention or its complementary sequence. Longer probes and primers are also within the scope of the present invention as well known in the art. Primers having more than 30. more than 40, more than 50 nucleotides and probes having more than 100, more than 200, more than 300, more than 500 more than 800 and more than 1000 nucleotides in length are also covered by the present invention. Of course, longer primers have the disadvantage of being more expensive and thus primers having between 15 and 30 nucleotides in length are Usually designed and used in the art. As well known in the art, probes ranging from 50 to more than 2000 nucleotides in length can used in the methods of the present invention. As for the % of identity descried above, non-specifically described sizes of probes and primers (e.g.16, 17. 31 , 24, 39, 350, 450, 550, 900, 1240 nucleotides....) are also within the scope of the present invention).

[0093] A "probe" is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e.. resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's "target" generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary" allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled.

[0094] By "sufficiently complementary" is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more residues (including abasic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions. Contiguous bases of an oligomer are preferably at least about 80% (81 , 82, 83. 84, 85, 86, 87, 88. 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes. Appropriate hybridization conditions are welt known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook ef al.. Molecular Cloning, A Laboratory Manual, 3 ed. (Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. 2000) at §§ 1.90-1.91. 7-37-7-57. 9.47-9.51 and 11.47-11.57, particularly at §§ 9.50-9.51 , 11.12-11.13. 11.45-11.47 and 11.55-11.57).

[0095] A detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to a probe oligonucleotide. One specific example of a detection step uses a homogeneous detection method such as described in detail previously in Arnold ef al. Clinical Chemistry 35:1588- 1594 (1989). and U.S. Patent Nos. 5,658,737 (Nelson et al.), and 5,118.801 and 5,312.728 (Lizardi et al.).

[0096] The types of detection methods in which probes can be Used include

Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g. protein detection by far western technology: Guichet et al., 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001 , EMBO 20(3): 510-519). Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to use a detection method which is amenable to automation. A non-limiting example thereof includes a chip or other support comprising one or more (e.g. an array) of different probes.

Labels [0097] In one embodiment, probes and primers of the present invention may be detectably labeled. A label or reporter group, as generally understood and used herein, means a molecule, which provides directly or indirectly a detectable signal. Various labels may be employed such as radiolabels (³²P. ³H, C, ^a5S etc.), biotynilated derivatives, enzymes (e.g. alkaline phosphatase, horseradish peroxidase) or fluorescers, (e.g. molecular beacons).

[0098] Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well-known methods (Sambrook et al.. 2000, supra). Non-limiting examples of labels include ³H, ^UC. ³²P. and ³⁵S. Non-limiting examples of detectable markers include ligands, ftuorσphσres, chemiluminescent agents, enzymes, and antibodies- Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotϊdes. It wilt become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

[0099] As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma ³²P ATP and polyhucleσtfde kinase, using the Klenow fragment of Pol I of -Ξ. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers In low-melt gels), using the SP6 T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.

[00100] A "label" refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g.. an amplified sequence). Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g.. covalent bonds or non-covalent interactions), whereas indirect labeling can occur through use a "tinker" or bridging moiety, such as additional oligonucteotide(s), which is either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionuclide. ligand such as biotin or avldin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent. phosphorescent or chemiluminescent compound, and fluorescent compound). Preferably, the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.

Amplification methods

[00101] "Amplification" refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, e.g.. transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as QfS-rep.icase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification Uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g.. Walker et al., U.S. Pat. No. 5,422,252). Another known strand-displacement amplification method does not require endonuclease nicking (Dattagupta et al., U.S. Patent No. 6,087,133). Transcription-mediated amplification is used in the present invention. Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase. (see generally Kwoh et al.. 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular Cloning - A Laboratory Manual, Third Edition. CSH Laboratories). As commonly known in the art. the oligos are designed to bind to a complementary sequence under selected conditions.

[00102] Polymerase chain reaction (PCR). PCR is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4.965,188 (the disclosures of aU three U.S. Patent are incorporated herein by reference in their entirety). In general, PCR involves a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following like, for example, EtBr staining of the DNA following gel electrophσresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications. Michael et al. Eds, Acad. Press, 1990).

[00103] Non-limiting examples of suitable methods to detect the presence of the amplified products include the followings: agarose or polyacrylamide gel, additions of DNA labeling dye in the amplification reaction (such as ethidiu bromide, picogreen, SYBER green, etc.) and detection with suitable apparatus (ftuorometer in most cases). Other suitable methods include sequencing reaction (either manual or automated); restriction analysis (provided restriction sites were built into the amplified sequences), or any method involving hybridization with a sequence specific probe (Southern or Northern blot, TaqMan™ probes, molecular beacons, and the like). Of course, other amplification methods are encompassed by the present invention. Molecular beacons are exemplified herein as one method for detecting the amplified products according to the present invention (see below).

[00104] In accordance with one embodiment of the present invention, the amplified product can either be directly detected using molecular beacons as primers for the amplification assay (e.g., real-time multiplex NASBA or PCR assays) or indirectly using, internal to the primer pair binding sites, a molecular beacon probe of 18 to 25 nucleotides long (e.g., 18, 19, 20, 21, 22, 23, 24, 25) which specifically hybridizes to the amplification product. Molecular beacons probes or primers having a length comprised between 18 and 25 nucleotides are preferred when used according to the present invention (Tyagi et al., 1996, Nature Biotechnol. 14: 303-308). Shorter fragments could result in a less fluorescent signal, whereas longer fragments often do not increase significantly the signal. Of course shorter or longer probes and primers could nevertheless be used. Proteins and polypeptides

[00105] As used herein, "protein" or "polypeptide" means any peptide- linked chain of amino acids, regardless of postranslationat modifications (e.g., phosphorylation, glycosylation, sulfatatiσn etc). A "MEK, MEK-CA, Erk1 or Erk2 protein" or a " MEK, MEK-CA, Erk1 or Erk2 polypeptide" is an expression product of the nucleic acids encoding same (e.g. MEK, MEK-CA, Erk1 or Erk2 gene) or a MEK, MEK-CA, Erk1 or Erk2 protein homolog that shares at least 75. 80. 85, 90. 95, 96, 97. 98, 99, 100%) amino acid sequence identity with MEK, MEK-CA, Erk1 or Erk2, and displays functional activity of native MEK, MEK-CA, Erk1 or Erk2 protein involved in the viability and function of the fully differentiated neurons of the present invention. For the sake of brevity, the units (e.g. 76, 77...81 , 82, ...91, 92%...) have not been specifically recited but are nevertheless considered within the scope of the present invention.

Activity

[00106] A "functional activity" of a polypeptide or protein of the present invention is any activity associated with a structural, biochemical or physiological activity of the protein (either structural or functional) of the present invention which is involved in the apoptosis pathway which converges on MEK through Erk1/2. For example, one non-limiting but critical function in accordance with one embodiment of the present invention is the phosphorylation function of MEK of Erk1/2.

[00107] The nucleic acids therapy and the pharmaceutical compositions of the invention can be administered by any means that achieve their intended purpose. For example, administration can be by subcutaneous, intravenous, intramuscular, intra-peritoneal, oral, ocular, nasal, or transdermal routes. [00108] the dosage administered will be dependent upon the age, health, disease to be treated and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[00109] Compositions within the scope of this invention include all compositions wherein the active ingredient (e.g. nucleic acid) is contained in an amount effective to achieve a delayed apoptosis of fully differentiated neurons, and more particularly to neurotrophic factor-responsive fully differentiated neurons. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.

[00110] Suitable formulations for parenteral administration include aqueous solutions of the MEK-CA nucleic acid in water-soluble form, for example, water-sαlubte salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension can also contain stabilizers.

t00111] In cases wherein oral administration was required, any of the above carrier or a solid carrier such as cellulose, sucrose, glucose, lactose, talcum, starch, magnesium carbonate, sodium saccharine, magnesium stearate and mannltol may be employed.

[00112] In some embodiments nucleic acids of the present Invention may be made resistant to endogenous nucleases (e.g. endσnucleases and exonucleases) and are therefore stable in vivo. Examples of modified nucleic acids molecules comprise methylsulfoπate, phosphoramidate. and phσsphothioate analogs of DNA.

[00113] A number of methods have been developed for deliverin nucleic acids into cells. In ah embodiment wherein the nucleic acids (or vector carrying such nucleic acid) of the present invention can be introduced into a chosen cell, methods such by micrσ'.njection, electropσration, transduction. DEAE-Dextran mediated transfection, lipofection, calcium phosphate mediated transfection or other procedures (e.g. commercial transfection kits such as fugehe and lipofectamine reagents) known to one skilled in the art (Molecular Cloning, A Laboratory Manual, Sambrook et al., Third Edition, Cold Spring Harbor Press, Plainview. New York (2000)).

Peptides and polypeptide preparation and synthesis

[00114] The peptide. polypeptide or peptide derivatives of the present invention are obtained by any method of peptide synthesis known to those skilled in the art, including synthetic (e.g., exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis) and recombinant techniques. For example, the peptides. polypeptides or peptides derivatives can be obtained by solid phase peptide synthesis, which in brief, consist of coupling the carboxyl group of the C-terminal amino acid to a resin (e.g., benzhydryla ine resin, chloromethylated resin, hydroxymethyl resin) and successively adding N-alpha protected amino acids. The protecting groups maybe any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. Such solid phase synthesis has been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85: 2149; Vale et al., 1981. Science, 213: 1394-1397, in US Patent Nos. 4, 305, 872 and 4,316, 891, Bodonsky et al., 1966. Chem. Ind. (London), 38:1597; Pietta and Marshall. 1970. Chem. Comm. 650. The coupling of amino acids to appropriate resins is also well known in the art and has been described in US Patent No. 4.244,946.

[00115] During any process of the preparation of the compound of the present invention, it may be necessary and/or desirable to protect sensitive reactive groups on any of the molecule concerned. This may be achieved by means of conventional protecting groups such as those described in Protective Groups In Organic Synthesis by T.W. Greene & P.G.M. Wuts, 1991, John Wiley and Sons, New-York; and Peptides: chemistry and Biology by Sewald and Jakubke, 2002. Wiley-VCH, Wheinheim p.142. For example, alpha amino protecting groups include acyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (BOG), cyclohexy xycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl- 9-methoxy-carbonyl (F σc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g., triphenyl methyl, benzyl). The amino acids side chain protecting groups include benzyl (For Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp. His), boc ( Arg, His, Cys) etc. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[00116] In one embodiment, the peptides of this invention, including the analogs and other modified variants, may generally be synthesized according to the FMOC protocol in an organic phase with protective groups. They can be purified with a yield of 70% with HPLC on a C 8 column and eluted with an acetonitrile gradient of 10-60%. Their molecular weight can then be verified by mass spectrometry.

[00117] Of course, peptides and polypeptides of this invention may be prepared in recombinant systems using polynucleotide sequences encoding the peptides. It is understood that a peptide of this invention may contain more than one of the above-described modifications within the same peptide. Also included in this invention are pharmaceutically acceptable salt complexes of the peptides of this invention or their derivatives.

[00118] Purification of the synthesized peptide, polypeptides or peptide derivatives is carried out by standard methods, including chromatography (e., ion exchange, size exclusion, affinity), centrifugation, precipitation or any standard technique for the purification of peptides and peptides derivatives. In one embodiment, thin-layered chromatography is employed. In another embodiment, reverse phase HPLC is employed. Other purification techniques well known in the art and suitable for peptide isolation and purification may be used in the present invention.

[001193 Where the processes for the preparation of the compounds according to the present invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their components enantiomers by standard techniques such as the formation of diastereσisσmeric pairs by salt formation with an optically active acid followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereomeric esters or amides, followed by removal of the chiral auxiliary. Alternatively, the compounds may be resolved using chiral HPLC column.

Peptide derivatives and peotldomlmetics

[00120] In addition to peptides consisting only of naturally occurring amino acids, peptidomimetics or peptide analogs are also encompassed by the present invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs ith properties analogous to those of the template peptide. The types of non-peptide compounds are termed "peptide mimetics" or peptidomimetics (Fauchere, J. 1986, Adv. Drug Res. 15: 29; Evans et al., 1987, J. Med. Chem. 30: 1229). Peptide mimetics that are structurally related to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as naturally occurring receptor-binding polypeptides but have one or more peptide linkages optionally replaced by linkages like _-_CH₂NH— , — CH₂S— ^■, -— CHr- CH*—, — CH=CH— (cis and trans), — CH₂SO— , — CH(OH)CH₂— , — COCH₂ — etc., by methods well known in the art (Spatσla A.F., Peptide Backbone Modifications, Vega Data, March 1983, 1(3): 267; Spatola et al., Life Sci 1986, 38:1243-1249; Hudson D- et at., Int. J. Pept. Res. 1979., 14: 177- 185; Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins. Weinstein eds, Marcel Dekker, New-York,). Such peptide mimetics may have significant advantages over natural polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficiency etc), reduced antigenicity and others.

t00121] It is often advantageous to utilize modified versions of peptides-

The modified peptides retain the structural characteristics of the original L-amino acid peptides that confer biological activity with regard to Erk1 and Erk2 (as well as MEK-CA), but are advantageously not readily susceptible to cleavage by protease and/or exopeptidases.

[00122] substitution of unnatural amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus. Such substitutions have been described and these substitutions do not affect biological activity. Examples of non-naturally occurring amino acids include o-.o- -disubstituted amino acids. N-alkyl amino acids, lactic acids. C-α-methyl amino acids, and β-methyl amino acids. Amino acids analogs useful in the present invention may include but are not limited to (-.-alanine. norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine. 2-aminoisobutyric acid, 6-amlnohexanoic acid, t-butylgtycine, phenylglycine, o-phosphoserine, N-acetyl serine, N- fαrmylmethiσnine, 3-methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with unnatural amino acids is routine and known in the art.

[00123] One other effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the peptides at either or both termini. Certain chemical modifications, in particular N- terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al. 1993). Other chemical modifications which enhance serum stability include, but are not limited to. the addition of an N-terminal alkyl group, consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and/or a C-terminal amide group.

Pharmaceutical compositions

[00124] In one embodiment, gene delivery vectors can be prepared as a pharmaceutically acceptable composition suitable for administration. Such pharmaceutical compositions comprise an amount of a gene delivery vector suitable for delivery an Erk1/2 activating polypeptide or nucleic acid-encoding same, and more particularly. MEK-CA. In an embodiment wherein the compositions is used to treat the eye. the composition is of course adapted to the mode and place of delivery, combined with a pharmaceutically acceptable carrier or excipient. In am eye-related embodiment preferably, the pharmaceutically acceptable carrier is suitable for intraocular administration. Non-limiting examples of pharmaceutically acceptable carriers include, saline or a buffered saline solution (e.g., phosphate-buffered saline). Various pharmaceutically acceptable excipients are also well known in the art. As used herein, "pharmaceutically acceptable excipient" includes any material which, in combination with an active ingredient of the present invention, which allows a preservation of biological activity. Preferably, the ingredient has no adverse effect on the patient (e.g., immune reaction or adverse side effect to the tissues surrounding the site of administration (e.g., within the eye). Non-limiting examples of pharmaceutically acceptable carriers include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples include, but are not limited to, any of the standard pharmaceutical excipients such as a saline, buffered saline (e.g., phosphate buffered saline), water, emulsions such as oil/water emulsion, and various types of wetting agents. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, hyaluronic acid, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[00 25] A composition of gene delivery vector of the invention may also be lyophilized using means well known in the art, for subsequent reconstitutlon and use according to the invention. Where the vector is to be delivered without being encapsulated in a viral particle (e.g., as "naked" polynucleotide), formulations for liposσmal delivery, and formulations comprising microencapsulated polynucleotides, may beneficial. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value may also optionally be present in the pharmaceutical composition.

[00126] The amount of gene delivery vector in the pharmaceutical formulations varies widely and can be adapted by the clinician or practitioner to which the present invention pertains (e.g., from less than about 0.1%, to at least about 2%, to as much as 20% to 50% or more by weight, and can be selected primarily by fluid volumes, viscosities, etc, in accordance with the particular mode of administration selected, the type of disease, the severity thereof, and patient's parameters.

[00127] The pharmaceutical composition can comprise other agents suitable for administration. For example in the case of glaucoma, pressure relieving drugs can be added to a pharmaceutical composition of the present invention.

[00128] The pharmaceutical compositions of the present invention can also be packaged in a kit format. Thus, broadly, the present invention also pertains to kits comprising various materials for carrying out the methods of the invention. In one embodiment, the kit comprises a vector encoding a MEK-CA polypeptide, the vector being adapted to deliver same to a subject having a disease or disorder associated with a compromised neuronal function or associated with the degeneration of fully differentiated neuron which are NF- responsive. In one particular embodiment, the neuronal cells which are targeted are in the eye of the subject. The kit can comprise the vector in a sterile vial, which may be labeled for use. The vector can be provided in a pharmaceutical composition. In one embodiment, the vector is packaged in a virus. The kit can further comprise a needle and/or syringe suitable for use with the vial or, alternatively, containing the vector, which needle and/or syringe are preferably sterile. In another embodiment, the kit comprises a catheter suitable for delivery of a vector to the eye, which catheter may be optionally attached to a syringe for delivery of the vector. The kits can further comprise instructions for use, e.g., instructions regarding route of administration, dose, dosage regimen, site of administration, and the like. Of course numerous examples of kits are known in the art and can comprise a number of vials, instructions, ingredients and the like.

[00129] The present invention relates to a kit for treating a disease or condition associated with the degeneration of fully differentiated neurons, or a predisposition to contracting same comprising a nucleic acid, a protein or a vector in accordance with the present invention. For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.

[00130] Composition within the scope of the present invention should contain the active agent (e.g. peptide, peptide derivative or peptidomimetics) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art. For the administration of polypeptide antagonists and the like, the amount administered should be chosen so as to avoid adverse side effects. The amount of the therapeutic or pharmaceutical composition which is effective in the treatment of a particular disease, disorder or condition will depend on the nature and severity of the disease, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 100 mg/kg/day will be administered to the subject. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat is divided by six.

[00131] Various delivery systems are known and can be used to administer peptides, peptide derivatives or peptidomimetics or a pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be administered by any suitable route including, intrevanous or intramuscular injection, intraventricular or iπtrathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally. or via intranasal, intradermal, sUblingual, vaginal, rectal or epidural routes.

[00132] Other delivery system well known in the art can be used for delivery of the pharmaceutical compositions of the present invention, for example via aqueous solutions, encapsulation in microparticules, lipososmes, or microcapsu.es.

[00133] In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system. In one embodiment polymeric materials can be used (see Smolen and Ball. Controlled Drug Bioavaiiability. Drug product design and performance, 1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology and toxicology series, 2003, 2^nd edition, CRRC Press), in another embodiment, a pump may be used (Saudek et at-, 1989. N. Engt. J. Med. 321: 574).

[00134] Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled tα a class of biodegradable polymers useful in achieving controlled release of the drug, for example, polylactic acid, pσlyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans.

[00135] As mentioned above, pharmaceutical compositions of the present invention comprise a genetic delivery vehicle, a polypeptide. peptide derivatives or peptidomimetic combined with a pharmaceutically acceptable carrier. The term carrier refers to diluents adjuvants, excipients or vehicles with which the peptide, peptide derivative or peptidomimetic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils including mineral oil, vegetable oil (e.g., peanut oil, soybean oil, sesame oil), animal oil or oil of synthetic origin. Aqueous glycerol and dextrose solutions as well as saline solutions may also be employed as liquid carriers of the pharmaceutical compositions of the present invention. Of course, the choice of the carrier depends oh the nature of the genetic delivery vehicle, the polypeptide, peptide derivative or peptidomimetic, its solubility and other physiological properties as well as the target site of delivery and application. For example, carriers that can penetrate the blood brain barrier are used for treatment, prophylaxis or amelioration of symptoms of diseases or conditions (e.g. inflammation) in the central nervous system. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro. 2003, 21^th edition. Mack Publishing Company. [00136] Further pharmaceutically suitable materials that may be incorporated in pharmaceutical preparations of the present invention include absorption enhancers, pH regulators and buffers, osmolarity adjusters, preservatives, stabilizers, antioxidants, surfactants, thickeners, emollient, dispersing agents, flavoring agents, coloring agents and wetting agents.

[00137] Examples of suitable pharmaceutical excipients include, water glucose, sucrose, lactose, glycol, ethanol. glycerol monostearate. gelatin, rice, starch flour, chalk, sodium stearate, malt, sodium chloride and the like. The pharmaceutical compositions of the present invention can take the form of solutions, capsules, tablets, creams, gels, powders sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides (see Remington: The Science and Practice of Pharmacy by Alfonso R- Gennaro, 2003. 21^th edition, Mack Publishing Company). Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulations are designed so as to suit the mode of administration and the target site of action (e.g., a particular organ or cell type).

[00138] The pharmaceutical compositions of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those that form with free amino groups and those that react with free carboxyl groups. Non-toxic alkali metal, alkaline earth metal and ammonium salts commohly used in the pharmaceutical industry include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present invention with suitable organic or inorganic acid. Representative salts include the hydrobromide, hydrσchloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysσlate, citrate, mateate, fumarate, tartrate. succinate, napsylate salts and the like.

[00139] Although the present invention has been described hereinabσve by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

EXAMPLE 1 Preparation of recombinant rAAV serotype 2 vectors [00140] One constitutively active MEK1 mutant used in our study was created by substitution of regulatory phosphorylation sites to acidic residues (S218D, S222D) and deletion of residues 44-55 N-terminal to the consensus catalytic core (MEK-CA). Biochemical in vitro assays showed that this mutant was several-fold more active than wild-type MEK1 [31].

[00141] The MEK-CA cDNA was inserted downstream of the hybrid chicken

B-actin/CMV enhancer promoter in the plasmid pXX-UF 2, a derivative of pTR- UF5 [32], containing the AAV terminal repeat sequences and a simian virus 40 polyadeπylation sequence. The helper plasmid pDG [33] that contains both the AAV genes [rep and cap) and helper genes required for AAV propagation was used to generate recombinant serotype 2 AAV. Vectors were packaged, concentrated and titered as previously described [34]. Control AAVs containing genes that encoded wild-type MEK1 (AAV.MEK-WT) or green fluorescent protein (AAV.GFP) were generated in identical fashion. Low-passage 293 cells were co- transfected with pXXUF12-MEK-CA and the pDG helper plasmid [33]. Upon cell harvesting, the virus was extracted by freezing and thawing the cells and the resulting supernatant was then clarified by low speed centrifugation. AAV was then purified on an affinity column of heparin and concentrated. The number of infectious particles/ml (ip/ml) was determined by infectious center assay as described [35] and was: 1.7x10¹⁰ ip/ml for AAV.MEK-CA. 3.7x10¹⁰ ip/ml for AAV.MEK-WT, and 3.0x10¹⁰ ip/ml for AAV.GFP. No helper adenovirus or wild-type AAV contamination was detected in these preparations. The MEK1 genes used here contained a N-terminal hemagglutinin (HA) tag to track expression of AAV- ediated MEK1 proteins in vivo.

EXAMPLE 2 Experimental animals and surgical procedures {00442} All procedures were in accordance with the ARVO Statement for the

Use of Animals in Ophthalmic and Vision Research. Because glaucoma is primarily an age-related disease, surgeries were performed in adult male Brown Norway rats, retired breeders, between 10-12 months of age (300-400 g), under general anesthesia by intraperitoneal injection of 1 ml/kg standard rat cocktail consisting of ketamine (100 mg/ml), xylazine (20 mg/m!) and acepromazine (10 mg/ml).

[00143] f) Intravitreal Injection of viral vector: Viral vectors (5 μl) were injected into the vitreous chamber of one eye using a 10-μl Hamilton syringe adapted with a 32-gauge needle. Contralateral, unoperated eyes served as controls. The tip of the needle was inserted in the superior hemisphere of the eye at a 45° angle through the sclera into the vitreous body. This route of administration avoided injury to eye structures, such as the iris or the lens, reported to promote survival and regeneration of RGCs [8, 36], Once the tip of the needle reached the iπtravitreal space, it was held in place for injection of the viral solution over a period of -2 min after which it was gently removed. The site of injection was then sealed with surgical glue (Indermiil, Tyco Health Care, Mansfield, MA, USA). We, and others, have observed that AAV-mediated transgene expression reaches a plateau between 3-4 weeks after administration of the vector into the rodent eye [24, 37-39] and persists thereafter [30]. Therefore, subsequent surgical procedures were performed over a period of 3-4 weeks after AAV administration (Fig. 1). The reason for delayed onset of AAV-mediated gene expression in vivo is unclear, but may arise from the need to convert single- stranded viral DNA to a double-strand prior to active transcription [40].

[00144] ll) Retrograde labelling of RGCs. For neuronal survival experiments. RGCs were retrogradely labeled with 3% Dil (1,1'-dioctadecyl- 3,3.3\3'-tetramethyt-indσcarbocyaπine perchlorate; Molecular Probes, Junction City, OR), a fluorescent carbocyanine marker that persists for several months without fading or leakage and does not interfere with the function of labeled cells [41 , 42]. Dil crystals were dissolved by sonication in 0.9% NaCl, containing 10% dimethyl sulfoxide (DM5O) and 0.5% Triton X-100. For retrograde labeling, both superior colliculi, the main targets of RGCs in the brain [43], were exposed and a small piece of gelfoam (Pharmacia and Upjohn Inc., Mississauga, ON) soaked in Dil was applied to their surface. Seven days after Dil application, the time required for labeling the entire RGC population, animals were subjected to ocular hypertension surgery as described below (Fig. 1). For co-labeling experiments, RGCs were retrogradely labeled with 2% FluoroGold (Fluorochrome, Englewood, CO) in 0.9% NaCl containing 10% dimethyl sulfoxide by application of the tracer to both superior colliculi.

[00 45] ill) Ocular hypertension surgery Unilateral and chronic elevation of

IOP was induced as previously described [44] using a method that involves injection of a hypertonic saline solution into an episcleral vein. All the animals involved in this study received only a single saline vein injection. The eye previously injected with a viral vector was selected for the procedure and a plastic ring was applied to the ocular equator to confine the injection to the limbal plexus. A microπeedle (30-50 μ in diameter) was used to inject 50 μl of sterile 1.85 M NaCl solution through one episcleral vein. The plastic ring temporarily blocked off other episcleral veins forcing the saline solution into the SchlemnVs canal to create isolated scarring. Following injection, the plastic ring was removed and the eyes were examined to assess the extent to which the saline solution traversed the limbal vasculature. Polysporin ophthalmic ointment (TMIMC Pfizer Canada Inc., ON, Canada) was applied to the operated eye and the animal was allowed to recover from the surgery. Animals were kept in a room with constant low fluorescent light (40-100 lux) to stabilize circadian IOP variations [45, 46],

[00146] Iv) Measurement f Intraocular pressure (IOP): IOP from glaucomatous and normal (contralaieral) eyes was measured in awake animals based on previous studies showing that general anesthetics, including ketamine and xylazine, caused marked reduction of the measured IOP [47]. A calibrated tonometer (TonoPen XL, Medtronic Solan, Jacksonville. FL) was used to measure IOP after application of one drop of proparacaine hydrochloride (0.5%. Alcσn Laboratories. Inc., Fort Worth, TX) per eye. Holding the tonometer exactly perpendicular to the comeal surface, 10-15 consecutive readings per eye were taken and averaged to obtain an accurate daily IOP measurement. IOP was measured daily for two weeks after ocular hypertension surgery, and then every other day for the entire duration of the experiment.

[00147] The onset of pressure elevation was defined as the day in which we first detected an increase in the IOP of the operated eye compared to the normal, fellow eye. The mean IOP (mm Hg ± S.E.M.) per eye was the average of all IOP readings since the onset of pressure elevation. The individual eye mean lOPs were then used to calculate the mean tOP for each experimental or control group. The maximum IOP measured in each individual eye, glaucomatous or normal contralaterat eye, was defined as the peak IOP and this value was used to estimate the m^an peak IOP (mm Hg ± S.E.M.) for each group. In addition, the positive integral IOP was calculated as the area under the IOP curve in the glaucomatous eye minus that of the fellow normal eye from ocular hypertension surgery to euthanasia. Therefore, integral IOP represents the total, cumulative IOP exposure throughout the entire experiment. Data analysis and statistics were performed using the GraphPad Ihstat software (GraphPad Software Inc., San Diego, CA).

[00148] v) Quantification of RGC soma and axons: RGC survival was quantified at 5 and 7 weeks after ocular hypertension surgery (Fig 1). Quantification of RGC bodies or axons was always performed in duplicate and in a masked fashion. For RGC density counts, rats were deeply anesthetized and perfused intracardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer and both eyes were immediately enucleated. Retinas were dissected, fixed for an additional 30 min, and flat-mounted on a glass slide with the ganglion cell layer side up. Under fluorescence microscopy, Dil-labeled neurons were counted in 12 standard retinal areas as described [48]. Groups included: i) animals sacrificed at 5 weeks after ocular hypertension surgery treated with AAV.MEK-CA (n= 3). AAV.MEK-WT (n=7) or AAV.GFP (n=7), and ii) animals sacrificed at 7 weeks after ocular hypertension surgery treated with AAV.MEK-CA (n=9). AAV.MEK-WT (n=8) or AAV.GFP (n=8).

[00149] For axonal counts, animals received an intracardial injection of heparin (1,000 u/kg) containing sodium nitropruside (10 mg/kg) followed by intracardial perfUsion with 2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer. Five weeks after ocular hypertension surgery, optic nerves were dissected, fixed in 2% osmium tetroxide, and embedded in epon resin. Semi-thin sections (0.7-μm-thick) were cut on a microtome (Reichert, Vienna. Austria) and stained with 1% toluidine blue. RGC axons were counted in five non-overlapping areas of each optic nerve section, encompassing a total area of 5,500 pm² per nerve. The five optic nerve areas analyzed included one in the center of the nerve, two peripheral dorsal and two peripheral ventral regions. The total surface area per optic nerve cross section was measured using the Northern Eclipse image analysis software, and this value was Used to estimate the total number of axons in each optic nerve. Groups included animals treated with AAV.MEK-CA (n*9). AAV.MEK- WT (n=5) or AAV.GFP (n=3). Data analysis and statistics were performed using the GraphPad Instat software (GraphPad Software Inc., San Diego, CA) by a oneway analysis of variance (ANOVA) test.

[00150] vl) Retinal I munohfstochemfstry: Rats were perfused intracardially with 4% PFA and the eyes were immediately enucleated. The anterior part of the eye and the lens were removed, and the remaining eye cup was immersed in the same fixative for 2 hr at 4^βC. Eye cups were equilibrated in graded sucrose solutions (10-30% in PB) for several hours at 4°C, embedded in O.C.T. compound (Tissue-Tek, Miles Laboratories, Elkhart, . IN) and frozen in a 2- methylbutaπe/liquid nitrogen bath. Radial retinal cryosections (16 μm) were collected onto gelatin-coated slides and processed. Sections were incubated in 10% normal goat serum (NGS), 0.2% Triton X-100 (Sigma) in PBS for 30 min at room temperature to block nonspecific binding. Monoclonal HA primary antibody (2 μg/ml, clone 12CA5, Roche Diagnostics Corporation, Indianapolis. USA) was added in 2% NGS, 0.05% Triton X-100 and incubated overnight at 4°C. Sections were then incubated with fluorophore-conjugated goat anti-mouse IgG (red, 4 gg/ml; Alexa 594, Molecular Probes) for 1 hr at room temperature, washed in PBS and mounted using an anti-fade reagent (SlσwFade, Molecular Probes, Eugene, OR).

[00151] For whole-retina immunostaining, the retina was carefully dissected out of the eye and transferred to a dish where it was permeabilized in PBS containing 2% Triton X-100 and 0.5% DMSO at 4^DC for 3 days. Tissue was then incubated in blocking solution (10% NGS in 2% Triton-X100 and 0.5% DMSO) for 1 hr at room temperature. Retinas were then incubated overnight with monoclonal neurofilament (NF) RT-97 antibody, which recognizes phosphorylated NF-H, (1:200; gift from Dr. J. Wood. McGill University) at 4°C followed by incubation with Alexa 594 red goat anti-mouse IgG secondary antibody (4 μg/ml). Fluorescent staining was examined Using a Zeiss Axioskop 2 Plus microscope (Carl Zeiss Canada, Kirkland, QC) or a Leica TCS-SP1 confocal microscope (Leica Microsystems, Heidelberg. Germany) and pictures were captured with a CCD video camera (Qirnsging, Mississauga. ON) and analysed with Northern Eclipse software (Empix Imaging, Mississauga, ON).

[00152] vlt) Western blot analysis: Retinas were quickly extracted and homogenized with an electric pestle (Kontes, Vinetand, NJ) in lysis buffer (20 mM Tris, pH 8.0. 135 mM NaCl, 1% NP-40. 0.1% SDS and 10% glycerol. supplemented with protease inhibitors) at 4^βc Retinal lysates were incubated for 30 min on ice before centrifugation at 10,000 rpm for 5 min and the supernatant collected. The protein concentration of retinal extracts was determined by the Lσwry method (Bio-Rad Life Science, Mississauga. Ontario, Canada). Samples (100-150 μg of protein) were separated by electrophoresis on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Life Science, Mississauga, Ontario. Canada). Non-specific binding was blocked by incubating blots in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.2% Tween 20 (TBST), and 5% lyophilized skim milk for 1h at room temperature. Membranes were incubated with the following primary antibodies: monoclonal phospho-Erk1/2 (10 μg/ml, BioSource Int.) or polyclonal Erk1/2 (2.3 μg/ml. BioSource Int.). Blots were washed in TBST and then incubated with anti-mouse or anti-rabbit peroxidase- linked secondary antibody (0.5 μg/ml, Amersham Pharmacia. Baie dOrfe, QC). Protein signals were detected using a chemiluminescence reagent (ECL, Amersham Biosciences) followed by exposure of blots to X-OMAT (Kodak) imaging film.

EXAMPLE 3 AAV.MEK-CA activates the Erk1/2 pathway in adult RGCs

For gene transfer experiments, recombinant AAV vectors containing genes encoding constitutively active (CA) or wild-type (WT) MEK1 were prepared. Viral vectors were injected intraocularly in intact and gtaucomatous rat eyes to examine MEK1 gene expression in retinal cells in vivo. To distinguish AAV-mediated MEK1 expression from endogenous MEK1, we used an antibody against the HA tag present only in MEK1 transgenes. Robust HA staining was observe in a large number of celts in the ganglion cell layer (GCL) of retinas treated with AAV.MEK- CA (Fig. 2A) or AAV.MEK-WT (Fig. 2D), but not in control eyes injected with AAV.GFP (not shown). Identical expression of AAV-mediated MEK proteins was observed at 4 and 10 weeks following administration of viral vectors, consistent with previous observations that AAV vectors mediate long-term transgene expression in the adult retina [24, 29. 30]. Of note, MEK-CA expressing RGCs retained a typical neuronal phenotype with a single axon projecting to the optic nerve head and an elaborated dendritic tree. These results indicate that MEK-CA expression preserves the morphology of surviving RGCs. We have previously demonstrated that RGCs are the main cellular targets for infection when AAV is administered into the vitreous chamber [24, 25]. To confirm this, we performed co-localization studies in retinas from eyes that received a single travitreal injection of AAV.MEK vectors followed by retrograde labeling of RGCs using FluoroGold applied to the superior cσlϋculus. Double- labeling experiments demonstrated that the vast majority of RGCs, visualized with FluoroGold (Figs. 2B. 2E), also produced virally-mediated MEK1 proteins (Figs. 2C, 2F). In addition, staining was also observed in RGC dendritic processes extending into the inner plexiform layer (IPL) (Figs. 2G-L). Thus, the diffuse HA staining detected in the IPL is likely due to the presence of MEK-CA in RGC dendrites. No signs of inflammation, cytotoxicity, abnormal growth or immune reaction were detected in any of the eyes following administration of AAVs. [00153] To establish the efficacy of AAV. MEK vectors to stimulate the Erk1 /2 pathway in vivo, we examined the levels of phosphorylated Erk1 and Erk2 in whole retinal homogenates using antibodies that specifically recognize the phosphorylated forms of these kinases. AAV.MEK-CA significantly increased Erk1 and Erk2 activation above the levels found in AAV.MEK-WT-treated retinas (Fig. 2M), consistent with an increase in MEK activity of the constitutively active mutant protein. We, and others, have shown that >75% of RGCs can be effectively infected with recombinant AAV [24. 49-51]. In a previous study, we showed that only a small number of displaced amacrine cells (-8%) in the ganglion cell layer were also infected by AAV, while no glia! cells were transduced [24]. Based on these findings, it is likely that changes in protein phosphorylation largely reflect changes in AAV-infected RGCs. Together; these results indicate that AAV.MEK- CA drives selective and sustained stimulation of the Erk1/2 pathway in adult RGCs in vivo.

EXAMPLE 4 Intraocular pressure elevation (IOP) in experimental and control groups

Unilateral elevation of IOP was induced in aging, male brown Norway rats after a single injection of hypertonic solution into one episcleral vein. This model of ocular hypertension leads to Inner retinal atrophy, optic nerve degeneration, and optic nerve head remodeling similar to that seen in human, age-related glaucoma [44]. Of the 72 animals that underwent ocular hypertension surgery, 68 were used for quantification of neuronal survival following different AAV treatments and 4 were used for immunostaining with an antibody against neurofila ent to visualize RGC axons.

[00154] Table 1 shows the IOP increase in experimental and control groups throughout the duration of the study. Baseline mean IOP in both eyes prior to ocular hypertension surgery was -27 mm Hg, which is a typical measurement in awake rats that are housed in a constant light environment to stabilize circadian IOP variations [45, 46]. Mean sustained pressure elevation among all groups was 17 mm Hg, well within the range of IOP increase observed in this model [44], and the retinal vasculature remained perfused in all eyes. Importantly, there was no significant difference in the mean, peak or integral IOP among the three experimental or control groups at 5 weeks or 7 weeks following induction of glaucoma (Table 1, Row: P Value. ANOVA). Given that the rate of RGC death and optic nerve damage is proportional to IOP increase in this mode! [44], the similar increase in IOP among all groups allowed reliable comparison of the neuroprotective effect of each viral vector treatment.

EXAMPLE 5 Erk1/2 activation protects RGCs from hypertension-Induced death

The widespread expression of AAV-mediated MEK-CA in RGCs and its ability to activate Erk1/2 in vivo prompted us to test Its effect on neuronal survival in glaucomatous eyes (Fig. 1). Following intraocular injection of viral vectors. RGCs were back labeled with the fluorescent tracer Dil. Unlike other retrograde markers that leak out of the cell bodies after several weeks, Dil has been shown to persist in RGCs in vivo for periods of up to 9 months without fading or leakage [42]. In addition, Dil does not interfere with the function of living neurons [41]. Episcleral vein injection was performed one week after Dil application to assure that all RGCs were labeled prior to intraocular pressure increase. Retinas were examined histologically at 5 and 7 weeks following ocular hypertension surgery to determine the density of surviving RGCs in all retinal hemispheres. Macrophages and microglia that may have incorporated Dil after phagocytosis of dying RGCs were excluded from our quantitative analysis based on their morphology and immunolabeling using specific markers as described [24].

[00155] Injection of AAV vectors was routinely performed in the superior

(dorsal) hemisphere of the eye, consequently, a higher density of RGCs expressing MEK-CA was always observed in this retinal hemisphere. Since it could be anticipated that the effect of MEK-CA on RGC survival would be most noticeable in retinal regions with the highest density of infected RGCs. Thus, we compared the effect of AAV.MEK-CA on RGC survival in the entire retina with that in the superior hemisphere (Fig 3). A single intraocular injection of AAV.MEK-CA increased RGC survival in the whole eye (Fig. 3A), but most notably in the superior hemisphere (Fig. 3B) at 5 and 7 weeks after hypertension surgery. For example, at 5 weeks after episcleral vein injection AAV.MEK-CA protected -77% of the total number of RGCs in the superior retina compared to ~38% with either AAV.MEK- WT or AAV.GFP (Table 2; ANOVA, **: PO.001). Remarkably, in some retinas up to 89% of RGCs were protected in the superior retinal hemisphere. Neuronal survival in this region following treatment with AAV.MEK-CA was still significant at 7 weeks post-surgery: -49% of the total number of RGCs remained alive in contrast to only 22% or 18% of neurons that survived with AAV.MEK-WT or AAV.GFP, respectively (table 2; ANOVA, *^*: P<0.001). This neuroprotective effect led to higher neuronal densities and better preservation of cellular integrity than with control vectors (Fig. 4).

Interestingly, AAV.MEK-CA induced stronger phosphorylation of Erk2 than Erk1. A more prominent Erk2 phosphorylation may be due to higher abundance of Erk2 in retinal cells, particularly in RGCST

EXAMPLE 6 AAV.MEK-CA treatment reduces RGC axon damage in glaucoma

Glaucoma is characterized by the degeneration of RGC axons in the optic nerve followed by the progressive loss of cell bodies [52, 53]. Hence, we investigated the effect of AAV.MEK-CA on RGC axon protection following hypertension damage. For this purpose, we examined RGC axons within the retina, which are unmyelinated, as well as in the optic nerve where axons are ensheathed in myelin. Figure 5 shows intraretinal axons visualized following staining of whole-mounted retinas with RT-97. an antibody that recognizes the phosphorylated 200-kDa neurofilament H subunit. Immunoreactive axons coursed in organized bundles toward the optic nerve head in normal retinas (Fig. 5A). In retinas treated with the control vector AAV.GFP, intraretinal axon bundles suffered significant fiber loss at 5 weeks after hypertension surgery (Fig. 5C). Many remaining fibers had a beaded appearance confirming the progressive axonal degeneration after glaucomatous injury. In contrast, treatment with AAV.MEK-CA remarkably preserved the overall structure of RGC Intraretinal axon bundles (Fig. 5B). [00156] To further investigate the protective effect of AAV.MEK-CA on RGC axons. we analyzed optic nerve segments from intact and glaucomatous eyes collected at 1-2 mm behind the globe, where all axons are yeϋnated (Fig 6). Optic nerve cross-sections from AAV.MEK-CA-treated eyes displayed a larger number of axonal fibers with normal morphology (Fig. 6B) compared to AAV.MEK- WT-treated control eyes, which showed extensive axon degeneration including disarray of fascicular organization and degradation of myelin sheaths (Fig. 6C). Quantification of RGC axons demonstrated that AAV-mediated MEK-CA protected 54% of the total number of axons in the optic nerve at 5 weeks after ocular hypertension surgery, compared to 35% and 32% of axons found in eyes treated with AAV.MEK-WT or AAV.GFP, respectively (Fig. 5D). Together, these results strongly suggest that activation of the Erk1/2 pathway via MEK-CA gene transfer protects RGC sorna and axons in this injury model.

[00157] Of note, MEK-CA expressing RGCs retained a typical neuronal phenotype with a single axon projecting to the optic nerve head and an elaborated dendritic tree. These results indicate that MEK-CA expression preserves the morphology of surviving RGCs. The correlation between neuronal survival and transgene product expression was also assessed. For example, at four weeks after axotomy, 86% of surviving RGCs also expressed rAAV-mediated MEK-CA (data not shown). Together, these results strongly suggest that activation of the MAPK pathway via MEK supports RGC survival in two different injury animal models.

^• EXAMPLE 7 AAV.MEK-CA neuroprotection of RGC and axon damage in glaucoma, predictive of neuroprotection for other diseases associated with degeneration of fully differentiated neurotrophic factor responding neurons

[00158] There is convincing evidence that the primary form of RGC death in glaucoma occurs by apoptosis [52, 54], but the precise molecular mechanisms that lead to RGC loss remain undefined. The elucidation of the intracellular signaling pathways that regulate RGC survival and death is paramount for the design of effective neuroprotective strategies. The Erk1/2 pathway is a central, evolutionarily conserved mechanism used by several peptide factors to elicit a broad spectrum of biological activities including cell survival and axon growth [55-57]. Here, we tested a gene therapy strategy using recombinant AAV to investigate the role of Erk1/2 signaling on the survival of adult rat RGCs in experimental glaucoma. Our results demonstrate that constitutive activation of Erk1/2 confers striking structural protection of RGC soma and axons at 5 and 7 weeks after ocular hypertension surgery. In contrast, AAV vectors carrying genes for either witd-type Erk1/2 or GFP promoted minimal neuroprotection. A major advantage of this approach over other strategies is that AAV serotype 2 mediates gene transfer to =>70% of RGCs and few displaced amacrine cells [24], but not other retina! cell types, hence Erk1/2 activation is largely restricted to a target neuronal population.

[00159] Glaucoma has been defined as an axσgenic disease, characterized first by the degeneration of RGC axons in the optic nerve followed by the progressive loss of cell bodies [53]. We performed complementary, but independent, quantitative analyses of the neuroprotective effect of AAV.MEK-CA on two major RGC compartments: soma and axons. Consistent with the idea that the primary site of degeneration in glaucoma is at the level of the axon, we found that all eyes had more pronounced axon loss than cell body loss. However, a single intraocular injection of AAV.MEK-CA effectively protected a similar proportion of RGC soma and axons within the optic nerve. The protection of all cellular compartments following hypertension damage is key for the preservation of appropriate neuronal function and vision in glaucoma. Functional studies in macaque monkeys subjected to experimental glaucoma demonstrated that only subtle visual field defects are detected with a loss of -≤50% of RGCs, whereas vision loss increased dramatically with more advanced glaucoma [58], Thus. structural protection of >50% of RGC soma and axons, as shown with selective stimulation of the Erk1/2 pathway, is likely to be sufficient for substantial preservation of visual function in glaucoma. Future studies are required to evaluate the functional state of the rescued neurons.

[00160] Two other studies have previously explored neuroprotective

AAV-based strategies in rat models of experimental glaucoma: one group investigated the effect of BDNF gene transfer [26], while the other Used gene transfer of the caspase inhibitor BIRC4 [59]. The mean IOP increase reported in these previous studies was lower than in our model; therefore it is difficult to directly compare these data. An advantage of our approach, however, is that direct stimulation of Erk1/2 in RGCs bypasses the use of exogenous neurotrophic factors which affect many different cell types and may have adverse side effects in the retina [17, 18, 22]. The method and genetic constructs of the present invention are thus more specific. In addition, Erk1/2 is an intermediary signaling component that blocks apoptotic cell death prior to caspase activation. Suppression of apoptosis using caspase inhibitors is an approach that has been explored with only modest success [60]. Mitochondria! dysfunction, which marks the point of no return during apoptosis, occurs even in the presence of caspase inhibition leading to impairment in ATP production and increased production of reactive oxygen species [61]. Thus, strategies that halt the commitment to die prior to irreversible mitochondria! dysfunction are likely to contribute to better functional outcome.

[00161] From a clinical perspective, intravitreal injection of AAV.MEK-CA may confer neuroprotection of RGCs and axons in patients affected by glaucoma Used in combination with other glaucoma treating or delaying drugs (e.g. IOP reducing drugs) Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies and this vector is increasingly being applied to human clinical trials including neurological conditions. [62-66]. The results presented herein raise the exciting possibility that AAV.MEK-CA may have potential as a therapeutic agent for the treatment of glaucoma and other optic nerve diseases In humans. EXAMPLE 8 AAV.MEK-CA leads to a specific stimulation of the Erk1/2 pathway and not to other signalling pathways stimulated by neurotrophic factors

To establish the efficacy of AAV.MEK vectors to stimulate the Erk1/2 pathway in vivo, we examined the levels of phosphorylated Erk1 and Erk2 in whole retinal homogenates using antibodies that specifically recognize the phosphorylated forms of these kinases (Fig. 11A). Because mostly RGCs are infected by AAV, changes in protein phosphorylation reflect changes that occur in these neurons. Low but detectable levels of phospho-Erk1/2 were observed in intact, uninjected retinas indicating basal activation of these kinases. AAV.MEK-CA significantly increased Erk1 and Erk2 activation above the levels found in intact or control retinas at 4 weeks after vector administration. Interestingly, AAV.MEK-CA induced more abundant phosphorylation of Erk2 than Erk1. A more modest, but clearly detectable, increase in phosphα-Erk2 was found in retinas injected with AAV.MEK- wt consistent with an increase in the pool of MEK1 protein available to phosphorylate Erk1/2. AAV.GFP-infected retinas showed phosho-Erk1/2 levels simitar to those found in intact retinas indicating that AAV infection by itself did not stimulate the Erk1/2 pathway.

[00162] To confirm the specificity of Erk1/2 activation in vivo, we examined the phosphorylation status of the following key signaling components following MEK-CA gene transfer; Akt, a critical downstream target of PI3K (Huang and Reichardt 2003), and Erk5, which has been shown to mediate neuronal survival (Cavanaugh 2004). There was no activation of Akt in the presence of AAV.MEK- CA (Fig. 11 B). Similarly, phosphorylation of Erk5 was not stimulated by AAV.MEK- CA (Fig. 11C). Together, these results indicate that AAV-MEK-CA drives selective and sustained stimulation of the MEK1/Erk1/2 pathway in adult RGCs in vivo.

[00163] Adeno-associated virus (AAV) containing genes that encoded constitutively active MEK (AAV.MEK-CA), wild-type MEK (AAV.MEK-wt) or green fluorescent protein (AAV.GFP) were injected into the vitreous of adult female Sprague-Dawley rats. For analysis of cell survival. RGCs were backtabeled with FluoroGold and quantified on whole-mounted retinas at 1. 2 and 4 weeks after optic nerve transection. To investigate nerve regeneration, RGC axons were labeled with the anterograde tracer cholera-toxin β-subυnit (CTβ) and regenerating axons were quantified in optic nerve sections at 2 weeks after micro-crush lesion.

Table 1. Intraocular pressure (IOP) elevation in glaucomatous eyes

Table 2. Survival of RGCs in glaucoma following in vivo gene transfer

RGCs/mm³ ± S.E.M. (% of intact contralateral retinas), n

Contralateral intact retinas; Superior = 1.774 ± 108 RGCs/m ² (100%). n=9 Alt -1.849 ± 39 RGCstmtn² (100%), n=9

CONCLUSION

[00164] Although glaucoma is a leading cause of blindness worldwide, at present, there are no effective neuroprotective strategies for the treatment of this disease. Current therapies for glaucoma, such as reduction of intraocular pressure (IOP), are often inadequate. Here we demonstrate that constitutive activation of the MEK gene product and more particularly expression of a MEK-CA nucleic acid sequence leads to RGC survival following optic nerve injury. Therefore, this invention opens the way to neuroprotective strategy to prevent the loss of injured RGCs.

[00165] Herein, we demonstrate that activation of the Erk1/2 signaling pathway leads to marked RGC neuroprotection in glaucomatous eyes and results in morphological preservation of cell bodies and axons. These data provide the prσof-of-principle that selective stimulation of key intracellular signalling pathways can be an effective strategy to delay neuronal death associated with aging, injury or disease of CNS. Of note, MEK-CA gene delivery markedly increased RGC survival at all times examined.

[00166] From a clinical perspective, intravitreai injection of AAV.MEK-CA may confer neuroprotection of RGCs in patients affected by glaucoma or at risk of developing same (e.g. as known by familial history, or early diagnosis). In one embodiment, the invention provides AAV.MEK-CA in a gene therapy approach to promote RGC survival in glaucoma. In vivo delivery of exogenous genes has the potential of treating glaucoma using the MEK-CA gene instead of drugs, or in another embodiment a combination of gene therapy (or protein-delivery based therapy of MEK-CA and/or Erk1/2). Because AAV infects almost selectively RGCs when introduced into the vitreous chamber, it precludes affecting other cells and reduces any secondary effects. In addition, because AAV-mediated MEK-CA gene expression is sustained over time it is likely that this therapy will have a longer effect than other treatments such as neurotrophic factors or glaucoma surgery. In addition as seen earlier, the present invention is not limited to the glaucoma mode! but finds utility in a number of neurodegenerative diseases.

[00167] RGCs are a prototypical fully differentiated central nervous system neuronal population, it is expected that the neuroprotective effect observed with AAV.MEK-CA is applicable to other retinal degenerative diseases such as age- related macular degeneration or retinitis pigmentosa and to other neurodegenerative diseases such as, but not limited to. Alzheimer's, multiple sclerosis or Parkinson's disease. Thus the present invention is not limited to , glaucoma and finds support in neuroprotective effects in a broad range of degenerative diseases in which fully differentiated neurotrophic factor-responsive neurons are dying or are functionally haπdicaped.

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Claims

WHAT IS CLAIMED IS:

1. A vector which expresses MEK nucleic acid sequence encoding a recombinant MEK product which is more active than a wild type MEK product, wherein said recombinant MEK is expressed to a level which is sufficient to enable an increase in activation of at least one of Erk1 and Erk2. thereby enabling neuronal survival of cells undergoing cell death.

2. The vector of claim 1. wherein said MEK product is constitutively active.

3. The vector of claim 1 or 2, wherein said MEK gene is expressed constitutively, or is inducibly expressed.

4. The vector of claim 1 , 2 or 3, wherein said MEK gene encodes a recombinant MEK product which is traceable andfor constitutively active, as compared to a wild type MEK product.

5. A method to stabilize or increase the survival of fully differentiated, post- mitotic neurons, comprising an increase thereinto of the activation of at least one of Erk1 and Erk2, whereby said increase in the activation of at least one of Erk1 and Erk2 stabilizes or increases the survival of said cells.

6. The method of claim 5, wherein said increase expression is carried out by providing said cell with constitutively active MEK or by activating said native MEK thereinto.

7. The method of claim 5, wherein said increase expression is carried out by introducing in said cell a vector encoding MEK-CA.

8. The method of claim 7, wherein a vector of one of claims 1 to 4 is used.

9. The method of one of claims 1 to 8, wherein said cells are neural cells.

10. The method of claim 9, wherein said cells are affected by a degenerative disease.

11. The method of one of claims 5 to 10, wherein said method is an in vivo method, whereby cells are treated ex vivo before re-introduction into an animal from which they are derived.

12. The method of one of claims 5 to 10, wherein said method is an In vivo gene therapy method.