WO2005080573A1 - Vecteurs viraux recombinants pour la promotion de la survie de cellules neuronales et leurs utilisations - Google Patents

Vecteurs viraux recombinants pour la promotion de la survie de cellules neuronales et leurs utilisations Download PDF

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WO2005080573A1
WO2005080573A1 PCT/CA2005/000225 CA2005000225W WO2005080573A1 WO 2005080573 A1 WO2005080573 A1 WO 2005080573A1 CA 2005000225 W CA2005000225 W CA 2005000225W WO 2005080573 A1 WO2005080573 A1 WO 2005080573A1
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mek
aav
erk1
vector
nucleic acid
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PCT/CA2005/000225
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Adriana Dipolo
Vincent Pernet
William W. Hauswirth
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Universite De Montreal
University Of Florida Research Foundation, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention broadly relates to neur ⁇ protection.
  • 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).
  • the invention relates to recombinant viral vectors to promote the survival of fully differentiated neur ⁇ tr ⁇ phic factor-responsive neurons.
  • the invention relates to recombinant viral vectors to promote the survival in glaucoma and other retinal diseases and Uses thereof.
  • 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).
  • 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 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].
  • IOP intraocular pressure
  • BDNF brain-derived neurotrophic factor
  • 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].
  • CNTF ciliary neurotrophic factor
  • AAV ade ⁇ o-associated virus
  • the present invention seeks to meet these needs and other needs.
  • the present invention relates to vectors and methods which aim to overcome the defects of the prior art.
  • the present invention relates to recombinant adeno-ass ⁇ ciated (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
  • 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.
  • 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 ax ⁇ nal processes, which is an essential requisite for proper neural function.
  • the present invention relates to an increase in
  • 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.
  • the neuroprotective strategy of the present invention is exemplified with several models of optic nerve injury.
  • 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.
  • rAAV adeno-associated virus
  • 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).
  • neurotrophins e.g. brain-derived neurotrophic factor: BDNF.
  • BDNF brain-derived neurotrophic factor
  • 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.
  • 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.
  • 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.
  • MEK-CA constitutively active MEK
  • 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).
  • 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).
  • 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.
  • diseases or conditions also include: optic neuritis and multiple sclerosis (Frohman et al., Lancet NeUr ⁇ l. 2005 Feb; 4(2):111-21; and Foro ⁇ zan et al.. Curr. Opin. Ophthalmol. 2002 Dec; 3(6):375-80); optic neuropathies (Johns et al., Semin.
  • RGCs and their axons are but one example of fully differentiated ne ⁇ trophic fact ⁇ r-(NF)resp ⁇ ns.ve cells.
  • 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.
  • 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.
  • the vector is a recombinant Adeno-Associated-Virus (rAAV).
  • rAAV Adeno-Associated-Virus
  • 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.
  • virus-free delivery can also be used.
  • 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 (Foll ⁇ t, et al., J. Gene Med.
  • 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.
  • 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.
  • 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.
  • the genetic construct is a rAAV construct comprising at least an activating portion of MEK-ca.
  • 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.
  • 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.
  • 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.
  • the phosphorylation of Erk1 and/or Erk2 needs to be increased as compared to wild-type levels, in order to have a neuroprotective effect.
  • 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.
  • increases from about 2 fold to about 10 fold are required to promote neuronal cell survival.
  • 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.
  • 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.
  • 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;
  • FIG. 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).
  • FG retrograde tracer FluoroGold
  • PS photoreceptors segments
  • ONL outer nuclear layer
  • OPL outer plexiform layer
  • INL inner nuclear layer
  • IPL inner plexiform layer
  • GCL ganglion cell layer
  • FIG. 3 shows AAV.MEK-CA protects RGCs from hypertension- induced death.
  • 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.
  • FIG. 4 shows AAV.MEK-CA protects RGC soma from hypertension-damage.
  • FIG. 5 shows Erk1/2 activation protects intraretinal RGC axons in glaucoma.
  • 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 ⁇ m;
  • FIG. 6 shows that AAV.MEK-CA treatment reduces optic nerve damage in glaucoma.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 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.
  • 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;
  • Figure 10C shows the phosphorylation status of Erk5, a critical factor having been shown to be involved in neuronal survival.
  • Figure 11 shows an alignment between human MEK-wt (SEQ ID NO: 1
  • TrkB retinal ganglion cells
  • MAPK mitogen-activated protein kinase
  • 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.
  • AAV adeno- associated virus
  • 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).
  • Ade ⁇ ovirus-associated virus-2 is a human parvovirus which is routinely used in gene therapy strategies.
  • AAV is a safe vector whose rescue mechanism is simple.
  • 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.
  • 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.
  • MEK and downstream effectors thereof are very highly conserved throughout evolution.
  • 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 m ⁇ sc ⁇ lus 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.
  • purified refers to a molecule or molecules having been separated from a component of the composition in which it was originally contained.
  • 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).
  • the term “crude” means molecules that have not been separated from the components of the original composition in which it was present.
  • the units e.g. 66. 67...81 , 82....91 , 92%.
  • 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.
  • 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.
  • 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.
  • the substituting amino acid 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, hydrophyl ⁇ city 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.
  • 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.
  • 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.
  • the functional activity is preferably the ability of the MEK-CA, or part thereof to ph ⁇ sph ⁇ rylate Erk1/2.
  • 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.
  • 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.
  • 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.
  • MEK-CA refers to a constitutively active MEK, which enables an increase in Erk1/2 activation which is sufficient to neuroprotect.
  • 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.
  • 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.
  • 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.
  • 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.
  • Erk1/2 refers to Erk1 and/or Erk2. or "at least one of Erk1 and Erk2".
  • Erk1 and Erk2 refers to Erk1 and/or Erk2. or "at least one of Erk1 and Erk2".
  • treatment 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, a ⁇ dtor may be therapeutic when it partially or completely stabilizes or cures the disease, condition or the adverse effect attributable to same.
  • Treatment 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)
  • 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, synapt ⁇ genesis or synaptic modification, etc) upon contact with such neurotrophic factor.
  • NF neurotrophic factor
  • 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.
  • nucleic acid motecule 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]).
  • MEK-wt nucleic add or MEK-wt polynucleotide refers to a native MEK.
  • MEK-CA nucleic acid sequence 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.
  • a "nucleic acid”, a “nucleic acid molecule” or a “polynucleotide” means a chain of two or more nucleotides such as RNA (rib ⁇ nucleotide) 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.
  • 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.
  • nucleic acid molecule 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, methylph ⁇ sphonate linkages or combinations thereof.
  • Sugar moieties of the nucleic acid may be rib ⁇ se or deoxyribose, or similar compounds having known substitutions, e.g.. 2' meth ⁇ xy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No.
  • 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 meth ⁇ xy backbone, or a nucleic acid including conventional bases and one or more base analogs).
  • recombinant DNA 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”.
  • 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.
  • cDNA Complementary DNA
  • mRNA messenger RNA
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyrib ⁇ nucleotides). 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.
  • 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).
  • 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).
  • AHele defines an alternative form of a gene, which occupies a given locus on a chromosome.
  • a “mutation” is a detectable change in the genetic material, which can be transmitted to a daughter cell.
  • a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
  • 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.
  • 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 endo ⁇ uclease 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Promoters 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.
  • 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.
  • 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.
  • the transgene may be operably linked to a heterologous promoter (constitutive [CMV, CBA promoters], or inducible [tet promoter].
  • the rAAV vector may have a polyadenylati ⁇ n sequence.
  • 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.
  • 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”).
  • IRS Internal Ribosome Entry Site
  • Recombinant AAV vectors of the present invention may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through 6.
  • the rAAV vector also contains additional adenoviral sequences, which assist for example in packaging the rAAV vector into virus particles.
  • 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 .
  • 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.
  • 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 NaH 2 PO 4 t0.02M EDTA.
  • 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).
  • RNA-DNA hybrids can also be formed and detected.
  • 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., ExpressHybTM from BD Biosciences Clonetech) using different annealing and washing solutions can also be used as well known in the art.
  • 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.
  • 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).
  • 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)).
  • a complementary sequence e.g., oligonucleotide probes
  • 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.)).
  • 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.
  • 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 ⁇ on- ⁇ mifing example thereof includes a chip or other support comprising one or more (e.g. an array) of different probes. Primers and Probes.
  • 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.
  • a primer can be designed so as to be complementary to a nucleic acid sequence of the present invention (MEK, Erk , or Erk2).
  • MEK, Erk , or Erk2 nucleic acid sequence of the present invention
  • 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").
  • any oligomer that can function as a primer can be modified to include a 5' promoter sequence and thus function as a promoter primer.
  • any promoter primer can serve as a primer, independent of its functional promoter sequence.
  • the design of a primer from a known nucleic acid sequence is well known in the art.
  • the oligos it can comprise a number of types of different nucleotides.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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).
  • 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.
  • 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.).
  • 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.
  • primers having between 15 and 30 nucleotides in length are Usually designed and used in the art.
  • probes ranging from 50 to more than 2000 nucleotides in length can used in the methods of the present invention.
  • % 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).
  • 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.
  • 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.
  • oligomer specifically hybridizes 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).
  • 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.).
  • RNA detection 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.
  • probes and primers of the present invention may be detectably labeled.
  • a label or reporter group means a molecule, which provides directly or indirectly a detectable signal.
  • Various labels may be employed such as radiolabels ( 32 P. 3 H, C, a5 S etc.), biotynilated derivatives, enzymes (e.g. alkaline phosphatase, horseradish peroxidase) or fluorescers, (e.g. molecular beacons).
  • probes can be labeled according to numerous well-known methods (Sambrook et al.. 2000, supra).
  • Non-limiting examples of labels include 3 H, U C. 32 P. and 35 S.
  • Non-limiting examples of detectable markers include ligands, ftuor ⁇ ph ⁇ res, chemiluminescent agents, enzymes, and antibodies-
  • Other detectable markers for use with probes include biotin and radionucleot ⁇ des. 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.
  • 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 32 P ATP and polyhucle ⁇ tfde kinase, using the Klenow fragment of Pol I of - ⁇ . 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.
  • radioactive dNTP e.g. uniformly labeled DNA probe using random oligonucleotide primers In low-melt gels
  • 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).
  • a 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.
  • 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 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).
  • 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).
  • 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.
  • oligos are designed to bind to a complementary sequence under selected conditions.
  • PCR Polymerase chain reaction
  • 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).
  • 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.
  • 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.
  • 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 electroph ⁇ resis, or using a detectable label in accordance with known techniques, and the like.
  • PCR techniques see PCR Protocols, A Guide to Methods and Amplifications. Michael et al. Eds, Acad. Press, 1990).
  • 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, TaqManTM probes, molecular beacons, and the like).
  • sequencing reaction either manual or automated
  • restriction analysis provided restriction sites were built into the amplified sequences
  • any method involving hybridization with a sequence specific probe Southern blot, TaqManTM probes, molecular beacons, and the like.
  • Molecular beacons are exemplified herein as one method for detecting the amplified products according to the present invention (see below).
  • 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.
  • protein or “polypeptide” means any peptide- linked chain of amino acids, regardless of postranslationat modifications (e.g., phosphorylation, glycosylation, sulfatati ⁇ n 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.
  • 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.
  • one non-limiting but critical function in accordance with one embodiment of the present invention is the phosphorylation function of MEK of Erk1/2.
  • nucleic acids therapy and the pharmaceutical compositions of the invention can be administered by any means that achieve their intended purpose.
  • administration can be by subcutaneous, intravenous, intramuscular, intra-peritoneal, oral, ocular, nasal, or transdermal routes.
  • 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.
  • 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.
  • active ingredient e.g. nucleic acid
  • Suitable formulations for parenteral administration include aqueous solutions of the MEK-CA nucleic acid in water-soluble form, for example, water-s ⁇ lubte salts.
  • 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.
  • the suspension can also contain stabilizers.
  • 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.
  • nucleic acids of the present Invention may be made resistant to endogenous nucleases (e.g. end ⁇ nucleases and exonucleases) and are therefore stable in vivo.
  • modified nucleic acids molecules comprise methylsulfo ⁇ ate, phosphoramidate. and ph ⁇ sphothioate analogs of DNA.
  • nucleic acids or vector carrying such nucleic acid
  • methods such by micr ⁇ '.njection, electrop ⁇ ration, 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)).
  • 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.
  • 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.
  • 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.
  • 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 ⁇ c), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g., triphenyl methyl, benzyl).
  • 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., fluoren
  • 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.
  • the peptides of this invention 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.
  • 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.
  • 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.
  • chromatography e., ion exchange, size exclusion, affinity
  • centrifugation e.g., centrifugation
  • precipitation e.g., a standard technique for the purification of peptides and peptides derivatives.
  • thin-layered chromatography is employed.
  • 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.
  • 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 diastere ⁇ is ⁇ meric 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.
  • 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.
  • paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological activity
  • paradigm polypeptide i.e., a
  • 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.
  • 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.
  • 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.
  • non-naturally occurring amino acids include o-.o- -disubstituted amino acids. N-alkyl amino acids, lactic acids. C- ⁇ -methyl amino acids, and ⁇ -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.
  • the synthesis of peptides with unnatural amino acids is routine and known in the art.
  • 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.
  • 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.
  • the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and/or a C-terminal amide group.
  • 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.
  • 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.
  • the pharmaceutically acceptable carrier is suitable for intraocular administration.
  • pharmaceutically acceptable carriers include, saline or a buffered saline solution (e.g., phosphate-buffered saline).
  • pharmaceutically acceptable excipient includes any material which, in combination with an active ingredient of the present invention, which allows a preservation of biological activity.
  • 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).
  • 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.
  • 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.
  • Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • 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.
  • the vector is to be delivered without being encapsulated in a viral particle (e.g., as "naked" polynucleotide)
  • formulations for lipos ⁇ mal 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.
  • 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.
  • the pharmaceutical composition can comprise other agents suitable for administration.
  • pressure relieving drugs can be added to a pharmaceutical composition of the present invention.
  • kits comprising various materials for carrying out the methods of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits 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.
  • 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.
  • 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.
  • active agent e.g. peptide, peptide derivative or peptidomimetics
  • Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art.
  • 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.
  • 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 i ⁇ trathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally. or via intranasal, intradermal, sUblingual, vaginal, rectal or epidural routes.
  • compositions of the present invention 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.
  • the pharmaceutical compositions of the present invention can be delivered in a controlled release system.
  • 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).
  • 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 t ⁇ a class of biodegradable polymers useful in achieving controlled release of the drug, for example, polylactic acid, p ⁇ lyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans.
  • compositions of the present invention comprise a genetic delivery vehicle, a polypeptide. peptide derivatives or peptidomimetic combined with a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro. 2003, 21 th edition. Mack Publishing Company.
  • compositions 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.
  • 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).
  • 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).
  • 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, hydr ⁇ chloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tys ⁇ late, citrate, mateate, fumarate, tartrate. succinate, napsylate salts and the like.
  • 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: 1.7x10 10 ip/ml for AAV.MEK-CA. 3.7x10 10 ip/ml for AAV.MEK-WT, and 3.0x10 10 ip/ml for AAV.GFP.
  • the MEK1 genes used here contained a N-terminal hemagglutinin (HA) tag to track expression of AAV- ediated MEK1 proteins in vivo.
  • HA hemagglutinin
  • Viral vectors (5 ⁇ l) were injected into the vitreous chamber of one eye using a 10- ⁇ l 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 i ⁇ travitreal space, it was held in place for injection of the viral solution over a period of -2 min after which it was gently removed.
  • 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].
  • 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).
  • 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.
  • 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 micro ⁇ eedle (30-50 ⁇ in diameter) was used to inject 50 ⁇ l 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.
  • IOP Intraocular pressure
  • 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.
  • 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).
  • 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.
  • rats were deeply anesthetized and perfused intracardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer and both eyes were immediately enucleated.
  • PFA paraformaldehyde
  • 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].
  • RGC axons were counted in five non-overlapping areas of each optic nerve section, encompassing a total area of 5,500 pm 2 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.
  • 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.
  • 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 (Sl ⁇ wFade, Molecular Probes, Eugene, OR).
  • 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 D C 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.
  • NF monoclonal neurofilament
  • 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 ⁇ c 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 L ⁇ wry method (Bio-Rad Life Science, Mississauga. Ontario, Canada).
  • Blots were washed in TBST and then incubated with anti-mouse or anti-rabbit peroxidase- linked secondary antibody (0.5 ⁇ g/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.
  • ECL chemiluminescence reagent
  • 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.
  • an antibody against the HA tag present only in MEK1 transgenes 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.
  • 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.
  • 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.
  • 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].
  • 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).
  • 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
  • Glaucoma is characterized by the degeneration of RGC axons in the optic nerve followed by the progressive loss of cell bodies [52, 53].
  • AAV.MEK-CA the effect of AAV.MEK-CA on RGC axon protection following hypertension damage.
  • 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.
  • 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).
  • 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.
  • Glaucoma has been defined as an ax ⁇ genic disease, characterized first by the degeneration of RGC axons in the optic nerve followed by the progressive loss of cell bodies [53].
  • a single intraocular injection of AAV.MEK-CA effectively protected a similar proportion of RGC soma and axons within the optic nerve.
  • 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 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.
  • Erk1/2 is an intermediary signaling component that blocks apoptotic cell death prior to caspase activation.
  • 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
  • AAV.MEK-CA induced more abundant phosphorylation of Erk2 than Erk1.
  • a more modest, but clearly detectable, increase in phosph ⁇ -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.
  • Adeno-associated virus 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.
  • RGC axons were labeled with the anterograde tracer cholera-toxin ⁇ -sub ⁇ nit (CT ⁇ ) and regenerating axons were quantified in optic nerve sections at 2 weeks after micro-crush lesion.
  • CT ⁇ anterograde tracer cholera-toxin ⁇ -sub ⁇ nit
  • IOP Intraocular pressure
  • 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).
  • 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).
  • AAV infects almost selectively RGCs when introduced into the vitreous chamber, it precludes affecting other cells and reduces any secondary effects.
  • 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.
  • the present invention is not limited to the glaucoma mode! but finds utility in a number of neurodegenerative diseases.
  • 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.
  • 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 ha ⁇ dicaped.
  • TrkB gene transfer protects retinal ganglion cells from axoto y- induced death in vivo. J. Ne ⁇ rosci. 22: 3977-3986.
  • Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury. Mol. Cell. Neum ⁇ ci. 24: 656-672.
  • Second- strand synthesis is a rate irniting step for efficient transduction by recombinant adeno-associated virus vectors. J. Virol. 70: 3227-3234.
  • Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J. Cell. Biol. 103: 171-187.
  • MAPK-activated protein kinases a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68: 320-344.

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Abstract

La présente invention a trait à l'apoptose. De manière plus spécifique, la présente invention a trait à des vecteurs viraux recombinants pour la promotion de la survie neuronale en glaucome et autres maladies rétiniennes. L'invention a trait à un vecteur exprimant une séquence d'acides nucléiques MEK codant pour un produit recombinant MEK qui est plus actif que le produit MEK de type sauvage, dans lequel ledit recombinant MEK est exprimé jusqu'à un niveau suffisant pour permettre un accroissement dans l'activation d'au moins un parmi l'Erk1 et l'Erk2, permettant ainsi la survie neuronale de cellules subissant l'apoptose.
PCT/CA2005/000225 2004-02-20 2005-02-21 Vecteurs viraux recombinants pour la promotion de la survie de cellules neuronales et leurs utilisations WO2005080573A1 (fr)

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WO2013029008A1 (fr) 2011-08-25 2013-02-28 Cornell University Codeur rétinien pour vision industrielle
US9180309B2 (en) 2010-02-26 2015-11-10 Cornell University Retina prosthesis
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US10426844B2 (en) 2008-05-20 2019-10-01 University Of Florida Research Foundation, Incorporated Capsid-mutated rAAV vectors and methods of use
US10515269B2 (en) 2015-04-20 2019-12-24 Cornell University Machine vision with dimensional data reduction
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US11771763B2 (en) 2010-04-05 2023-10-03 Eos Neuroscience, Inc. Methods and compositions for decreasing chronic pain
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US20100221228A1 (en) * 2006-01-24 2010-09-02 The University Of Chicago y134.5 Deficient HSV and the MAPK Pathway
US10426844B2 (en) 2008-05-20 2019-10-01 University Of Florida Research Foundation, Incorporated Capsid-mutated rAAV vectors and methods of use
US10561841B2 (en) 2010-02-26 2020-02-18 Cornell University Retina prosthesis
US9180309B2 (en) 2010-02-26 2015-11-10 Cornell University Retina prosthesis
US9220634B2 (en) 2010-02-26 2015-12-29 Cornell University Retina prosthesis
EP3088044A1 (fr) 2010-02-26 2016-11-02 Cornell University Prothèse de rétine
EP3669933A1 (fr) 2010-02-26 2020-06-24 Cornell University Prothèse de rétine
US10039921B2 (en) 2010-02-26 2018-08-07 Cornell University Retina prosthesis
US11771763B2 (en) 2010-04-05 2023-10-03 Eos Neuroscience, Inc. Methods and compositions for decreasing chronic pain
US9302103B1 (en) 2010-09-10 2016-04-05 Cornell University Neurological prosthesis
US9925373B2 (en) 2010-09-10 2018-03-27 Cornell University Neurological prosthesis
US12097268B2 (en) 2010-10-15 2024-09-24 Eos Neuroscience, Inc. Modulation of neural pathways
US10303970B2 (en) 2011-08-25 2019-05-28 Cornell University Retinal encoder for machine vision
US9547804B2 (en) 2011-08-25 2017-01-17 Cornell University Retinal encoder for machine vision
US10769483B2 (en) 2011-08-25 2020-09-08 Cornell University Retinal encoder for machine vision
US11640681B2 (en) 2011-08-25 2023-05-02 Cornell University Retinal encoder for machine vision
WO2013029008A1 (fr) 2011-08-25 2013-02-28 Cornell University Codeur rétinien pour vision industrielle
US10515269B2 (en) 2015-04-20 2019-12-24 Cornell University Machine vision with dimensional data reduction
US11430263B2 (en) 2015-04-20 2022-08-30 Cornell University Machine vision with dimensional data reduction
US10617770B2 (en) 2015-04-24 2020-04-14 University Of Florida Research Foundation, Incorporated AAV vector for treatment of Friedreich's ataxia
US11446395B2 (en) 2015-04-24 2022-09-20 University Of Florida Research Foundation, Incorporated AAV vector for treatment of Friedreich's ataxia
WO2016172659A1 (fr) * 2015-04-24 2016-10-27 University Of Florida Research Foundation, Inc. Vecteur de virus adéno-associé pour le traitement de l'ataxie de friedreich
WO2022182983A1 (fr) * 2021-02-26 2022-09-01 Icahn School Of Medicine At Mount Sinai Méthode de réduction de la dégénérescence des cellules ganglionnaires de la rétine

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